Optimisation of Artemia biomass production in salt - PhD

Optimisation of Artemia biomass production in salt ponds in
Vietnam and use as feed ingredient in local aquaculture
Nguyen Thi Ngoc Anh
Promoter:
Prof. Dr. Patrick Sorgeloos
Laboratory of Aquaculture & Artemia Reference Center
Faculty of Bioscience Engineering, Ghent University
Co-promoter:
Dr. Nguyen Van Hoa
College of Aquaculture & Fisheries, Can Tho University, Vietnam
Dean:
Prof. Dr. ir. Guido Van Huylenbroeck
Rector:
Prof. Dr. Paul Van Cauwenberge
Examination Committee and Reading Committee (*):
Prof. Dr. ir. Jacques Viaene (Chairman)
Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent
University. [email protected]
Prof. Dr. Peter Goethals (Secretary)
Department of Applied ecology and environmental biology, Faculty of Bioscience
Engineering, Ghent University. [email protected]
Prof. Dr. Patrick Sorgeloos (Promoter), Department of Animal Production,
Faculty of Bioscience Engineering, Ghent University), [email protected]
Prof. Dr. ir. Peter Bossier (Department of Animal Production, Faculty of Bioscience
Engineering, Ghent University), [email protected]
*Dr. Nguyen Van Hoa (Co-promoter), College of Aquaculture & Fisheries, Can Tho
University, Vietnam. [email protected]
*Prof. dr. Johan Mertens (Department of Biology, Faculty of Sciences, Ghent University)
[email protected]
*Prof. Dr. Josse De Baerdemaeker (Laboratory of Agricultural Machinery & Processing,
Department of Agro-Engineering and Economics, Katholic University of Leuven).
[email protected]
*Dr. Roeland Wouters (INVE Technologies, Belgium), [email protected]
Nguyen Thi Ngoc Anh
Optimisation of Artemia biomass production in salt ponds in
Vietnam and use as feed ingredient in local aquaculture
Thesis submitted in fulfilment of the requirements for the degree of Doctor
(PhD) in Applied Biological Sciences
Dutch translation of the title:
Optimalisatie van Artemia biomassa productie in zoutpannes in Vietnam and gebruik als
voeder-ingredient in lokale aquacultuur
To cite this work:
Anh, N.T.N. (2009) Optimisation of Artemia biomass production in salt ponds in Vietnam
and use as feed ingredient in local aquaculture. PhD thesis, Ghent University, Belgium.
The author and the promoters give the authorisation to consult and to copy parts of this
work for personal use only. Every other use is subject to the copyright laws. Permission to
reproduce any material contained in this work should be obtained from the author.
ISBN 978-90-5989-308-5
This study was funded by Vietnamese government, PhD scholarship of the Vietnamese
Overseas Scholarship Program (322 project), and the CWO scholarship of the Faculty of
Bioscience Engineering, Ghent University, Belgium.
Acknowledgements
First of all, I would like to express my deep gratitude to my promoter, Prof. dr. Patrick
Sorgeloos for giving me the opportunity to study at Ghent University. His scientific
orientation, encouragement, and support during my four year study, especially his patience
in correcting the papers and final thesis drafts during his already busy time.
Special thank to my co-promoter Dr. Nguyen Van Hoa (Can Tho University, Vietnam) for
his scientific guidance, encouragement and experience on Artemia research.
My sincere thanks go to my supervisors Dr. Gilbert Van Stappen and Mathieu Wille for
their devoted and thoughtful revision and recommendations in the preparation and
completion of all chapters of the thesis.
I sincerely thank Prof. Dr. Josse De Baerdemaeker (Catholic University of Leuven,
Belgium), Nguyen Thuan Nhi (College of Technology, Can Tho University) for giving me
the basic knowledge and critical suggestions in the design of the experimental solar drier,
and Dr. Vu Quang Thanh for instruction in indoor drying techniques and allowing me to
utilize the drying machines and equipment, and Phan Thanh Dung for his help during the
drying experiment in the College of Technology, Can Tho University.
I am grateful to Prof. Dr. Truong Quoc Phu, Dr. Tran Thi Thanh Hien, Dr. Vu Ngoc Ut and
Duong Thuy Yen (College of Aquaculture and Fisheries, Can Tho University) for their
proper suggestions on experimental designs, feed formulation and for providing me with
facilities and space to perform the feeding trials.
I am greatly indebted to Peter Baert for his valuable recommendations with data-processing
on the Artemia experiments and also willing to help me whenever needed.
I am especially thankful to the members of the examination and reading committee, Prof.
Dr. ir. Jacques Viaene; Prof. Dr. Patrick Sorgeloos, Prof. Dr. ir. Peter Bossier, Prof. Dr.
Johan Mertens, Prof. Dr. Peter Goethals (Ghent University), Prof. Dr. Josse De
Baerdemaeker (Catholic University of Leuven), Dr. Nguyen Van Hoa (Can Tho University)
and Dr. Roeland Wouters (INVE Technologies NV, Belgium) for their critical reviews and
extremely valuable suggestions to improve this thesis.
My warmest thanks go to Magda Vanhooren, who kindly helped me whenever needed.
I am greatly indebted to Geert Van de Wiele and Anita De Haese for HUFA and proximate
composition analyses of the Artemia samples.
I deeply thank the staff of ARC: Dorina Tack, Alex Pieters, Marc Verschraeghen, Jean
Dhont, Bart Van Delsen, Kristof Dierckens, Marijke Van Speybroeck, Christ Mahieu,
Sebastiaan Vanopstal, Brigitte Van Moffaert, Tom Baelemans, Jorg De Smyter, for their
help with administrative matters.
Special acknowledgements are due to Prof. Dr. Nguyen Anh Tuan, the rector of Can Tho
University and Prof. Dr. Nguyen Thanh Phuong, the dean of the College of Aquaculture &
Fisheries, for allowing me to study abroad.
Many thanks to Dr. Duong Nhut Long, Dr. Nguyen Van Kiem, Dr. Tran Ngoc Hai and Dr.
Ngo Thi Thu Thao from the College of Aquaculture & Fisheries for their support and
providing me enough free time to accomplish this thesis.
I owe special thanks to my colleagues Huynh Thanh Toi for his endless kindness and all his
help in Ghent and in Vietnam, Nguyen Thi Hong Van for her provision of scientific
journals, encouragement and supporting experimental equipment, Tran Huu Le and Le Van
Thong for helping me with filtering chlorophyll a samples, transporting water samples to
Can Tho for analysis and biomass collection.
I greatly appreciate Le Van Nhieu, Phan Thanh Phuoc, Giang Van Nghiep, Giang Van Hay,
Nguyen Duyen Hai, Nguyen Thi Phuong, Tran Thi Yen who have been devoted hard
workers in Bac Lieu salt works and for their enthusiasm and efficient support during my
field study in Vinh Hau station, Bac Lieu province.
Many thanks to my PhD colleagues: El-Magsodi Mohamed, Natrah Ikhsan, Kartik Sri
Barua, Gunasekara Asanka, Dang To Van Cam, Le Hong Phuoc, Dinh The Nhan, Nguyen
Duy Hoa, Nhu Van Can, Ho Manh Tuan and others for their encouragement and support
during my study in Ghent.
To all Vietnamese students in Ghent, I thank you for your moral support during my stays in
Ghent. I would like to express my warmest feelings to all my friends and my colleagues in
various institutions and universities, Can Tho University and College of Aquaculture and
Fisheries, who always were concerned about my PhD completion.
I am very grateful to the Ministry of Education & Training, Vietnamese Government for
providing me with a scholarship to pursue my PhD study and the Faculty of Bioscience
Engineering, Ghent University, Belgium through the CWO scholarship for the defence of
this thesis.
My great gratefulness goes to my grandmother, my aunts, my brothers and sisters who
always encouraged me to finish my PhD, especially my mother who always gave me all
physical and moral support, but unfortunately does not live anymore.
I wish to dedicate this thesis to my husband Phan Huu Tam, who has sacrificed a lot during
my four years intensive study. This thesis is a present for him.
TABLE OF CONTENTS
Chapter 1 General introduction .......................................................................................... 1
Chapter 2 Literature review ................................................................................................ 7
Chapter 3 Culture of Artemia biomass
Section I Effect of partial harvesting strategies on Artemia biomass production in salt
works ......................................................................................................... 31
Section II Effect of different food supplements on proximate compositions and
Artemia biomass production in salt works ................................................ 47
Secttion III Effect of different ratios of N:P on primary productivity: its combination
with feeding strategies for Artemia biomass production in salt ponds...... 69
Chapter 4 Drying Artemia biomass
Section I Total lipid and fatty acid contents of Artemia biomass dried using different
drying techniques .................................................................................... 109
Section II Effect of solar drying on lipid and fatty acid composition of dried Artemia
biomass .................................................................................................... 117
Chapter 5 Application of Artemia biomass for target aquaculture species
Section I Formulated feeds containing fresh or dried Artemia biomass as live food
supplement for larval rearing of black tiger shrimp, Penaeus monodon 151
Section II Effect of fishmeal replacement with Artemia biomass as protein source in
practical diets for the giant freshwater prawn Macrobrachium rosenbergii
................................................................................................................. 141
Section III Effect of different forms of Artemia biomass as a food source on survival,
molting and growth rate of mud crab, Scylla paramamosain ................. 157
Section IV Substituting fishmeal with Artemia meal in diets for goby
Pseudapocryptes elongatus: effects on survival, growth and feed
utilization ................................................................................................. 173
Chapter 6 General discussion and conclusions .............................................................. 207
Chapter 7 References ....................................................................................................... 216
Summary/Samenvatting .................................................................................................. 239
Curriculum vitae .............................................................................................................. 247
i
List of abbreviations
Σ
Total
°C
Degree Cencius
ANOVA
Analysis of variance
AOAC
Association of Official Analytical Chemists
APHA
American Public Health Association
ARA
Arachidonic acid
BHT
Butylated hydroxytoluene
C:N
C-to-N ratio
C1
First crab stage
Ca
Calcium
CF
Commercial feed
cm
Centimeter
CMI
Cumulative mortality index
DA
Dried Artemia
DHA
Docosahexaenoic acid
DIN
Dissolved inorganic nitrogen
DRP
Dissolved reactive phosphorus
DW
Dry weight
EFA
Essential fatty acid
EPA
Eicosapentaenoic acid
FA
Fresh/frozen Artemia
FAME
Fatty acid methyl easters
FAO
Food and agriculture organization
FFA
Free fatty acid
FM
Fish meal
g
Gram
GW
Green water
h
Hour
HA
Hot air
HUFA
Highly unsaturated fatty acid
L
Liter
ii
LIA
Linoleic acid
LNA
Linolenic acid
min
Minute
MKD
Mekong delta
ml
Milliliter
mm
Millimeter
MoFI
Ministry of Fisheries
MUFA
Mono-unsaturated fatty acid
MW
Microwave
N:P
N-to-P ratio
P
Phosphorus
PL
Polar lipid
PL 15
Postlarvae 15
PM
Pig manure
PUFA
Poly-unsaturated fatty acid
RB
Rice bran
SB
Soybean meal
SE
Standard error
SFA
Saturated fatty acid
TAG
Triacylglycerols
TAN
Total ammonia nitrogen
TN
Total nitrogen
TP
Total phosphorus
VIAE
Vietnam Institute of Agricultural Engineering
VND
Vietnam dong
WW
Wet weight
iii
CHAPTER
General introduction and thesis outline
1
2
Chapter 1
General introduction
Populations of the brine shrimp Artemia (Crustacea, Anostraca) are typical inhabitants of
extreme environments, such as hypersaline inland lakes, coastal lagoons, and solar salt
works, distributed all over the world, and characterized by communities with low species
diversity and simple trophic structures (Lenz, 1987; Lenz and Browne, 1991). Artemia can
be found in a great variety of habitats in terms of water chemistry (Lenz, 1987; Bowen et
al., 1988), altitude (Triantaphyllidis et al., 1998; Van Stappen, 2002) and climatic
conditions, from humid-subhumid to arid areas (Vanhaecke et al., 1987).
The first use of Artemia nauplii, hatched from cysts, is known from the 1930s when this
zooplankton organism was used as a suitable food source for fish larvae in the culture of
commercially important species (Sorgeloos, 1980b; Léger et al., 1986). Since then, Artemia
has been found to be a suitable food for diverse groups of organisms of the animal
kingdom, especially for a wide variety of marine and freshwater crustaceans and fishes
(Sorgeloos, 1980b). Also decapsulated Artemia cysts, juvenile and adult Artemia have
increasingly been used as appropriate diets for different fish and crustacean species
(Sorgeloos et al., 1998; Dhont and Sorgeloos, 2002; Lim et al., 2003).
Since the early 1990s cyst consumption has increased exponentially as a consequence of the
rapidly expanding shrimp and marine fish industries (Sorgeloos et al., 2001; Dhont and Van
Stappen, 2003). On the other hand, the limited supply of Artemia cysts, originating from
natural harvests, may lead to a serious bottleneck in many aquaculture developments
(Lavens and Sorgeloos, 2000b). In particular, in South East Asia where no natural
populations of Artemia occur, therefore diversification of Artemia sources has been
considered a possible solution to sustain the fast growing aquaculture industry. This
strategy has been performed by the exploration of natural harvesting from new Artemia
sites such as China (Xin et al., 1994), Iran (Van Stappen et al., 2001), Mexico and Chile
(Castro et al., 2006) etc. Furthermore, man-made introduction of Artemia into saltworks
and man-made ponds has also contributed to supplement cyst supply. This approach has
been conducted during the last couples of decades in several countries with a monsoon
climate. For instance, Philippines (De Los Santos et al., 1980), Thailand (Tarnchalanukit
and Wongrat, 1987), Vietnam (Quynh and Lam, 1987; Brands et al., 1995) and other
countries such as India, Sri Lanka, Iran (Hoa et al., 2007).
1
Chapter 1
In Vietnam, Artemia production is successfully conducted on a seasonal basis in the
coastal areas of the Mekong Delta, southern Vietnam (Brands et al., 1995; Baert et al.,
1997). To date this region is an important supplier of high-quality Artemia cysts that are
used in domestic aquaculture as well as for export. This activity has had significant positive
socio-economic impacts for the local rural populations (Hoa et al., 2007; Son, 2008). In
practice, cysts produced during the previous culture season are used to establish, by
inoculation, a new population of Artemia. This practice may favour the accumulation of
adaptations to the new environment (Frankenberg et al., 2000). This Artemia culture system
is referred to as semi-intensive (Tackaert and Sorgeloos, 1991) and static (Quynh and Lam,
1987; Brands et al., 1995). Semi-intensive refers to small seasonal man-managed ponds in
which Artemia is inoculated at high densities (between 60 and 100 nauplii L-1). Ponds are
managed intensively (i.e. inoculation of selected strains, manipulation of primary and
secondary production, predator control, etc.) but most of the management procedures are
empirical. Furthermore, Artemia production in Vietnam has largely focused on cyst
production, and all techniques and methodologies developed to optimize Artemia
production have used maximal high-quality cyst production as their primary target (Brands
et al., 1995; Baert et al., 1997; Hoa et al., 2007).
Artemia is a non-selective particle feeder, feeding on microalgae, detritus and bacteria,
where the only limiting factor is the size of the ingested particles (Van Stappen, 1996;
Fernández, 2001; Dhont and Sorgeloos, 2002). Although the feeding and filtration biology
of Artemia has been studied in laboratory tests (Coutteau and Sorgeloos, 1989; Evjemo and
Olsen, 1999; Fernández, 2001), up to now, this type of study has not been extended to the
field, and there is very little information on optimal Artemia biomass production in salt
works. Artemia biomass is an excellent food source in aquaculture as it converts detritus
and phytoplankton into high-quality proteins, thus extracting nutrients from the aquatic
environment (Sorgeloos, 1985). Artemia biomass is valorised as a high-quality feed for
ornamental fish (Lim et al., 2001; 2003), as a nursery food for marine fish, shrimp, prawn
and crab (Merchie, 1996; Sorgeloos et al., 1998; Dhont and Sorgeloos, 2002), as an overall
high-protein ingredient for aquaculture feeds, and as maturation trigger in shrimp
broodstock (Naessens et al., 1997; Wouters et al., 2002).
In pond systems, the success of Artemia cyst and biomass production relies on the
favourable growth of the Artemia population after inoculation. This growth is, amongst
others, significantly influenced by the food management of the culture ponds. The final
2
Chapter 1
yield of Artemia biomass can also be considerably affected by various technical aspects,
such as harvesting strategies (Brands et al., 1995; Baert et al., 1996, Anh and Hoa, 2004).
Hence, substantial research is required to (1) optimize culture techniques, in particular in
relation to the effects of organic and inorganic fertilizers on the production of microalgae as
a natural food for Artemia, (2) on the use of supplementary feeds and (3) on adequate
harvesting strategies. Moreover, there is a need for (4) research into the possible
applications of Artemia biomass products in Vietnamese aquaculture. Farming of highly
valuable aquaculture species in the Mekong delta has been studied for several species such
as Penaeid shrimp (Nghia et al., 1997a,b; Phuong et al., 2008), freshwater prawn
Macrobrachium rosenbergii (Thang, 1995; Lan et al., 2006). Similar work exists for the
mud crab Scylla spp. (Dat, 1999; Ut et al., 2007a,b) and for different types of polyculture of
marine and freshwater species (Rothuis et al., 1998; Minh et al., 2001; Lan et al., 2003).
Recently, Vietnamese aquaculture activities have been expanding with the culture of new
target marine aquatic species such as swimming crab (Portunidae), cobia (Rachycentridae),
grouper (Serranidae), goby (Gobiidae), eel (Anguillidae), Areola babylon (Buccinidae), etc.
(MoFI, 2006). These new species offer opportunities for diversification in the use of
Artemia, including live juveniles and adults as well as frozen or dried Artemia biomass.
This indicates that there is a high potential market for Artemia biomass not only in the
Mekong Delta but also along the coast line of central Vietnam (Hoa et al., 2007).
Research objectives
The general objectives of this thesis are firstly to improve Artemia pond management in
terms of the supply of natural and supplementary foods, and by adaptation of biomass
harvesting strategies. It also aims to develop a simple and cheap processing technique for
Artemia biomass, resulting in a product which is suitable for application in local
aquaculture operations in the Mekong Delta.
3
Chapter 1
The specific objectives and the thesis outline are as follows:
Chapter 1 (General introduction and thesis outline) describes an outline covering the
main topics of this thesis.
Chapter 2 (Literature study) presents the biology and ecology of Artemia, and gives an
overview of aquaculture as well as the history of Artemia study in Vietnam. It comprises
general geographic and climatological information on the site where the field research has
been conducted. It describes the general principles of Artemia biomass pond production in
this area, and its various applications in local aquaculture. It also provides a summary of
drying methods currently used in food and feed processing technology.
Chapter 3 (Culture of Artemia biomass) describes the experimental work aiming to
optimize Artemia biomass production in salt ponds. This chapter consists of three parts:
- Effect of partial harvesting strategies on Artemia biomass production in salt works
(Section I)
- Effect of different food supplements on proximate compositions and Artemia biomass
production in salt works (Section II)
- Effect of different ratios of N:P on primary productivity: its combination with feeding
strategies for Artemia biomass production in salt ponds (Section III)
Chapter 4 (Drying Artemia biomass) describes tests aiming to work out a simple and
cheap drying method for Artemia biomass, resulting in a product with appropriate quality
for use in aquafeeds. It comprises two parts:
- Total lipid and fatty acid contents of Artemia biomass dried using different drying
techniques (Section I)
- Effect of solar drying on lipid and fatty acid composition of dried Artemia biomass
(Section II)
4
Chapter 1
Chapter 5 (Application of Artemia biomass for some target aquaculture species)
evaluates the potential uses of different Artemia biomass preparations as feeds in the
larviculture and nursery phases of the important cultured species in the Mekong delta. It
contains four parts.
- Formulated feeds containing fresh or dried Artemia biomass as live food supplement for
larval rearing of black tiger shrimp, Penaeus monodon (Section I)
- Effect of fishmeal replacement with Artemia biomass as protein source in practical diets
for the giant freshwater prawn Macrobrachium rosenbergii (Section II)
- Effect of different forms of Artemia biomass as a food source on survival, molting and
growth rate of mud crab Scylla paramamosain (Section III)
- Substituting fishmeal with Artemia meal in diets for goby Pseudapocryptes elongatus:
effects on survival, growth and feed utilization (Section IV)
Chapter 6 (General discussion) restates and discusses the overall results of the
experiments conducted in this thesis. Based on the discussion, the general conclusions are
drawn and prospective research topics are proposed.
Chapter 7 (References) contains all the bibliographic citations mentioned in this thesis.
5
Chapter 1
6
CHAPTER
Literature study
Chapter 2
Literature study
1. Overview of aquaculture in Vietnam
Globally, aquaculture is the fastest growing food-producing sector, with a total production
of almost 63 million metric tonnes in 2005, more than five times the amount produced in
1985. Asian countries play an important role in the development of the aquaculture sector,
accounting for more than 90% of global aquaculture production. Three quarters of global
aquaculture production is generated from Asian countries, with China and Vietnam ranking
as the first and third global producers. Among the top ten Asian producers, Vietnam
experienced the fastest growth between 1985 and 2005, which has raised concerns among
several stakeholders concerning the sustainability of the aquaculture sector in Vietnam
(Corsin, 2007).
Vietnam has great potential for aquaculture development including marine, brackish and
fresh waters, all of which are widely available throughout much of the country. There are
3,260 km of coastline, 12 lagoons, straits and bays, 112 estuaries, canals and thousands of
big and small islands scattered along the coast. On the land, an interlacing network of
rivers, canals, irrigation and hydroelectric reservoirs has created a great potential of water
surface with an area of about 1.7 million ha (Final report of Ministry of Fisheries (MoFI)
and World Bank, 2005).
In 2006, the total area of water surface used for aquaculture in Vietnam was 1,050 thousand
ha, which represents a 64% increase over the 641.9 thousand ha used in 2000. A variety of
species are cultivated in these waters, but shrimp and catfish are by far the most prevalent.
Total aquatic production increased almost 7% in 2006, while aquaculture production
increased 14.6% (Huong and Quan, 2007). The rapid development of the aquaculture sector
achieved during the last two decades has been a direct result of the sector diversifying its
farming practices and adapting to the production of exportable species at increased levels of
intensification. In 2007 the total aquaculture area accounted for more than one thousand
hectares and it was reported that the total aquaculture area and productivity have increased
2.1 and 2.5 times, respectively, compared to that in 1996. A similar tendency was also
found in the Mekong Delta (MKD) in South Vietnam (Figure 1). In addition, the total
output value of aquaculture in 2006 was 47,446.9 billion VNDs, which corresponds with a
7
Chapter 2
4.03 times increase, compared with 11,761 billion VNDs in 2000 (General Statistics Office,
2008; www.gso.gov.vn/default/news).
5,000,000
1,200,000
4,000,000
800,000
3,000,000
600,000
2,000,000
400,000
Productivity (ton)
Culture area (ha)
1,000,000
1,000,000
200,000
0
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Total area
MKD area
Total productivity
MKD productivity
Figure 1. Aquaculture culture area and productivity in Vietnam in general and in the
Mekong delta between 1996 and 2007 (Source: Data from General Statistics Office,
Vietnam, 2008)
The Mekong Delta has an area of 3.9 million ha, accounting for 12% of the total area of
Vietnam. Agriculture occupies 83% of the total delta so it plays an important role in the
development of the economy in Vietnam (Ni et al., 2003). In addition, this delta is the most
important region in Vietnam for both fisheries and aquaculture, accounting for 43 and 67 %
of the nation’s total production, respectively, and for 57 % of the total export values in
2003. The Mekong Delta has also the most diversified aquaculture farming activities and
has a large potential for increased aquaculture production (MoFI, 2005). The culture
systems include pond, fence and cage culture of catfish (Pangasius) as well as several
indigenous species such as snakehead fish (Channa), climbing perch (Anabas) and giant
freshwater prawn (Macrobrachium). Moreover, integrated farming systems such as ricecum-fish, rice-cum-prawn and mangrove-cum-aquaculture are broadly practiced across this
region (Minh et al., 2001; MoFI, 2006). Particularly, the Vietnamese Government has
promulgated a long term planning to develop sustainable aquaculture in the country,
namely “Decision No. 10/2006/QÐ-TTg dated Jan. 11, 2006.” The objectives of this plan
are the following: aquaculture production in 2010 will be about 2 million tonnes, including
8
Chapter 2
0.98 million tonnes from fresh water aquaculture and 1.02 million tonnes of marine and
brackish water aquaculture; 1.1-1.4 million ha of water bodies will be exploited for
aquaculture activities, of which there are 0.6 million ha of freshwater area and 0.7 million
ha of brackish water and marine areas. In parallel, applied research, education and training
activities have been developed to meet the need for the sustainable and effective
development of the fisheries sector, particularly in aquaculture during the period 20052010.
2. Trends in the use of fishmeal in Vietnam
Since aquaculture is developing rapidly in Vietnam, the future demand for fish meal (FM)
as an ingredient in aquafeeds is expected to increase dramatically. FM availability in
Vietnam is low and FM produced domestically is mostly of poor quality because of
inadequate preservation of trash fish on board. Consequently, Vietnam only uses
domestically produced FM for livestock and some freshwater fish for grow-out feed as it is
generally of low quality. FM for higher quality feed for fish fingerlings and crustaceans is
imported and represents about 90% of the total FM used. Fish oil for aquafeed manufacture
is also imported (Edwards et al., 2004).
A prognosis made by MoFI (2006) shows that about 150,000-200,000 tonnes of FM will be
required over the next decade for aquaculture, two to three times the present level of use.
However, the price of imported FM continues to rise and therefore the development of
Vietnamese aquaculture will be influenced strongly by the price for FM and oil on the
international market (Edwards et al., 2004). Moreover, Huong and Quan (2007) reported
that the aquatic feed industry is scrambling to keep pace with increased demand for
commercially made feed from the booming aquaculture industry in the Mekong Delta
where the major aquaculture activities in Vietnam are located. Latest available statistics
indicate that Vietnam’s 39 industrial aquatic feed producers in 2001 had a production
capacity of about 50,000 tonnes year-1. This would only satisfy about 40% of today’s
aquaculture feed demand. More and more the trend among farmers is to replace traditional
home-made feed with industrial feed, hence the higher demand for industrial fish feed. Feed
cost may contribute to more than 50% of the total cost of an aquaculture operation.
Moreover, the MoFI target of 200,000 tons of marine fish by 2010 would in theory require
at least 2 million tonnes of trash fish based on current practices, which is unattainable
without investments in more efficient feeds and feeding practices. Consequently, due to the
9
Chapter 2
need to reduce the dependence of the aquaculture industry upon a wild and finite food
resource, feed manufacturers and researchers alike have spent considerable time and effort
in trying to find dietary replacements for FM and fish oil within compound aquafeeds. This
has contributed to a reduction in input costs for production (MoFI, 2005). The development
of alternative dietary protein and lipid sources as partial or total replacements of FM in the
formulated aquafeeds, have been intensively studied and applied. Different sources of
materials can be used, especially cheap raw materials which are locally available, such as
terrestrial animal by-products, fish offal from processing and agricultural by-products,
(FAO, 2002).
3. Overview of main target aquaculture species
Some target aquaculture species in the Mekong delta with high economic values on the
home and export markets are briefly described below:
Black tiger shrimp (Penaeus monodon)
Shrimp culture is considered as one of the most lucrative industries because of its high
market price and great demand on the international market. The black tiger shrimp Penaeus
monodon is the most prominent farmed crustacean products in international trade, and has
driven a significant expansion in aquaculture in many developing countries in Asia. Global
aquaculture production of P. monodon increased gradually from 21,000 tonnes in 1981 to
650,000 tons in 2005 (http://www.fao.org/fishery/culturedspecies/Penaeus_monodon).
In Vietnam, P. monodon is the most popular cultured species, accounting for 290,987
tonnes out of the total shrimp production of 386,596 tons in 2007. Farming of this species
has developed rapidly and has proven its success by producing an export turn-over of
appropriately 1.3-1.5 billion US dollars, annually (General Statistics Office, 2008). In the
Mekong delta, marine shrimp farming has been operated in different systems and levels of
intensification, including extensive, improved extensive, semi-intensive, intensive,
integrated shrimp-mangrove, and rotational rice-shrimp culture systems (Nghia et al.,
1997c; Sinh et al., 2006). The extensive and improved extensive culture systems were most
applied, accounting for 90% and intensive culture accounting for 10% of the total shrimp
culture area in 2005. Productivity was 0.2-0.3; 2.5-3.0 and 5.0-7.0 tonnes ha-1 crop-1 for the
improved extensive, semi-intensive and intensive shrimp farming practices, respectively
10
Chapter 2
(MoFI, 2005; 2006). Total culture area and productivity increased by 3.1 and 6.7 times
respectively from 1999 to 2007, with an average annual increase of 35.3% (General
Statistics Office, 2008). However, the rapid expansion of shrimp farming has brought about
some problems such as lack of high quality shrimp postlarvae and knowledge of culture
techniques. Moreover, the existing irrigation systems do not meet the need for appropriate
operation of shrimp farming. As a result, the frequent occurrence of diseases is a major
constraint to the sustainability in the culture regions (MoFI, 2006). To reduce this risk,
alternative culture species and diversification of species have been taken into account.
Recently, white shrimp Litopenaues vannamei has become the important cultured shrimp
species, the production of the white shrimp in Vietnam was 6,268 tons in 2005 (MoFI,
2006).
Mud crab (Scylla spp.)
Exploitation of the world’s mud crab resources has increased dramatically over the past 30
years and aquaculture of mud crab, Scylla spp., contributes largely to the world production
of the genus (FAO FIGIS database; www.fao.org/ES/ess/figis.asp). Moreover, mud crabs
are large size animals with high nutritional value, and are of high commercial value in
Southeast Asian countries (Keenan, 1999).
In Vietnam, capture fisheries of mud crab is about 1400 tonnes annually and large part
obtained from aquaculture and approximately 6,000 tonnes of crab was exported to China
in 2004, accounting for 25 millions US dollars (MoFI, 2005). Mud crab has been the first
candidate as alternative to shrimp culture because it is the only species that has been
cultured traditionally in the coastal area besides shrimp (Dat, 1999; Ut, 2003). The natural
potential for mud crab culture development in Vietnam is great; with 858,000 ha of marine
and brackish water area available for shrimp and mud crab culture (Lindner, 2005).
Moreover, Thach (2007) reported that the total area of semi-intensive and intensive mud
crab culture has been estimated to be well in excess of 40,000 ha. Mud crab Scylla
paramamosain is the most prevalent species used in aquaculture farms in the Mekong delta
(MKD), and has become increasingly popular over the past decades (Nghia et al., 2001; Ut,
2003). In 1995, the culture area and production of crab in the Mekong delta were 3086 ha
and 1644 tonnes (Tuan et al., 1996) whereas in 2004 they were estimated to be about 7,500
ha and over 7,000 tonnes (MoFI, 2005). Many farmers have indicated that their income
from crab is more reliable than that from shrimp (Ut et al., 2007c).
11
Chapter 2
The diverse culture systems used to farm crabs are similar to the range of methods used for
shrimp aquaculture: polyculture systems in which two or more aquatic species are raised
together are quite common in the Mekong delta: grow-out of crabs from juvenile to
marketable size in open extensive mangrove forest-aquaculture-fishery farms or semiintensive pond units; fattening of immature females to maturity; fattening of ‘thin’ crabs;
and soft-shell crab production (Dat, 1999; Ut et al., 2007c). A 2005 survey of the eight
eastern coastal provinces in the MKD indicated that crab productivity was highest (1,008
kg ha-1) in monoculture and lowest (75 kg ha-1) in mangrove-shrimp integrated culture (Ut
et al., 2007c). In the improved extensive systems of mud crab stocked at a density of 1 crab
5-10 m-2, yields are usually about 200-300 kg ha-1 crop-1 while in intensive aquaculture
systems in which mud crab is stocked at 1-2 crab m-2 1.5-2.0 tonnes ha-1 crop-1 can be
obtained, although yields of about 1 ton ha-1 are more common (Lindner, 2005; Thach,
2007). However, in previous years mud crab farming relied entirely on wild seed stock and
the main obstacle for the development of mud crab culture is the availability of hatcheryreared seed (Keenan, 1999; Xuan, 2001; Ut, 2003).
Nowadays, mud crab reproductive technology has been improved remarkably; over 100
millions of mud crab seeds are produced from more than 150 mud crab hatcheries yearly.
The seeds are reared on 115,276 ha of land and produce about 480 tonnes of commercial
mud crab per year. Seed production techniques have been perfected and the supply is
considered reliable. Most crab aquaculture production now relies on commercial hatcheryreared stocks Therefore, expansion of mud crab aquaculture is realistic (Lindner, 2005;
Thach, 2007). Nonetheless, hatchery-produced seed supplied to farmers is typically in the
form of small postlarval crabs between 4 and 8 mm carapace width, resulting in low
survival and productivity (Ut et al., 2007a). Thus a nursery period is a necessary
intermediate step in crab production between hatchery and grow-out, to grow postlarvae to
a size appropriate for transport and release into large extensive to intensive production
systems (Ut et al., 2007a; Rodriguez et al., 2007). Additionally, with respect to economic
efficiency, utilizing locally available feeds for nursery of crab postlarvae to achieve high
survival and good growth also play an important role as crabs are characterised by their
high cannibalism.
12
Chapter 2
Goby (Pseudapocryptes elongatus)
The goby, Pseudapocryptes elongatus is a commercially important species for food in
Japan and Taiwan (Ip et al., 1990); It is also a highly valuable fish for domestic
consumption as well as for exportation in Vietnam (Dinh et al., 2004), It is well adapted to
a wide range of environments so that recently it has become an important alternative
species for integrated brackish water aquaculture (Dinh et al., 2004; Khanh, 2006).
According to the Agriculture Statistics, the alternative goby-shrimp or goby-salt culture
models have widely been applied in the coastal provinces of the Mekong delta such as Soc
Trang, Ca Mau, Ben Tre and Tra Vinh. This can help the farmers to improve their income
and concurrently contribute to diminish the vulnerability of shrimp culture. The production
areas have increased noticeably from a few hectares in 2000 to 1,500 ha in 2008, in which
Soc Trang and Bac Lieu made up 800 ha (www.vnn.vn/kinhte/2008/10/807666/). In
extensive goby culture in salt works in the rainy season, the yield was 0.5-0.7 tonnes ha-1
crop-1 and the income was 20-25 millions VND ha-1 (Bac Lieu Extension Service, 2007).
Survey data also show that the semi-intensive and intensive culture of goby in shrimp
ponds has been developing rapidly in the coastal provinces of the Mekong Delta in recent
years: at stocking densities of 30-100 fish m-2, after 4-5 months of culture, productivity was
in the range of 3-6 tonnes ha-1 crop-1, and net income was between 50 and 100 millions
VND ha-1 crop-1 (Nhon, 2008). Nevertheless, the constraint in goby culture is that the seed
supply completely relies on the wild; the quality of fingerlings is usually unstable and their
size is small causing high mortalities and low productivity (Khanh, 2006; Chung, 2007).
Hence, nursery of goby using local available high quality feed to attain larger size fry with
good quality for stocking in grow-out ponds is essential. This approach could contribute to
improve profitability for farmers and the development of the farming of this species.
Giant freshwater prawn (Macrobrachium rosenbergii)
There has been a very rapid global expansion of freshwater prawn farming since 1995. The
total global production of Macrobrachium is estimated to reach 0.8 - 1.0 million tonnes
year-1 by the end of this decade; most of which is produced in Asia in which China is the
leader, followed by Vietnam and India (New, 2005). Currently, the giant freshwater prawn
(Macrobrachium rosenbergii), which is indigenous to the Mekong river Delta, Vietnam, is
becoming an increasingly important target species for aquaculture (Phuong et al., 2006b).
The total culture area and production of M. rosenbergii in the Mekong Delta were 4,759 ha
13
Chapter 2
and 3,358 tonnes in 2003 (MoFI, 2005) and 9,077 ha and 9,514 tonnes in 2006 (Sinh,
2008). This species is cultured in ponds, pens and integrated or alternated with paddy rice
production; alternate culture of rice with prawn is considered to have high potential to raise
the income among impoverished farmers and to contribute to enhance rural development in
Vietnam (Khanh and Phuong, 2005; Lan et al., 2008). The productivity of prawn culture
varies with the culture practices employed and ranges from 100-887 kg ha-1 crop-1 for
integrated rice-prawn culture systems, to 384-1,681 kg ha-1 crop-1 for alternate rice-prawn
culture (Khanh and Phuong 2005), and 1,400-1,600 ha-1 year-1 for pen culture (Son et al.,
2005).
Advantages of freshwater prawn farming in rice fields are lower stocking densities, lower
investment and feed costs as well as absence of major diseases as associated with marine
shrimp farming (Phuong et al., 2006a). Besides, M. rosenbergii prawn can grow well in
brackish waters having salinity up to 10 g L-1 (New, 2002; Cheng et al., 2003), thus prawn
culture has been considered as an alternative to shrimp in low salinity areas and coastal
saline soils in order to reduce the threats of disease outbreaks in tiger shrimp, P. monodon.
Increase in prawn culture has led to a growing demand for postlarvae (PL) from hatcheries
and most prawn farmers desire to stock PL with larger size to reduce mortality and shorten
the grow-out period. Feed is the single largest cost item for M. rosenbergii culture, as it
constitutes 40-60% of the operational costs (Phuong et al., 2003; Mitra et al., 2005). Hence,
nursery of prawn PL using locally available protein sources as an ingredient in practical
diets to improve cost-effectiveness with higher survival and better growth is necessary.
4. Biology and ecology of Artemia
The genus of the brine shrimp Artemia consists of several bisexual species, identified by
reproductive isolation, and numerous parthenogenetic populations (Triantaphyllidis et al.,
1998; Abatzopoulos et al., 2002). The systematic classification of the genus is as follows:
Phylum: Arthropoda
Class: Crustacea
Subclass: Branchiopoda
Order: Anostraca
Family: Artemiidae
Genus: Artemia, Leach 1819
14
Chapter 2
Artemia is a cosmopolitan organism, inhabiting coastal lagoons as well as inland salt lakes
where there are no or few predators and competitors. In these hypersaline environments
which are not tolerable by other filter feeders, brine shrimp survive thanks to their
physiological adaptations. Artemia distribution is not continuous; the populations are found
throughout the tropical, subtropical and temperate climate zones (Persoone and Sorgeloos,
1980; Van Stappen, 1996). The geographical isolation of Artemia populations has led to
numerous geographical strains that have adapted to conditions that fluctuate widely with
regard to temperature, salinity and ionic composition of the biotope (Bowen et al., 1985,
1988). Artemia can be found at altitudes from as low as sea level up to almost 4,500 meters
e.g. Tibet (Abatzoupolos et al., 1998; Van Stappen, 2002) and in climatological conditions
ranging from humid, sub-humid to arid regions (Vanhaecke et al., 1987).
Figure 2 summarizes the life cycle of Artemia. The fertilized eggs in the brood pouch of the
female develop into either free-swimming nauplii (ovoviviparous reproduction) or,
alternatively, when reaching the gastrula stage, they are surrounded by a thick shell and are
deposited as cysts, which are in diapause (oviparous reproduction) (Jumalon et al., 1981).
Oviparity and ovoviviparity are found in all Artemia strains, switching of reproductive
mode in the natural environment can be expected to vary depending on the environmental
conditions (Lenz, 1987). Other factors such as temperature, salinity, photoperiod and brood
number also potentially contribute to a shift of mode of reproduction (Berthélémy-Okazaki
and Hedgecock, 1987). Females can change in-between two reproduction cycles from
oviparity to ovoviviparity or the other way round, but the entire progeny in a specific brood
will be either cysts or nauplii. The cysts usually float in the high salinity waters and are
blown ashore where they accumulate and dry. As a result of this dehydration process the
diapause mechanism is generally inactivated; cysts are in a state of quiescence and can
resume their further embryonic development when hydrated in optimal hatching conditions
(Van Stappen, 1996).
The postembryonic development continues through about 15 molts in total, and the
organism reaches the adult stage after about two weeks, depending on environmental
conditions (Sorgeloos, 1980a; Clegg and Conte, 1980). Under optimal conditions brine
shrimp can live for several months, grow from nauplius to adult in only 8 days time and
reproduce at a rate of up to 300 nauplii or cysts every 4 days (Van Stappen, 1996; Hoa,
2002). Adult Artemia have an elongated body (up to about 10 mm in length in the bisexual
populations and up to 20 mm in some polyploid parthenogenetic populations) with two
15
Chapter 2
stalked complex eyes, a linear digestive tract, sensorial antennae and 11 pairs of functional
thoracopods. Female Artemia can be recognized by the presence of the uterus between
cephalothorax and abdomen. The male can be differentiated by muscular graspers
(modified 2nd antennae) in the head region, occurring from instar X onwards (Sorgeloos et
al., 1980a; Jumalon et al., 1981; Criel and Macrae, 2002).
Figure 2. Life cycle of Artemia (Jumalon et al., 1981)
5. Use of Artemia in aquaculture
Over the past two decades the brine shrimp Artemia has become a key resource in the
industrial expansion of fish and crustacean larviculture. Annual consumption of Artemia
cysts has increased from a few tons in the mid seventies to over 2,000 tons in recent years
(Sorgeloos, 2001).
The nutritional value of Artemia spp. varies highly among geographical sources and even
from batch to batch; especially Artemia is characterized by low contents of some essential
fatty acids (Léger et al., 1986; Lavens and Sorgeloos, 2000a; Sorgeloos et al., 1998; 2001).
Hence, appropriate techniques have been developed to improve the hatchery use and
maximize the nutritional value of Artemia nauplii, taking advantage of the indiscriminate
filter-feeding behaviour of Artemia (Van Stappen, 1996; Fernández, 2001; Dhont and
Sorgeloos, 2002; Lin and Shi, 2002). Apart from the application of cyst decapsulation
(Garcia-Ortega et al., 2000; Lim et al., 2002) and nauplius cold storage techniques
(Merchie, 1996), Artemia has been used as vehicle for enrichment with selected fatty acids,
vitamins, essential nutrients (Léger et al., 1986; Lavens and Sorgeloos, 2000a; Sorgeloos et
16
Chapter 2
al., 1998; 2001; Camargo et al., 2005) and therapeutic agents (Cook et al., 2003; Gomes et
al., 2007). These developments contributed to the fast expansion of the industrial farming
of several aquaculture species all over the world.
Although Artemia are mostly used under the form of freshly hatched nauplii, more and
more use is made of the juvenile and adult Artemia known as biomass, collected from
natural salt lakes, salinas, man-managed pond productions and intensive culture systems for
use in shrimp nursery and maturation facilities (Léger et al., 1986; Dhert et al., 1993;
Merchie, 1996; Sorgeloos et al., 1998; Dhont and Sorgeloos, 2002). Furthermore, the
nutritional value of on-grown and adult Artemia is superior that of freshly-hatched nauplii,
as they have higher protein content and are richer in essential amino acids and fatty acids
(Léger et al., 1998; Bengtson et al., 1991; Lim et al., 2001; Dhont and Sorgeloos, 2002). In
recent years, the development of new aquaculture species with life-stage specific
requirements has meant diversification in the use of Artemia to include live juvenile and
adults as well as frozen or dried Artemia biomass (Browdy et al., 1989; Dhert et al., 1992b;
1993; Naessens et al., 1997; Olsen et al., 1999; Wouters et al., 2002; Smith et al., 2002;
Lim et al., 2003).
As more attention is given to the use of on-grown Artemia as a cheaper alternative to the
use of nauplii, simple cost-effective production techniques have been developed. The use of
the right size of on-grown Artemia for feeding ensures a better energetic balance in food
intake and assimilation, thereby improving the performance of the fish and shrimp (Dhont
et al., 1993; Merchie, 1996; Lim et al., 2003). Furthermore, its palatability induces a good
and fast feeding response. These characteristics, coupled with the use of bioencapsulation
techniques to enhance the quality of the on-grown Artemia, make this organism an
optimum diet for nursery of the fish (Dhert et al., 1993; Merchie, 1996; Sorgeloos et al.,
2001; Dhont and Sorgeloos, 2002; Lim et al., 2003).
Kim et al. (1996) found that adult Artemia are highly palatable feed items for juvenile
salmonids. First-feeding coho salmon, experienced significantly improved growth when fed
live adult Artemia compared with live nauplii that was likely related to the differences in
size between the two. Based on energy values of 8 kcal g-1 for lipid and 5 kcal g-1 for
protein, the capture of an adult Artemia provided a fish with 240 times more energy than
did the capture of a nauplii. Nonetheless, most of the time, juvenile Artemia are used
instead of adults just before weaning (Lee and Litvak, 1996; Olsen et al., 1999). The study
17
Chapter 2
of Ritar et al. (2003) assessed the effect of Artemia prey size on the survival and growth of
rock lobster larvae. They reported that survival and growth of newly-hatched lobster larvae
cultured to stage III were lower when fed 0.8 mm Artemia than 1.5 mm or 2.5 mm Artemia.
In addition, survival and growth were higher between stages III and V when fed 2.5 mm
Artemia than 1.5 mm Artemia. However, stage VI larvae grew to a similar size at stage VIII
when fed 1.5 mm or 2.5 mm Artemia. Similar observation was reported by Lim et al.
(2003) the cultured on-grown Artemia with size range from 0.45 mm at inoculation to an
average length of about 5 mm was considered suitable for all sizes of freshwater
ornamental fish species of up to 10 cm total length, i.e. discus juveniles displayed a better
feeding response to the on-grown Artemia and showed a better growth performance and
higher survival than fish fed Moina or frozen bloodworms.
In postlarval stages of penaeid shrimp and clawed lobster, live Artemia biomass has been
shown to provide excellent nutrition (Conklin, 1995; Wickins and Lee 2002), although the
cost of its purchase or production is prohibitive for large-scale hatchery use, the traditional
alternative has been to feed frozen adult Artemia, which supports growth rates
approximately 60% of that of live Artemia (Conklin, 1995; Tlusty et al., 2005a). Moreover,
survival and growth of P. vannamei postlarvae fed ensiled Artemia biomass were
comparable to PL fed frozen Artemia form (Abelin et al., 1991). The study conducted by
Naegel and Rodriguez-Astudillo (2004) illustrated that dried Artemia is a well-suited feed
for postlarval shrimp, Litopenaeus vannamei.
As mentioned earlier, adult Artemia has higher nutrition value compared to nauplii and also
appears containing hormonal substances. According to Naessens et al. (1997), reproductive
hormones of Artemia contribute to the shrimp endocrinological cycle. This could be true in
organisms that share the same hormones as Penaeid shrimp. The role of hormonally active
substances has been suggested for Artemia biomass in the reproductive stage. Thus freshfrozen Artemia biomass (usually boosted with specific nutrients) has been reported to
stimulate ovarian maturation, increase spawn frequency and improve larval quality
(Browdy et al., 1989; Naessens et al., 1997; Wouters et al., 1999). Additionally, it was
observed that incorporating freeze-dried Artemia biomass into an artificial broodstock diet
increased diet ingestion, improved gonad maturation in female and male L. vannamei, and
increased spawning performance (Wouters, 2001).
18
Chapter 2
The investigations implemented by Gandy et al. (2007), found that replacement of
bloodworms with enriched adult Artemia as a feed for Farfantepenaeus aztecus
broodstocks resulted in higher hatch and larval survival rates (nauplius 1 to zoea 1) (55.0%
vs. 46.9% and 44.8% vs. 37.2%), respectively. In addition, the life span of ablated females
fed adult enriched Artemia was 8 and 40 days longer than ablated females fed bloodworms
for the first and second studies, respectively. Other results showed that the spent spawners
of Penaeus monodon, fed herbal enriched Artemia supplementation showed a better
reproductive performance and larval quality (Babu et al., 2008).
In Vietnam, previous investigation on the nutritional value of different forms of Artemia
biomass was reported by Brands et al. (1995), live Artemia biomass can be used as a
complete replacement of trash fish (fresh or cooked) for nursing of penaeid postlarvae.
Although the survival did not show any relation with the amount of biomass feeding,
growth of nursed shrimps displayed an increasing trend with the amount of biomass
feeding. Moreover, the two forms of processed Artemia biomass, frozen and ensiled, which
were tested in nutritional bioassays with various aquatic species, only frozen Artemia
biomass showed an intermediate potential for application. According to recent researches,
live Artemia biomass can be used as a feed for nursing mud crabs Scylla paramamosain
from instar I up to 60 days, reaching a size of 35 mm carapace width and 10.5 g body
weight (Ut et al., 2007a). Le et al. (2008) found that live Artemia biomass was a very
favourite food of sea-bass (Lates calcarifer) during 4 week-nursing in earthen ponds.
Similar observation was made by Van et al. (2008), five types of Artemia biomass that were
obtained from different culture conditions consisting of four live biomass and a frozen. All
of which were suitable food sources for nursing of tiger shrimp PL (Penaeus monodon) and
ornamental fighting fish (Betta splendes). Preliminary observations for brackish water fish,
the goby (Pseudapocryptes elongatus) broodstocks fed frozen Artemia biomass showed a
better maturation than animals fed Tubifex or fresh shrimp in captivity conditions
(unpublished data).
In practice, Artemia cyst used as a live food in larviculture of prawn and shrimp accounted
for more than 50% of total operating cost of hatchery (Phuong et al., 2006b). On the other
hand, Artemia cyst prices have noticeably increased in recent years, resulting in
augmentation of the hatchery cost; hence cheaper alternative diets with comparable
nutritional quality to partially replace live food are needed to maintain the cost
competitiveness of shrimp in the local market in Vietnam.
19
Chapter 2
6. History of Artemia study in Vietnam
6.1. Geographic areas of field study
The area of Vinh Hau salt works, is situated at latitude of 9o38'9"N and longitude of
105o51'45"E and belongs to Bac Lieu province, South of Vietnam (Figure 3).
Field study
Figure 3. Location of the study area in the Mekong Delta of Vietnam.
Similar to other coastal areas in the Mekong Delta, saltworks in Bac Lieu shows typical
clayish soil characteristics. It has a semi-tidal regime and is dominated by a rainy southwest monsoon from end of April until October (85% of the annual rainfall), and a dry
north- east monsoon from November until April (15% of the annual rainfall). Diurnal
temperatures fluctuate between 21 and 34oC, the maximum temperature is recorded in
April-May, often exceeding 36°C. Sunshine and radiation vary with the seasons: highest
monthly averages occur towards the end of the dry season, from February to May (8-10
hours day-1 and 450-550 cal m-2, respectively) and are lowest from August to
September/October (5-7 hours day-1 and 360-400 cal m-2, respectively). Salinity of the area
fluctuates seasonally, and highest salinities are recorded from April to May, at the end of
the dry season. Nevertheless, incoming salinity from the main seawater supply canal is
generally below 30 g L-1. Furthermore, as Artemia ponds are usually shallow and heavy
20
Chapter 2
rains quickly dilute the pond salinity, pond culture of Artemia is not feasible during the
rainy season.
6.2. Overview of Artemia culture in Vietnam
Since 1983, a KWT project was established between the Faculty of Fishery (Can Tho
University, Vietnam) and the Dutch NGO Komitee voor Wetenschap en Techniek (Brands,
1992). This program was founded with, as one of the objectives, to develop the larviculture
of the giant fresh water prawn (Macrobranchium rosenberii) so that hatchery-produced
juveniles of prawn could be distributed to local farmers. However, successful larviculture
of the fresh water prawn requires Artemia nauplii as live larval food. Since Artemia is not
naturally distributed in Vietnam and Artemia cysts were imported at high prices, it was
decided to test and develop Artemia culture techniques in the solar saltworks during the dry
season. Therefore, in 1985 an Artemia Project was initiated with the support of KWT and a
field research station was set up at Vinh Tien Shrimp- Salt Cooperative, Vinh Chau district,
100
1100
90
1000
80
900
800
70
700
60
600
50
500
40
400
30
Culture area (ha)
Total production (ton wet weight); yield (kg/ha/crop)
Soc Trang province by the Faculty of Fishery.
300
20
200
10
100
0
0
86
87
88
89
90
91
92
93
94
95
Culture area
96
97
98
99
Total production
2000 2001 2002 2003 2004 2005 2006 2007 2008
Cyst yield
Figure 4. Evolution of Artemia cyst production in the Mekong Delta- in the period
1986-2008 (Source: College of Aquaculture and Fisheries, Can Tho University, Vietnam).
In Cam Ranh Bay (Central Vietnam) Artemia was first inoculated in 1983 (Quynh and
Lam, 1987) and the first introduction with Artemia franciscana from San Francisco Bay
(SFB, USA) was made in 1986 into Vinh Chau salt fields, southern Vietnam (Rothuis,
1987). Over the years this strain showed its ability to adapt to the new habitat with high
21
Chapter 2
water temperatures of Vinh Chau salt-fields as cyst yields gradually improved exceeding
the amounts obtained with the original SFB (Hoa, 2002).
Since then, the interest in the seasonal culture of Artemia in view of the possibility of
harvesting cysts has grown and the know-how was transferred to a few salt farmers. This
alternative farming system was successful and resulted in higher profits for farmers
compared to their traditional low income from the salt production. In 1990, about 1.4
tonnes of raw cysts were collected from a culture area of 16 ha, which made the product
available for commercialization (Brands et al., 1995). By 2001, the area covered by
production sites increased up to over one thousand hectares of salt-fields in Vinh Chau and
Bac Lieu coastal lines, yielding almost 50 tonnes of raw cysts (Figure 4).
This region is nowadays an important supplier of high quality cysts for domestic use and
partially for export. However, from 2002 onwards the development of Artemia culture has
encountered some limitations due to a dramatic increase in production area without
appropriate planning, the limited knowledge of the farmers in pond management; the large
variation in economical effect within the region and the instability of the output market,
apart from the effect of the unusual weather conditions (Nam et al., 2008). Resolving these
problems will be helpful for the sustainable development of the Artemia production in this
region.
6.3. Perspectives for production of Artemia biomass in Vietnam
Aside from the successful production of Artemia cysts, culture of Artemia biomass for local
use has also been viewed as a possible integrated economic activity, where Artemia cystscum-salt production is combined with a further diversification of Artemia products
(biomass), thus providing for more flexibility in the integrated systems (Brands et al., 1995;
Quynh, 1995). These authors reported that partial harvesting Artemia biomass in the large
ponds also allowed cyst collection at a rate of 30 to 75% of the cyst production ponds
without biomass harvesting (Brands et al., 1995; Anh et al., 1997a; Anh and Hoa, 2004).
Moreover, at the same time the nutritional value of different forms of Artemia biomass was
assessed as a replacement, total or partial, of other traditional aquaculture feeds used in
Vietnam. This strategy was performed for a variety of cultured species through a series of
laboratory bioassay and earthen pond culture tests (Brands et al., 1995).
22
Chapter 2
Economic analysis in previous studies shows that biomass production could increase the
benefit from Artemia cyst production up to several times, depending on the cyst farm-gate
price and the Artemia standing stock. The salterns in Vinh Chau and Bac Lieu areas had
economic potential. Other less remote salt production areas have the same potential,
especially those that are in close proximity to urban areas, and/or where salt production is
artisanal and consists of small ponds that are not suitable for efficient cyst production.
These ponds can be used to culture Artemia biomass contributing to the farmer’s income
(Brands et al., 1995). Although Artemia biomass culture has not fully developed at the
current time, the diversity of highly economic cultured species require excellent quality
foods with life stage specific for hatchery and nursery phases that indicate a good chance
for near future development of Artemia biomass production in the country.
7. Culture technique of Artemia biomass in salt works
According to Brands et al. (1995) and Anh et al. (1997a), culture system and pond
management of both Artemia biomass- and cyst-oriented production are similar, and they
only have a difference in the main product: biomass (live animals) versus cysts (dormant
eggs). The general principles of pond production of Artemia biomass in salt ponds are
briefly described as follows:
Site selection
Artemia is cultured in coastal salt works, where seawater is available during the culture
period and can easily be concentrated to produce highly saline water through the
evaporation of seawater. The soil should not allow leakage and seepage so that the water
level is maintained and the animals will not be able to escape with the water current. Vinh
Chau and Bac Lieu salt fields are suitable locations for Artemia ponds.
Culture season
Artemia culture takes place in the dry season from December to May, or longer depending
on the weather and salinity in the culture ponds.
Culture system
Artemia culture uses mainly the static system in which all ponds are managed separately. It
consists of a reservoir, fertilization ponds and Artemia ponds with a surface area ratio of
23
Chapter 2
20%, 25%, and 50-60%, respectively; and a supply canal. However, this ratio can vary
depending on the particular region, the tidal regime and water depth of the reservoir and
fertilization ponds.
Pond design
The production system is located alongside a canal as there must be a water intake/drainage
structure by which high salinity or fresh seawater can be pumped in and out anytime as the
need arises. The culture ponds are converted and modified from the existing salt fields.
Suitable sizes of Artemia biomass ponds can range from 0.05 to 0.5 ha. For new ponds,
they should be designed as follows: the peripheral ditch should be 2-3 m wide and 0.3-0.6
m deep, the dikes should be raised to 0.5-0.8 m in height and a supply canal should have a
width of 1-1.5 m. The pond must be constructed in such a way that it can hold at least 40
cm of water depth from the platform.
Pond preparation
The preparation of the culture ponds starts in December when the rainy season is over. All
ponds are drained completely and the pond bottom and canals are scraped and sun dried for
about 5 to 7 days. About 10-15 kg of lime 100m-2 should be spread over the pond bottom
and the pond is then left to dry for about 2 to 3 days if the pH is low (pH <8). Derris root
was applied at 1 kg 100m-3 to kill predators before inoculating. Saline water used to fill the
ponds is filtered through a 500 μm nylon screens to eliminate fish eggs and larvae.
Saline preparation
After drying the pond bottom, water is allowed into the ponds and left to evaporate. The
whole surface area can be used for water evaporation. It takes about 3 to 4 weeks to reach a
salinity of ≥ 80g L-1 for inoculation of the first pond. For the next ponds it will take about 3
to 7 days for inoculation. At the start of the culture period, the water level in the ponds is
about 4-5 cm above the platform and is then gradually increased up to >40 cm during the
culture period. In order to increase the availability of the natural food for Artemia, culture
ponds should be fertilized with urea and super phosphate at the rate of 1 and 0.2 g m-2 two
days before inoculation to stimulate phytoplankton bloom.
The optimal conditions for Artemia cyst hatching incubation are as follows (Van Stappen,
1996):
24
Chapter 2
•
Salinity: 30-35 ppt
•
Temperature: 28 - 30 oC
•
Illumination: 1000 lux (neon light is 30- 40 cm above incubation container)
•
Artemia cyst density: 3-5 g L-1
•
Continuous aeration
•
Incubation time: 24 hours
Suitable pond conditions for Artemia inoculation are:
•
Salinity: ≥80 g L-1
•
Water depth: 4-5 cm (above the platform; 30-40 cm from the pond bottom)
•
Turbidity: 25-30 cm
•
Water colour: green brown or green
•
Stocking density: 60-100 nauplii L-1
•
Time for inoculation: early morning or late evening
Management of the fertilization pond
Pig or chicken manure can be applied in fertilization ponds (salinity of 30-40 g L-1) at an
initial rate of 0.8- 1.2 and 0.4-0.6 tonnes DW ha-1, respectively, and every three weeks
consecutively (usually applied 3-4 times during the culture season). Manure is combined
with inorganic fertilizers (urea and phosphates with a ratio of N:P between 5 and 10), at an
amount of 2-5 g m-3 1-2 times week-1 to stimulate the growth of microalgae. Few days after
fertilization the water will turn greenish- brown or greenish in colour and turbidity will
reach 15 to 20 cm. This “green water” is then pumped into the Artemia ponds. Only 75%
of the green water from the fertilization ponds is used as the rest will be inoculum to make
green water later.
Management of the Artemia ponds
To obtain high productivity of Artemia biomass, an appropriate pond management is
performed such as optimal salinity and temperature, and abundant food to maintain a fast
growth of the population (good recruitment rate).
-
Water supply: The enriched water from the fertilization pond is pumped every 2
days, increasing the water level with 2-5 cm, to provide food for Artemia and to
25
Chapter 2
compensate for evaporation and seepage of water. The amount of water can be
adjusted depending on turbidity, salinity of culture ponds and fertilization ponds.
The water level in the ponds is kept as high as possible to prevent high temperatures.
-
Supplementary feeding: The agricultural by-products and organic manures (pig or
chicken manure) can be directly applied into the culture ponds as supplementation
when there is a shortage of natural food. Manure is used at a rate of 200-300 kg DW
ha-1 week-1 during the first month. The amount is decreased to 50% in the following
months, and this treatment may be stopped later according to the condition of the
pond water. Rice bran or soybean meal are added at a feeding rate of around 20 to
30 kg ha-1 day-1 during the first 3-6 weeks of culture, and in-between the interval of
green water supply beyond this period, until the end of the culture.
-
Raking in culture ponds: Daily raking of the pond bottom and the peripheral ditch
provide for physical action to prevent the development of algal mats (lab-lab) on the
pond bottom, as well as for a re-suspension of organic particles, making them
available as food for Artemia.
-
Water exchange and renewal: After 1.5-2 months of inoculation, salinity may be
high (≥120 g L-1) and water quality could become deteriorative. At that time, 3050% of water in the pond can be exchanged to improve its quality, to provide more
food and maintain the renewal of the Artemia population.
Biomass harvesting
Artemia biomass is partially collected after 3 weeks of culture; a net with a mesh size of
about 1 mm is used to collect adult animals. A smaller mesh size is recommended to
harvest juvenile and sub-adult stages. Collection is mostly done in late morning or in the
afternoon when most animals are found near the surface of the pond. The frequency of
harvest depends on the need, thus it can be done daily or twice a week at an amount
between 25 and 50 kg ha-1 day-1. If the Artemia ponds are managed properly, production
can reach up to 0.7-1 tonnes ha-1 month-1, within a 4 to 5 months period. It has to be noted
that under- or over-harvesting of biomass may result in low productivity.
8. Drying methods and their effects on the quality of dried products
8.1. General information on drying methods
26
Chapter 2
Drying is a common technique for preservation of food and other products, including fish,
shrimp and meat, and it represents a very important aspect of food processing. The basic
essence of drying is to reduce the moisture content of the product to a level that prevents
deterioration within a certain period of time, normally regarded as the safe storage period.
The major advantage of drying food products is the extending of the shelf life of dried
products; the removal of water from foods provides microbiological stability and reduces
deteriorative chemical reactions. Also, the process allows a substantial reduction in terms of
mass, volume, packaging requirement, storage and transportation costs (Pigott and Tucker,
1990; Brennand, 1994; Ekechukwua and Norton, 1999).
Several drying technologies have been applied, such as oven drying, hot air drying,
microwave drying, solar and conventional sun drying etc., or combinations of some of these
methods. They are utilized depending upon the desired quality and flavour of the dried
products, the initial moisture content and the composition of products (Barbosa-Cánovas
and Vega-Mercado, 1996; Sumnu, 2001; Chua and Chou, 2003; Raghavan et al., 2005).
Oven drying is the simplest way to dry food at home because it needs almost no special
equipment and does not depend on the weather; however oven drying can be used only on a
small scale because of its energy costs; its drying time is two or three times longer
compared to other mechanical dryers (Brennand, 1994; Swanson, 2003). Furthermore,
convective hot air drying is a traditional method of food preservation, but also has long
drying times and low energy efficiency. Such limitations have led to the application of
newer technologies, including microwaves, in the drying of foodstuffs (Nijhuis et al., 1998;
Maskan 2001; Gowen et al., 2006). Although using microwave drying alone has a higher
energy efficiency and a shorter drying time compared to the conductive hot air drying
(Maskan, 2001; George et al., 2004), one major disadvantage associated with microwave
drying is uneven heating (Oliveira and Franca, 2002). Therefore, combination of
microwave power with convective hot air drying or other drying methods to overcome this
limitation has been recommended (Sharma and Prasad 2001; George et al., 2004; Chua and
Chou, 2005; Gowen et al., 2006). Combining microwave energy with convective drying has
resulted in considerable reductions in drying times compared to convective drying alone
(Funebo and Ohlsson 1998; Maskan 2000; Sharma and Prasad 2001; Bilbao-Sainz et al.,
2005).
The mechanical drying methods are one of the most energy intensive operations and
account 12-20% of the energy consumption in the industrial sector (Raghavan et al., 2005);
27
Chapter 2
moreover, they also need technical skills. As the sun is the cheapest source of renewable
energy, sun drying is still the most common method used to preserve agricultural products
in most tropical and subtropical countries, despite the problems and the risk of
contamination involved, such as high food losses ensuing from inadequate drying, fungal
attacks, insects, dust and other weather effects (Ong, 1999). Other researchers have also
been using solar-energy drying as an alternative to the traditional open sun drying in
developing countries (FAO, 1992; Sodha and Chandra 1994; Chua and Chou, 2003).
Moreover, the construction details and operational principles of different solar dryers have
been reviewed by Ekechukwu and Norton (1999) and Farkas (2004), who confirmed that
using solar energy is a promising solution for meeting the technical, economical, and
environmental demands as well acceptable quality products raised by the drying process.
Moreover, Purohit et al. (2006) emphasized the importance of developing low-cost solar
drying systems, preferably using local materials and skills in which natural convection solar
drying methods could be operationally superior and economically competitive to natural
open sun drying, especially when applied for drying food products in thin layers (Sodha
and Chandra, 1994; Pangavhane et al., 2002).
Vietnam is located in a tropical monsoon climate zone, especially in the south of the
country, has a natural advantage of abundant solar radiation that indicate high potential
renewable energy for drying agriculture products. According to the Vietnam Institute of
Agricultural Engineering (VIAE), in the rural areas of Vietnam sun drying has been the
most common method for drying of products, but it often results in poor quality if the
product has been dried too slowly. In large farm and grain processing plants, artificial
dryers heated with oil and coal stoves (furnaces) are also used but with high cost and
environmental pollution. In 1998, VIAE installed a solar dryer at household level for drying
fish and shrimp. They reported that using the solar dryer reduces drying time by 65% when
compared to open sun drying, resulting in higher product quality and reduction of costs and
of pollution (http://xttm.agroviet.gov.vn).
8.2. Quality of dried products
During drying, changes associated with physical and biochemical structure of dried
products are inevitable because the food is subjected to thermal, chemical and other
treatments. Hence, different drying methods would have a direct impact on nutrient
availability of the fresh products (Pigott and Tucker 1990). Previous researchers found that
28
Chapter 2
drying time and temperature can be considered the most important operating parameters
affecting dried product quality (Maskan, 2001; George et al., 2004; Gowen et al., 2006).
According to several studies, the lipid content and its quality of dried fish, shrimp and meat
of animals showed a high susceptibility to deterioration after drying, and the degree of
destruction depended on the different drying methods (Liou and Simpson, 1989; Pigott and
Tucker 1990; Bórquez et al., 1997; Toldrá, 2006; Unusan, 2007). Paleari et al. (2003)
reported that a decrease of fat content during processing has been shown in cured and dried
products from different animal species. The prolonged direct incidence of sunlight in case
sun drying may accelerate lipid oxidation (Pigott and Tucker 1990; Mottram, 1998).
Additionally, the dried reindeer meat showed higher values of total saturated fatty acids
whereas total unsaturated fatty acids were lower compared with fresh meat (Sampels et al.,
2004). Nonetheless, Liou and Simpson (1989) found that no statistical differences were
recorded in the total percentage of saturated and unsaturated fatty acids between fresh
Artemia and Artemia dried by freeze, vacuum or hot air drying. Bórquez et al. (1997; 2003)
reported that drying time increases resulting in higher losses of n-3 fatty acids in fish and
appropriate drying temperature is the most important variable, affecting both processing
time and product quality.
On the other hand, lipolysis and lipid oxidation have been considered to be key processes in
food processing procedures (drying, smoking, curing etc.). Toldrá and Flores (1998)
suggested that lipolysis is one of the main processes of lipid degradation in fresh meat
during processing; high temperatures and prolonged drying and ripening favour lipid
oxidation in the processed products. Moreover, lipolysis indicates an enzymatic release of
free fatty acids (FFA) from both triglycerides and phospholipids, and is thought to increase
lipid oxidation, since FFA is very sensitive to oxidation (Gray and Pearson, 1984; Motilva
et al., 1993; Coutron-Gambotti and Gandemer, 1999). Longer stages and higher
temperatures result in a higher FFA content of the hams and other parameters have obtained
less attention (Gandemer, 2002). Other studies illustrated that lipolysis causes an increase
in FFA and diacylglycerols and a correlated decrease in triacylglycerols (TAG) (Moltilva et
al., 1993; Garcia-Regueiro and Diaz, 1989). They are also congruent with the results of
Sampels et al. (2004) who found that FFA displayed a threefold increase during drying,
suggesting that lipolysis occurs before or during drying, whereas polar lipids and TAG
decreased in the dried reindeer meat and were significantly different from both smoked and
fresh meat.
29
Chapter 2
30
CHAPTER
Culture of Artemia biomass
Chapter 3
Chapter 3
Section I
Effect of partial harvesting strategies on Artemia biomass
production in salt works
Chapter 3
Chapter 3
Abstract
The effect of partial harvest strategies on production of Artemia biomass was evaluated for
twelve weeks under Vietnamese salt farm conditions. Initial stocking density was 100
nauplii L-1. After 3 weeks of inoculation, Artemia adults were partially harvested at
intervals of 1, 3, 6 and 9 days starting with an initial quantity of 30 kg ha-1 day-1 at first
harvest, then the quantity of harvestable biomass was adjusted according to the standing
stock present in the culture pond, combined with the time needed to harvest these quantities
and with the weight of biomass harvested in each pond. The results showed that in most
cases total densities were not significantly different among harvesting frequencies (P>0.05).
However, relatively higher Artemia adult density and its standing stock were better
maintained in the 3-day than in 1-day interval, and significantly higher compared to the
other two harvesting frequencies. Total biomass yields were highest (1587 kg ha-1) in the 3days harvesting interval followed by 1, 6 and 9-days harvesting interludes, corresponding
with 1323, 1091 and 975 kg ha-1, respectively. However, no statistical difference was
observed between the 1-day and 3-days interval as well as between the 6-days and 9-days
harvest schemes (P>0.05). The results of this study suggest that partial harvest of Artemia
biomass done every 3 days appears to be an appropriate strategy to enhance biomass
productivity.
Keywords: Artemia biomass, harvesting interval, density, total yield
1. Introduction
Artemia biomass can be collected from natural lakes, or produced in tanks and solar salt
ponds for use as food in fish and crustacean farming (Baert et al., 1996; Dhont and
Sorgeloos, 2002). In Vietnam, apart from the successful production of Artemia cysts in salt
works, the culture of Artemia biomass for local use has been practiced since the late 1980s
(Brands et al., 1995). It was found that Artemia biomass production is influenced by several
factors such as water depth, pond size, food availability in the culture pond, etc. (Brands et
al., 1995; Anh et al., 1997a). Furthermore, under optimal culture conditions the partial
harvest strategy can be one of the important factors affecting the final yield of Artemia
biomass. For example, in pilot-scale system where biomass was harvested as live feed for
shrimp nursery requirements, Artemia biomass productivity was 997 kg ha-1 crop-1 while
31
Chapter 3
1643 kg ha-1 crop-1 was achieved by daily harvesting between 25 and 35 kg ha-1 day-1 from
the experimental ponds (Brands et al., 1995).
According to several investigations, partial harvesting of the standing stock of cultured
species (fish or shrimp) over the course of the growing season would decrease competition
and thereby increase individual growth rates and total yield (Bjorndal, 1988; Brummett
2002). D’Abramo et al. (2000) found that by the establishment of selective harvest
strategies of prawn, Macrobrachium roesnbergii, the overall production per unit of time or
space can be increased. A similar observation was made by Rodriguez et al. (2003);
survival and net production of mud crab Scylla olivacea reared in ponds were more
sensitive to the harvesting regime than to the diet. Moss et al. (2005) tested partial
harvesting in a super-intensive recirculating shrimp production system. Their results
illustrated that a well-managed partial harvesting schedule could improve the overall
productivity and profitability of shrimp mariculture under conditions of density-dependent
growth.
A previous study indicated that partial harvest of Artemia biomass, conducted every three
days, results in a higher yield than daily harvesting although a significant difference was
not observed (Anh and Hoa, 2004). Nonetheless, a comparison between the two harvesting
frequencies in the study by these authors may not lead to the optimal harvest strategy.
Therefore, the present study examined multiple schedules of partial harvesting in small
ponds to determine whether partial harvesting strategy could increase the productivity of
Artemia biomass in the Vietnamese salt ponds.
2. Material and Methods
2.1. Culture system and pond management
The experiment was carried out at the field station at Vinh Hau village, Bac Lieu province
(southeast coast of the Mekong Delta, Vietnam), over a period of twelve weeks.
Twelve earthen ponds were newly constructed (each pond had a central platform and a
ditch around the perimeter of the platform) with an area of 300m2 each (20m x 15m); the
peripheral ditch was 2 m wide and 0.3 m deep. The fertilization pond was placed next to the
culture system and green water “microalgae” as a natural food were pumped into the
Artemia ponds via the supply canal (corresponding with a water level increase of 2-5 cm)
once every two days throughout the culture period.
32
Chapter 3
The ponds were drained and dried for 7 days before stocking. Agricultural lime was applied
at 10 kg 100 m-2, and saline water was filled to a depth of 4-5 cm above the platform at
inoculation and the water level was then gradually increased up to >40 cm during the
culture period.
The green water in the fertilization pond was produced by adding organic fertilizer (pig
manure was applied at a rate of 1.0 and 0.5 ton DW ha-1 initially and every four weeks
consecutively) and inorganic fertilizers (urea and di-ammonium phosphate, DAP at an
amount of 3-7 g m-3 week-1 with a ratio of N:P=5).
All experimental ponds were stocked with 100 nauplii L-1 at salinities of 83-85 g L-1. Daily
management of culture ponds was similar for all treatments and aimed at maximizing
biomass yields (Anh and Hoa, 2004). Pig manure and fine rice bran were used as
supplementary feed in the culture ponds with a range of 100-200 kg DW ha-1 week-1 and
15-30 kg ha-1 day-1, respectively.
2.2. Experimental setup
The twelve ponds were randomly allocated to one of four treatments with different
harvesting frequencies comprised of 1-day, 3-days, 6-days and 9-days harvesting intervals
with an initial amount for each harvesting run as follows:
•
30 kg ha-1 1 day-1
•
90 kg ha-1 3 days-1
•
180 kg ha-1 6 days-1
•
270 kg ha-1 9 days-1
2.3. Harvesting strategy
After 3 weeks of inoculation, adult Artemia was partially collected (Anh and Hoa, 2004).
Offspring release was observed after 11 days of culture, after which the later generations
contributed to the Artemia population recruitment.
At the beginning of partial harvesting, the amount of Artemia biomass to be harvested was
fixed at 30 kg wet weight ha-1 day-1 (0.9 kg 300 m² pond-1 day-1). However, in order to
achieve an optimal biomass yield, the quantity of harvestable biomass was adjusted based
33
Chapter 3
on the standing stock present in the culture pond, combined with the time needed to harvest
these quantities and with the weight of biomass harvested in each pond. For instance, if the
time of the current harvest was three times longer than the previous one, and if the
harvested biomass was <17 kg ha-1 day-1 (<0.5 kg pond-1 day-1) the collection was stopped
during 6 to 9 consecutive days (Anh and Hoa, 2004).
2.4. Harvesting procedure
In this experiment, a scoop net shaped as an isosceles triangle (0.6m x 0.9m, 1.2 mm mesh
size) was towed horizontally under the water surface at places where the Artemia
population concentrated, and mainly retained adult and sub-adult stages. Harvest was
mostly done in the late morning, between 9:00 and 11:00.
Excess water in harvested Artemia biomass was removed with a cloth and the wet weight
was recorded with a 5 g precision balance. Artemia biomass yield was calculated every 18
days as well as the total biomass collected during 12 weeks of culture (kg wet weight ha-1).
2.5. Data collection
Water temperature (mercury thermometer) was measured at 7:00 and 14:00 h, salinity
(refractometer, Atago, Japan) at 7:00 and turbidity (Secchi disk) at 14:00 h on a daily basis.
Weekly sampling was conducted to estimate density and population composition of
Artemia. Five point samples per pond were collected using a square plankton net (mesh size
100μm, surface area 0.25m2), towed vertically through the water column at each sampling.
Samples were distributed in the ditch using the fixed random sampling method (Baert et al.,
2002). Samples were fixed in 4% formalin solution and analyzed in the lab where the
average number of adults, juveniles and nauplii (individuals L-1) were determined in each
pond.
Survival was determined based on the samples taken for density on days 1 (week 0) and 7.
The number of animals in each sample was compared to the initial stocking density, which
allowed the calculation of the survival of Artemia in the culture ponds. Particularly, a larger
mesh size (1 mm) net was used to sample for assessment of the survival on day 14 (only
evaluating the original population inoculated). Individual weight of Artemia adults (wet
weight) was recorded weekly from week 3 until the termination of the experiment, by
taking five samples (about one gram each) of Artemia with a net of 1.2 mm mesh size in
34
Chapter 3
each pond, removing extra water by tissue-paper, weighing (0.01g precision balance) and
counting the number of animals in each sample, which allowed to calculate the mean
individual wet weight of the Artemia adults.
The estimation of standing stock of Artemia adults (kg pond-1 or kg ha-1) was calculated as
follows: standing stock = adult density x pond volume x individual wet weight.
Forty females with full brood sac were randomly taken from the pooled samples collected
from different places in each pond. The brood sacs were immediately cut off and preserved
in a 4% formalin solution; then the offspring (nauplii) was counted under a binocular
microscope to determine fecundity (brood size). The number of females producing nauplii,
observed among forty specimens, was calculated as percentage of ovoviviparity. Fecundity
and ovoviviparity were evaluated on a weekly basis.
2.6. Statistical analysis
The data of survival and percentage of ovoviviparity were normalized through a square root
arcsin transformation before statistical analysis. For all treatments, results were analyzed
statistically with one-way ANOVA analysis of variance to find the overall effect of the
treatment (SPSS, version 13.0). Duncan test was used to identify significant differences
between the experimental sample means at a significance level of P<0.05.
3. Results
3.1. Abiotic factors
Abiotic factors in all experimental ponds were similar. Mean salinity was in the range of 75
-114 g L-1, water depth increased from 5 to 45 cm from the platform. Turbidity fluctuated
within the range 25-55 cm. Water temperature tended to increase from February to the end
of April with observed ranges of 22-29°C and 30-40°C at 7 am and 2 pm, respectively.
Especially high temperatures in the afternoon usually occurred from week 8 onwards with a
range of 36-40°C (Figure 1).
35
Chapter 3
7 am
2 pm
41
Temperature (°C)
38
35
32
29
26
23
20
0
1
2
3
4
5
6
7
8
8
10
12
11
Experimental period (week)
Figure 1. Daily fluctuations of temperature (°C) in the Artemia ponds during harvesting
experiment.
3.2. Survival
The survival after 1 day of inoculation was estimated to be over 80% and tended to decline
with the culture period. Survival values were in the range of 68-74% and 54-58% for weeks
1 and 2, respectively (Figure 2). There was no significant difference among treatments
(P>0.05).
1-day
3-days
100
Survival (%)
90
6-days
9-days
80
70
60
50
40
0
1
7
Day after inoculation
14
Figure 2 Mean survival of Artemia after 14 days of inoculation (mean±SE)
36
Chapter 3
3.3. Artemia abundance and population composition
In Figure 3, abundance data are summarized. Artemia densities of the inoculated population
were in the range of 80-83 and 68-74 animals L−1, consisting only of nauplii and being
dominated by juveniles after twelve hours and one week of inoculation, respectively.
T otal number
Density (individual/L)
210
Naup lii
180
150
(a)
Juvenile
Adult
120
90
60
30
0
0
1
2
3
4
5
6
7
8
9
10
11 12
Density (individual/L)
210
180
150
120
90
(b)
60
30
0
0
1
2
3
4
5
6
7
Density (Individual/L)
210
9
10
11
12
(c)
180
150
120
90
60
30
0
0
1
2
3
4
5
6
7
210
Density (Individual/L)
8
8
9
10 11 12
(d)
180
150
120
90
60
30
0
0
1
2
3
4
5
6
7
8
9
10 11 12
Exp erimental period (week)
Figure 3. Variations in density and population composition of Artemia during the
harvesting experiment. (a) 1-day, (b) 3-days, (c) 6-days, (d) 9-days harvesting intervals.
Columns and error bars stand for mean values and standard error.
37
Chapter 3
In this study, offspring was observed following 11 days of culture. Therefore, from week 2
onwards, the population composition consists of three size classes i.e. nauplius, juvenile
and adult stages. Between weeks 2 and 4, Artemia adults were predominant and rapidly
declined over the harvesting period; adult densities reached 4-9 individuals L-1 by the end
of the experiment.
When comparing the adult abundances, statistical differences were not detected (P>0.05) at
week 3 (starting of harvesting) and week 12 (at the end of the experiment). Nevertheless,
most samples in the 1-day and 3-days harvesting interval showed significantly higher
values (P<0.05) than in the 6-days and 9-days harvesting frequency (Table 1). Total
densities from inoculation to week 6 indicated small changes and remained between 57 and
78 individuals L-1, and then steadily increased throughout the culture period as a
consequence of the noticeable increase of nauplii, and juvenile numbers (Figure 3). The
higher number of nauplii found in the 9-days harvesting interval on week 11 and 12 was
responsible for the increase in total density up to more than 200 animals L-1 (Figure 3 d). In
general, there were no statistical differences (P>0.05) in total densities among the
treatments from inoculation until 10 weeks of culture except for a significant difference
between week 11 and 12 with a higher density in the 9-days interval compared to the
others (P<0.05).
3.4. Reproduction
With regard to the reproductive mode, Artemia females in the four harvest treatments
followed a similar pattern with ovoviviparity percentages in the range 8.9 -13.3% at first
spawning (week 2), declining to 3.2-6.7% in week 3. From week 4 onwards the proportion
of ovoviviparous females increased dramatically and the highest values were observed in
week 12, ranging from 43.9% to 49.4% (Figure 4a). The mean brood size at first spawning
was low (32-40 nauplii female-1), peaked between week 3 and 4 (52-66 nauplii female-1)
and then gradually decreased over the remaining culture period (Figure 5).
38
Chapter 3
Table 1. Weekly estimates of individual weight (wet weight), density and standing stock of
Artemia adults (mean±SE) for the different harvesting intervals. Values with different
letters within a column are significantly different (P<0.05).
Individual weight
(mg)
Adult density
(individuals L-1)
Standing stock
pond-1 (kg)
Standing stock
ha-1 (kg)
Week 3
1-day
3-days
6-days
9-days
12.20±0.11
11.86±0.16
11.96±0.15
12.13±0.15
37.9±5.2a
38.7±6.0a
31.5±4.1a
33.2±4.6a
35.8±0.7
35.2±3.1
32.9±2.0
33.5±1.7
1193±84a
1173±96a
1097±63a
1117±81a
1-day
3-days
6-days
9-days
12.02±0.14
11.79±0.09
11.80±0.10
11.96±0.11
31.2±4.7a
29.0±3.7a
27.8±5.5a
32.3±6.9a
34.0±1.8
31.7±0.9
31.0±1.5
36.5±1.3
1133±99a
1057± 85a
1033±98a
1217±94a
1-day
3-days
6-days
9-days
11.84±0.19
11.72±0.11
11.64±0.07
11.79±0.10
20.2±3.3bc
24.9±3.6c
15.3±3.5a
13.8±2.9ab
23.9±4.8
28.8±2.3
17.7±3.9
16.6±3.4
794±132ab
960±108b
595±124ab
553±114a
1-day
3-days
6-days
9-days
11.72±0.11
11.74±0.09
11.68±0.12
11.69±0.10
17.0±2.5ab
17.6±2.7b
10.7±3.1a
13.9±2.4ab
20.9±1.6
22.2±2.7
13.6±2.1
18.0±1.2
704±70b
733±88b
451±72a
599±59ab
1-day
3-days
6-days
9-days
11.61±0.07
11.75±0.08
11.73±0.17
11.59±0.14
18.8±3.4b
12.9±2.7ab
7.4±1.8a
11.3±2.6ab
25.0±4.2
17.3±2.4
9.9±1.8
14.8±4.3
834±131b
570±73ab
328±77a
488±145ab
1-day
3-days
6-days
9-days
11.64±0.07
11.76±0.10
11.62±0.09
11.60±0.08
15.2±2.1b
17.3±2.5b
9.5±1.6a
5.0±2.4 a
22.0±1.6
24.8±2.2
13.7±1.3
7.3±2.1
731±57b
816±74b
453±43a
241±103a
1-day
3-days
6-days
9-days
11.68±0.06
11.75±0.10
11.51±0.08
11.62±0.09
12.7±1.9b
14.6±2.3b
4.6±2.1a
6.8±2.8a
20.1±1.7
23.4±4.5
7.3±2.4
10.8±2.6
669±69bc
770±125c
244±90a
359±84ab
1-day
3-days
6-days
9-days
11.37±0.05
11.32±0.09
11.16±0.04
11.17±0.06
9.3±1.2b
15.7±2.6c
3.4±1.5a
7.1±1.4ab
15.4±1.4
26.1±2.1
8.7±1.3
11.8±3.8
530±63b
896±48c
291±92a
381±122ab
1-day
3-days
6-days
9-days
11.21±0.09
11.12±0.09
10.99±0.07
10.95±0.06
8.7±1.9bc
10.0±1.7c
3.3±2.6a
4.9±2.2ab
14.7±1.9
17.1±1.7
5.5±2.0
8.5±2.2
481±62b
565±59b
183±69a
275±63a
1-day
3-days
6-days
9-days
11.05±0.14
10.90±0.09
10.81±0.05
10.72±0.06
4.1±2.9a
4.3±2.6a
2.6±1.9a
3.2±1.7a
10.8±1.2
8.0±1.4
4.9±1.5
6.0±1.7
210±41b
265±48b
154±45a
207±57ab
Week 4
Week 5
Week 6
Week 7
Week 8
Week 9
Week 10
Week 11
Week 12
39
Ovoviviparity (%)
Chapter 3
50
1-day
40
3-days
6-days
30
9-days
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
Experimental period (week)
Figure 4a. Average percentage of ovoviviparity during the harvesting experiment
Nauplii abundance (Ind./L)
150
y = 3.2243x - 6.0783
2
R = 0.7909
120
90
60
30
0
0
10
20
30
Ovoviviparity (%)
40
50
Figure 4b. Linear correlation between nauplii abundance and percentage of ovoviviparity
1-day
3-days
6-days
9-days
Brood size (nauplii/female)
80
70
60
50
40
30
20
10
1
2
3
4
5
6
7
8
Experimental period (week)
9
10
11
12
Figure 5. Variations in brood sizes (mean ±SE) of Artemia females during the harvesting
experiment.
40
Chapter 3
3.5. Individual weight and standing stock of Artemia adults
The individual weight, density and standing stock of Artemia adults is shown in Table 1.
The results indicated that the mean individual weight of Artemia adults was similar in all
treatments, ranging from 10.7 to 12.2 mg wet weight. However, their weight tended to
become smaller as the culture period progressed.
The standing stock of Artemia adults followed the same pattern as the adult density.
Between weeks 3 and 4, maximum estimated values were in the range 1033-1217 kg ha-1
and were similar among treatments. From week 5 onwards, data showed a steady decline
throughout the harvesting period and the lowest values (154-265 kg ha-1) were found by the
end of culture.
Moreover, in most cases when biomass harvesting was conducted with 1-day and 3-days
harvesting interval the standing stock of adults in the pond remained significantly larger
(P<0.05) as compared to harvesting with 6-days or 9-days harvesting interlude. On the
other hand, when comparing two harvesting intervals significant differences in standing
stock were not observed for the 1-day versus 3-days and for the 6-days versus 9-days
interval in the majority of cases (Table 1).
3.6. Biomass yield
The Artemia biomass yield for the different harvesting strategies is given in Table 2. When
considering the biomass harvested every 18 days, yields were highest (461- 538 kg ha-1)
and lowest (94-204 kg ha-1) in the first and the fourth 18 days, respectively with no
significant differences among harvesting intervals. For the second period of 18 days, the
yield in the 3-days harvesting interlude was significantly higher than in the 6-days and 9days harvesting interval, and a similar result was observed for the third 18 days (P<0.05).
Although biomass harvesting done every day resulted in lower biomass production
compared to that for the 3-days harvesting interval, a significant difference was not
observed (P>0.05).
Linear regression analysis showed that the biomass yield was positively correlated with
both density of adults and standing stock of adults presented in the culture ponds, as
described by the equation as follows:
Y= 2.892X1 + 0.365 X2 + 47.097, (R² = 0.835, P<0.01)
41
Chapter 3
Where Y: biomass yield (kg ha-118 days-1)
X1: adult density (individuals L-1)
X2: adult standing stock (kg ha-1)
Table 2. Average Artemia biomass yield (mean±SE) for the different harvesting intervals
(kg ww ha-1). Values with different letters in a row are significantly different (P<0.05).
Harvesting strategies
1-day
3-days
6-days
9-days
Total biomass yield
1323±116bc
1587±128c
1091±101ab
975±112a
Yield of first 18 days
538±12a
526±16a
461±23a
468±31a
Yield of second 18 days
382±34ab
481±31b
313±23a
287±39a
Yield of third 18 days
253±28bc
377±37c
194±14ab
126±22a
Yield of fourth 18 days
150±49a
203±63a
122±43a
94±25a
Total biomass yields were highest in the 3-days interval treatment followed by the 1-day, 6days and 9-days harvesting periodicity. The statistical analysis indicated that there was no
significant differences between the 1-day and 3-days treatment as well as the 6-days versus
the 9-days harvesting interval (P>0.05).
4. Discussion
According to several researchers, with affordable techniques and machines, partial
harvesting of aquaculture species could be a potential avenue to utilize more efficiently the
grow-out capacity of ponds, tanks, cages, or raceways in aquaculture operations. Especially
in semi-intensive and intensive farms with the eventual goal of enhancing profitability the
premise of partial harvesting is that animal growth would be delayed when the carrying
capacity is reached or simply that growth would be depressed with increasing density and
biomass (Bjorndal, 1988; Fu et al., 2000; Brummett, 2002; Saiti et al., 2007). In addition,
when growth is density dependent, partial harvest of the standing stock of cultured species
(fish or shrimp) over the course of the growing season would decrease competition and
thereby increase individual growth rates and total yield. Thus, well-designed partial
harvesting schemes can improve the profitability of aquaculture operations (Yu and Leung,
2006). Besides, recruitment and harvesting clearly affect the dynamics of exploited fish
42
Chapter 3
populations, interacting with the population’s age and size structure, and finally producing
the dynamics observed in the field (Huusko and Hyvärinan, 2005).
4.1. Effect of partial harvesting strategies on the density and composition of Artemia
population
The results in the present investigation showed that different partial harvesting strategies of
biomass caused a significant effect on Artemia density and population composition. At the
time of first harvest (week 3), the population was dominated by Artemia adults. In this
study, using the scoop net with mesh size of about 1.2 mm mainly retained adult and subadult stages, which resulted in a sharp reduction of adult numbers in all harvest schemes.
On the contrary, the quantities of nauplii and juveniles exhibited a progressive increase,
which was higher for nauplii than for juveniles, leading to the augmentation of total density
during the culture period (Figure 3) also because the proportion of ovoviviparous females
increased with the culture period (Figure 4a). Furthermore, linear regression indicated that
the nauplii density was positively related to the percentage of ovoviviparity (R²= 0.79;
P<0.05) (Figure 4b). The result of the present study was consistent with the findings
obtained by Anh and Hoa (2004) for the production of Artemia biomass. A similar
phenomenon was observed for Artemia cyst production in the salt ponds: when animals
were stocked at optimal density, the number of Artemia in the culture ponds increased
quickly and largely consisted of larval stages and juveniles although Artemia adults were
maintained to collect cysts (Baert et al., 1997). In the present experiment, abundance of
Artemia adults greatly dropped during biomass harvesting, the recruitment of new adult
generations is necessary to ensure a continuum in the biomass production. This may be
hampered by the fact that the conditions for development of the nauplii to the adult stage
for the offspring of the later generations are usually inferior to the parental population
(inoculated population). Because Artemia pond conditions at the later culture period could
be sub-optimal e.g. less food, more food competition, crowding, higher temperature, etc.
which results in slower growth and maturation, as well as lower fecundity.
It is noted that when weekly evaluating the Artemia density and population composition,
data showed huge variations among sampling points in the same pond, since the Artemia
population is characterized by a very heterogeneous distribution, and noticeably influenced
by environmental factors such as light, temperature and wind among others (Wear and
Haslett, 1987; Camargo et al., 2002; Baert et al., 2002). This might trigger a random error
43
Chapter 3
of these values. Additionally, Haslett and Wear (1985) reported that accurate quantitative
Artemia density estimation in large shallow ponds is uncertain due to its rapid reproduction
and gregarious behaviour. Consequently, determination of maximal harvesting rates of
Artemia is complicated, which makes accurate sampling and accurate population estimates
very difficult (Wear and Haslett, 1987). Moreover, measurement of Artemia productivity is
time consuming, requiring population dynamic and biometric data, which are often difficult
to gather (Barata et al., 1996).
Actually, Artemia biomass harvesting is a procedure, which relates more to the
management of a fishery resource. Therefore, the determination of harvesting strategies is
highly empirical and any change in procedure is reflected in the results. Additionally, it was
difficult to determine the accurate endpoint of time needed to collect biomass because of
variations in standing stock and the population distribution (scattered or congregated) at
any point of time in the culture ponds (Brands et al., 1995; Anh et al., 1997a; Anh and Hoa,
2004).
With reference to the reproductive characteristics, the percentage of ovoviviparity was
relatively high at the first spawning and generally declined at the second brood, but then
tended to increase throughout the rest of the culture period. Moreover, the brood size was
small at the first brood and peaked between weeks 3 and 4, then rapidly decreased in time,
which is commonly seen in Artemia culture ponds (Baert et al., 1997; Anh and Hoa, 2004).
According to Tackaert and Sorgeloos (1993), the recruitment rate of the population may be
high in ponds where the dominant reproduction mode is ovoviviparity and low in cystproducing ponds. However, in this experiment a higher proportion of females reproduced
ovoviviparously resulting in enhancement of density, which caused sub-optimal conditions
for growth of animals and reduced fecundities. A similar finding was reported in the study
by Baert et al. (1997), where the later generations had lower survival rates and produced
smaller broods compared to the parental populations, once the second generation started to
compete significantly with the parental population. Generally, from the biological point of
view, the parameters influencing the Artemia production in the salt ponds are the standing
stock of adults and of actively reproducing females, the fecundity, the generation time and
the lifespan (Brands et al., 1995; Baert et al., 1997). However, in biomass-oriented
production, the recruitment and renewal of the adult population may be very limited when
ovoviviparous reproduction greatly dominates (Anh et al., 1997a; Anh and Hoa, 2004). In
this situation, with a dramatic increase in density of the dominating larval and juvenile
44
Chapter 3
stages, their growth is seriously impeded, due to effects of intergenerational competition in
the culture ponds.
4.2. Effect of partial harvesting strategies on Artemia adult abundance and biomass yield
As mentioned earlier the population density increasing with time may cause intraspecific
competition and sub-optimal production conditions; consequently the average individual
weight of the adult becomes smaller. Additionally, as adult animals were regularly
collected during the culture period, as a consequence the standing stock of Artemia adults
showed a considerable decrease towards the end of the culture season (weeks 10, 11 and
12). Furthermore, the biomass yield had a positive correlation with the adult density and
standing stock of adults (R²= 0.835, P<0.01), which may explain why biomass yields
reducing with the culture period were found in all harvest treatments.
It is apparent that the biomass production rate of Artemia populations is positively related
to the standing biomass (Barata et al., 1996). Significant higher values of the adult
abundance and its standing stock in the 3-days harvesting interval were usually observed
during the experimental period. Nonetheless, there was no significant difference between
the 1-day and 3-days harvesting interval as well as between the 6-days and 9-days
harvesting frequency. Hence, the results obtained are similar to those for average total
biomass yield with the highest figure being achieved in the 3-days harvesting interval
where the biomass production was 17%, 31% and 39% higher compared to the 1-day, 6days and 9-days harvesting frequencies, respectively. These results allow to conclude that
biomass harvesting in salt ponds conducted periodically once every 3 days appears to be
more efficient than other harvesting frequencies. According to Allen et al. (1984), using
partial removal of the standing stock to keep the pond near, but below the carrying capacity
during a major portion of the grow-out period, should increase the efficiency of the inputs
into the culture systems. Moreover, appropriate partial harvesting strategies may result in
minimized competition for food and in turn may have allowed smaller animals to grow
faster leading to enhanced productivity, since total biomass yield in the 3-days harvesting
interlude exhibited slight improvement compared to daily harvesting. Analogous results
were reported by Anh and Hoa (2004), who demonstrated that total biomass yield was
slightly higher (2536 kg ha-1) in the 3-days interval than in daily harvesting (2121 kg ha-1)
after 16 weeks of culture in the salt ponds.
45
Chapter 3
For that reason, in case Artemia biomass is cultured as direct live food for aquaculture
animals, daily collection of biomass can be recommended. If biomass production is utilized
for other purposes (processing, drying, freezing) harvesting should be done every three
days, because it can save time needed for biomass collection. However, it should be noted
that patterns of recruitment, maturity and growth of aquatic animals affect optimal
harvesting strategies and harvesting strategies should be explored on a species- or
population-specific basis rather than by relying on general results (Clark, 1991; Fu et al.,
2000).
46
Chapter 3
Chapter 3
Section II
Effect of different food supplements on proximate
compositions and Artemia biomass production in salt works
Nguyen Thi Ngoc Anh1,2, Nguyen Van Hoa2, Gilbert Van Stappen1 and Patrick
Sorgeloos1
1
Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience
Engineering, Ghent University, Belgium
2
College of Aquaculture and Fisheries, Can Tho University, Vietnam
Paper published in Journal of Aquaculture 286, 217-225
Chapter 3
Chapter 3
Abstract
Experiments were conducted to evaluate the effect of different pond supplements on
Artemia biomass production in earthen ponds. Twelve experimental ponds (300m²) were
randomly designed. Green water as a natural food source for Artemia was supplied once
every two days to each treatment. The treatment without supplementation was referred to as
the control (GW), the second treatment (GW+PM) consisted of green water supplemented
with pig manure; in the third treatment (GW+PM+RB) green water was supplemented with
pig manure and rice bran. In the final treatment (GW+PM+SB) green water was
supplemented with pig manure and soybean meal.
Three weeks post-inoculation, growth performance in terms of Artemia length and weight
in the three groups receiving supplemental feed were significantly higher than in the control
(P<0.05). A similar pattern was observed for maturation rate and fecundity of the brine
shrimp adults. After twelve weeks of culture, the value for biomass production in the
control was lower (1.06 ton ha-1) as compared to the three supplemental groups. No
statistical difference in biomass production was found among the groups fed
complementary feeds (P>0.05) although PM+RB and PM+SB gave better results (2.21 and
2.44 ton ha-1, respectively) than PM alone (1.79 ton ha-1). At the same culture period, the
proximate composition of Artemia biomass was similar in all treatments. However, these
values slightly changed over the culture period (i.e. protein and lipid levels were in the
range 54.9-57.8% and 10.7- 11.6 %, respectively, at week 3 and 49.4-50.6% and 9.4-10.2%
at week 12), indicating a fair drop in protein and lipid contents in the last week of culture
while ash content slightly increased. The Artemia biomass in this study shows a proximate
composition comparable with data reported in other studies, and can be considered as a
suitable food source for aquatic species. Our results show that the combination of pig
manure and rice bran or soybean meal can be applied in the cultivation of Artemia in salt
ponds.
Keywords: Artemia biomass; proximate compositions; pig manure; rice bran; soybean
meal
1. Introduction
Artemia is an excellent live food source in larviculture of crustaceans and fish. Both
Artemia nauplii and adults have the great advantage of satisfying the nutritional
47
Chapter 3
requirements of various aquatic species (Sorgeloos, 1980a; Léger et al., 1986; Sorgeloos et
al., 1998; 2001). Compared with freshly-hatched nauplii, the nutritional value of on-grown
and adult Artemia is superior, as they have higher protein content and are richer in essential
amino acids (Léger et al., 1986; Bengtson et al., 1991; Naessens et al., 1997; Lim et al.,
2001). Furthermore, Artemia biomass appears containing hormonal substances and has
been included in maturation diets providing additional benefits, such as induction and
reinforcement of sexual maturation, and increased fertilization rates of fish and shrimp
(Naessens et al., 1997, Wouters et al., 2002; Gandy et al., 2007). However, the nutritional
value of Artemia biomass is not constant, but varies both geographically and temporally
(Léger et al., 1986; Sorgeloos et al., 1998).
It is commonly accepted that Artemia is a non-selective filter feeder and their foods are
microalgae, protozoa, bacteria, organic detritus, etc. They graze small food particles
ranging in size from 1 to 50 μm (D’Agostino, 1980; Van Stappen, 1996; Dhont and
Sorgeloos, 2002). However, Fernández (2001) concluded that the size of food for Artemia
must be in the range 6.8-27.5 µm, with the optimum at about 16.0 µm. Large-pond
extensive cultures and populations in the natural habitat typically depend on available
natural microalgae as a food source (Wear and Haslett, 1987, Wurtsbaugh and Gliwicz,
2001; Zmora et al., 2002), while small-scale semi-intensive and intensive cultures utilize
mostly by-products from the agriculture and food processing industries, consisting of
organic manures, rice bran, corn bran, soy protein, whey etc. as sole feeds or supplemental
feeds. These foods support the growth and reproduction of Artemia. When these foods are
decomposed, they turn into fertilizers which can serve to produce natural food for Artemia
such as bacteria, yeast, algae and different kinds of micro-organisms (Ronsivalli and
Simpson, 1987; Brands et al., 1995; Baert et al., 1997; Teresita et al., 2003; Zmora and
Shpigel, 2006).
In Vietnam seasonal culture of Artemia franciscana in salt works has been studied since the
mid-eighties. Through this research effort the commercial Artemia cyst production in the
coastal salt farms of the Mekong Delta has been developed into a successful activity
providing considerable socio-economic benefits (Brands et al., 1995; Anh et al., 1997b).
Later, production of Artemia biomass was also investigated to establish an integrated
economic activity, where Artemia cysts and salt production, combined with a further
diversification of Artemia components (adding biomass as possible Artemia product) could
provide for more flexibility in the integrated systems (Brands et al., 1995; Quynh, 1995).
48
Chapter 3
In practice, Artemia production relies on the food chains in the pond, which in turn is a
complex phenomenon. Traditionally there has been the widespread use of chicken manure
in Artemia ponds as direct food or in fertilization ponds to stimulate algal growth, followed
by pumping “enriched water/green water” to the culture ponds. However, it is difficult to
maintain a sufficient algal bloom to provide natural food for Artemia; therefore inert diets
such as rice bran, soybean meal etc. have been applied in the culture pond as food
supplement (Brand et al., 1995; Baert et al., 1997).
Unfortunately, outbreaks of avian influenza H5N1 occurred from late 2003 onwards among
poultry stocks in Vietnam and other countries in eastern Asia and have caused a serious
danger to human health; in some provinces in Vietnam the disease is still existing. Hence,
utilization of alternative organic manure sources had to be considered. According to Vu et
al. (2007), pig manure can provide nutrients for crop and fish production and as input for
biogas production. A large proportion of the total pig manure production is not utilized and
is discharged into public sewage systems, rivers and lakes. Other studies found that
successful culture is possible in fish ponds fertilized with either raw pig manure or
biodigester effluent, without supplementary feeding (Long et al., 2003; Lan et al., 2004).
Therefore, the use of pig manure as potential source of organic manure for Artemia culture
can contribute to a reduction of the pressure on the environment.
In recent years, Vietnamese aquaculture activities are expanding with the culture of new
target aquatic species such as mud crab, mud skipper, marine snails etc. These new species
offer opportunities for diversification in the use of Artemia, including live juveniles and
adults as well as frozen or dried Artemia biomass. This indicates that there is a high
potential market for Artemia biomass not only in the Mekong delta but also along the coast
line in central Vietnam (Hoa et al., 2007). Therefore, the study of different pond
supplements affecting the production and quality of Artemia biomass is necessary to
enhance its controlled production in solar salt ponds and to meet the increasing demand for
Artemia in aquaculture.
2. Material and methods
2.1. Culture system
The experiment was carried out at the field station at Vinh Hau village, Bac Lieu province
(southeast coast of the Mekong Delta, Vietnam), over a period of twelve weeks (from
February 20 to May 15, 2006).
49
Chapter 3
Two series of six earthen ponds were constructed (each pond had a central platform and a
ditch around the perimeter of the platform) with an area of 300m2 each (20m x 15m); the
peripheral ditch was 2 m wide and 0.4 m deep. The fertilization pond was located next to
the culture system and green water was pumped into the Artemia ponds via the supply
canal. The green water (microalgae) production was enhanced by adding organic fertilizer
(pig manure was added at a rate of 0.8 and 0.4 ton DW ha-1 initially and every three weeks
consecutively) and inorganic fertilizers (urea and super phosphate at an amount of 3-5 g m-3
every 3- 4 days with a ratio of N:P=10) (for details see Baert et al., 1996).
2.2. Experimental setup
In this study, different combinations of supplementations were tested for their effects on the
production and proximate composition of Artemia biomass reared in the earthen ponds. All
experimental ponds were supplied with “green water” as a natural food source once every
two days throughout the culture period. The different treatments were as follows:
o GW: control: only green water, without supplementation;
o GW+PM: green water, supplemented with pig manure;
o GW+PM+RB: green water, supplemented with pig manure and rice bran;
o GW+PM+SB: green water, supplemented with pig manure and soybean meal.
Each treatment counted three replicate ponds, randomly chosen from the 12 available
ponds.
For details on feed supplementation, see ‘Feeding procedures’ below.
2.3. Pond preparation
The ponds were prepared following the usual pre-stocking procedure of an Artemia culture,
which includes draining the ponds after the end of the rainy season, scraping the pond
bottom and canals, elevating all dikes to maintain pond water level at least 40 cm from the
platform, and drying the pond bottom for about 1-2 weeks. Derris root was applied at 1 kg
100m-3 to kill predators. Saline water used to fill the ponds was filtered through a 500 μm
nylon screens to eliminate fish eggs and larvae (Baert et al., 1996; Anh et al., 1997a).
At the start of the culture period, the water level in the ponds was about 4-5 cm above the
platform and then gradually increased up to >40 cm during the culture period. In order to
increase the availability of the natural food for Artemia, culture ponds were fertilized with
50
Chapter 3
urea and super phosphate at the rate of 1 and 0.2 g m-2 two days before inoculation to
stimulate phytoplankton bloom.
2.4. Artemia inoculation
Artemia cysts collected from the previous year were used for our experiment. Hatching
conditions were according to Van Stappen (1996): seawater of 35g L-1 salinity, constant
illumination and aeration, temperature 25-28 °C. Freshly-hatched nauplii were inoculated in
the late afternoon. Ponds were inoculated when water salinity exceeded 80g L-1, at a
stocking density of about 100 individuals L-1.
2.5. Pond management
Daily raking of the pond bottom and the peripheral ditch provided for increased turbidity of
the water and physical action to prevent the development of algal mats (lab-lab) on the
pond bottom as well as a re-suspension of organic particles on which Artemia can feed. The
green water from the fertilization pond was pumped every 2 days, increasing the water level
with 2-5 cm, to provide food for Artemia and to compensate for evaporation and seepage of
water. The amount of water could be adjusted depending on turbidity, salinity of culture
ponds and fertilization ponds. After 7 weeks of culture, about 10-20% of pond water was
exchanged weekly to minimize the increase in salinity and to remove weak Artemia nauplii
that accumulated in the corner of the pond. Discharged water from the Artemia ponds was
filtered over a 500µm screen to prevent the escape of adult and juvenile Artemia.
2.6. Feeding procedures
The pig manure was purchased from Bac Lieu animal husbandry farm. The rice bran (2nd
grade rice meal) and soybean meal were bought from a local market.
Pond supplementations started from day 5 after inoculation. Pig manure was applied at 250
and 125 kg DW ha-1 week-1 for single feeding and co-feeding, respectively. Rice bran and
soy bean meal was added at a rate of 20 kg ha-1 day-1. Feeding was done once daily between
16:00-17:00 h to provide extra food for Artemia in the evening during the first 6 weeks of
culture, and in-between the interval of green water supply beyond this period, until the end
of the culture.
51
Chapter 3
Pig manure was stored in a plastic bag, which was perforated and submerged under water
for 2-3 days and then daily moved around the Artemia pond to allow its gradually spreading
over the pond bottom. Rice bran (about 10-20% of particle sizes ≤ 50µm) was incubated in
freshwater for 24 hours before application, soybean meal (particle sizes range between 20
and 50 µm) or rice bran was diluted with water and then manually distributed over the pond
surface.
2.7. Data collection
Water temperature (mercury thermometer) was measured at 7:00 and 14:00 h, salinity
(hand refractometer, Atago, Japan) at 7:00 and turbidity (Secchi disk) at 14:00 h on a daily
basis.
Survival was determined at days 5 and 11 (only evaluating the original population
inoculated). Nine point samples per pond were collected using a square plankton net (mesh
size 100μm, surface area 0.25m2) that was towed vertically through the water column at
each sampling in the early morning (Baert et al., 2002). Samples were fixed in Lugol’s
solution, counted, and the number of animals per liter was calculated. These data were
compared to the initial stocking density, which allowed the calculation of the survival of
Artemia in the culture ponds.
Growth in length and weight of Artemia were recorded at days 5, 14 and 21. For
determination of the total length of Artemia, 30 animals were randomly taken from the
samples collected for determination of survival (see above) after they had been pooled.
Length was measured from the anterior part of the head to the end of the telson, using a
dissection microscope equipped with a micrometric ruler.
Wet weight of Artemia was determined by taking five samples (about one gram each) of
Artemia in each pond, removing extra water by tissue-paper, weighing (0.00g precision
balance) and counting the number of animals in each sample, which allowed to calculate
the mean individual wet weight of the Artemia.
For determination of total length and individual weight of Artemia on day 14 and 21, a
scoop net was used with large mesh size, 1.0 and 1.2 cm, respectively, to collect only the
original population, and not its offspring (in this experiment Artemia females started
producing offspring after 11-12 days following inoculation).
52
Chapter 3
Maturation rate was assessed based on the samples taken for individual weight on day 14.
In each sample females were subdivided into immature and mature individuals on the basis
of the presence or absence of ovarian development. The number of mature females, divided
by the total number of females and multiplied by 100 gave the maturation rate.
Sampling for evaluating the fecundity followed the same procedure as for growth
parameters. Forty females with full brood sack were randomly taken from the pooled
samples collected from five different places in each pond. The brood sacks were
immediately cut off and preserved in a 4% formalin solution; then the offspring (nauplii)
was counted under the binocular to determine fecundity. The number of females producing
nauplii, observed within forty specimens, was calculated as percentage ovoviviparity.
Fecundity and ovoviviparity were evaluated on a weekly basis. The purpose of this study
was to evaluate the production of Artemia biomass; therefore only ovoviviparous
reproduction was taken into account.
The proximate composition of Artemia was determined on day 5 and weeks 3, 6, 9 and 12.
From week 3 onwards, only adult Artemia was sampled. Live Artemia biomass was
transported from the culture site to the laboratory, washed with tap water to remove salt and
dust, and stored in the deep freezer (-80°C) for later analysis. The proximate composition
was determined according to the methods recommended by AOAC (1995); the content of
carbohydrates was estimated by subtraction of the percentage protein, lipid, ash and fiber
from the total dry weight.
Nitrogen, carbon and total phosphorus, lipid, fiber and dry weight of supplemental feed (pig
manure, rice bran and soybean meal) were analyzed at three different times. In particular,
pig manure was analyzed before application, and after 1 and 2 months of storage at room
temperature.
Algal density in the fertilization pond was determined weekly. Six different point samples
were collected using a 5 L-plastic beaker; all samples were pooled and mixed in a big
bucket and two sub-samples (1 litre) were taken out and preserved in 4% formaldehyde. For
counting, 100 µL of sample was spread evenly on a Malassez lame and algal cells were
counted under a Nikon microscope at 40 x magnification. Three counts were performed on
each sample.
2.8. Biomass harvesting
53
Chapter 3
Harvesting of Artemia biomass was based on the study of biomass harvest strategies of Anh
and Hoa (2004). Adult Artemia was partially collected on day 21, and harvest was
conducted every three consecutive days at an amount of 75-150 kg wet weight ha-1 per 3
days (2.25-4.50 kg pond-1). Depending on the time needed to harvest these quantities and
on the weight of biomass harvested in each pond, the amount of harvestable biomass was
adjusted in order to achieve an optimal biomass yield; i.e. if within 8-15 minutes harvested
biomass was <17 kg ha-1 (<1.5 kg pond-1) the collection was stopped for six consecutive
days. A scoop net shaped as an isosceles triangle (0.6m x 0.9m, 1.2 mm mesh size) was
towed horizontally under the water surface at places where the Artemia population
concentrated. Excess water in harvested Artemia biomass was removed with a cloth and the
wet weight was recorded with a 5 g precision balance. Biomass yield was calculated based
on total biomass collected during 12 weeks of culture (ton ww ha-1).
2.9. Statistical analysis
The data of survival and percentage of ovoviviparity and maturation rate were normalized
through an arcsine transformation before statistical treatment (Sokal and Rohlf, 1995). For
all treatments, results were analyzed statistically with one-way ANOVA analysis of
variance to find the overall effect of the treatment (STATISTICA, version 6). Tukey’s HSD
test was used to identify significant differences between the experimental sample means at
a significance level of p<0.05. Unequal numbers of replicates were only used for the
parameter "fecundity", as only ovoviviparous females were taken into account here. In
particular, Duncan test was used for detecting significant differences between the average
Artemia biomass yields.
3. Results
3.1. Chemical composition of the supplementation products and algal density in the
fertilization pond
The chemical composition of the three supplementation products is presented in Table 1.
The carbon level in rice bran (RB) and soybean meal (SB) was almost twice the value for
pig manure (PM). Nitrogen in SB was two times higher than in PM and RB. The C/N ratio
was highest in RB (23.1), followed by SB and PM (13.4 and 11.4). However, total
phosphorus in SB was lower than in PM, with RB showing an intermediate value. Ash
content in PM was 23 and 4 times higher than in RB and SB, respectively. Fibre in PM and
RB was also much higher as compared to SB.
54
Chapter 3
Table 1. Chemical composition of the three supplementation products (% dry matter).
Pig manure
Rice bran
Soybean meal
Dry matter
51.50 ± 4.55
84.43±1.57
87.72±0.96
Carbon
27.08 ± 6.43
46.77±3.41
58.89±2.87
Nitrogen
2.40±0.65
2.03±0.14
4.40±0.18
C/N ratio
11.35±0.68
23.07±0.94
13.39±0.59
Total phosphorus
2.18±0.60
1.84±0.21
1.00±0.10
Lipids
0.68±0.23
12.55±0.74
3.97±0.41
Ash
41.86±6.69
9.73±0.55
1.79±0.08
Fiber
14.89±4.87
13.14±1.35
1.67±0.06
Values are average and standard deviation of three replicates.
The average algal density in the fertilization pond is shown in Table 2. Low densities of
algae were observed between weeks 1- 4 and week 9, 11 and 12 (7-20 x106 cells L-1) while
higher algae abundance occurred from week 5 to 8 and 11 (42-84 x106 cells L-1). Generally,
development of algae in the fertilization pond was highly dependent on weather conditions.
Table 2. Average algal density in the fertilization pond during the experimental period.
Algal density (x 103 cells L-1)
Experimental period (week)
Week 1
6,990 ± 1,325
Week 2
11,363 ± 2,163
Week 3
20,137 ± 6,256
Week 4
13,828 ± 4,403
Week 5
63,982 ± 10,774
Week 6
83,774 ± 16,293
Week 7
41,770 ± 6,177
Week 8
61,989 ± 12,841
Week 9
21,012 ± 4,407
Week 10
28,359 ± 8,400
Week 11
62,868 ± 8,026
Week 12
17,535 ± 1,620
55
Chapter 3
3.2. Abiotic factors
There was almost no difference in abiotic factors among the ponds. Mean salinity was in
the suitable range from 70 to 95 g l-1, water depth increased from 5 to 40 cm from the
platform. Turbidity fluctuated within the range 25-50 cm. Water temperature tended to
increase from February to May with observed ranges of 23.5-29.5°C and 31.5-35.5°C at 7
am and 2 pm, respectively. However, extreme temperatures in the afternoon often occurred
throughout the last three weeks of culture (April-May) with an average of 36.6-37.8°C.
3.3. Survival and growth rate
The effect of different supplementations on survival, growth and maturation rate is shown
in Table 3. Survival on days 5 and 11 was estimated to be around 69-75% and 52-54%,
respectively. There was no significant difference among treatments (P>0.05). Length at day
5 did not reveal any significant differences among treatments (P>0.05). However, at days
14 and 21 total length values in the three supplementation groups were similar among each
other, but significantly higher than in the control group (P<0.05). Individual weight at day 5
was statistically equal in all treatments, ranging from 3.1 to 3.2 mg. On days 14 and 21,
individual weight showed the same effect as the total length. In addition, there was a trend
that Artemia co-fed pig manure with rice bran (GW+PM+RB) or soy bean meal
(GW+PM+SB) performed better than those receiving pig manure alone (GW+PM), and a
slight growth advantage was shown by the Artemia co-fed SB as compared to the group cofed RB. Moreover, the maturation rate on day 14 showed the same effect as the individual
weight: the control (83.6%) was significantly lower than the other three groups given
supplemental feeds (95.2-99.7%).
3.4. Reproduction
Figure 1 indicates the fecundity curve versus the culture period for all treatments. During
the first four weeks following inoculation, the difference in fecundity, expressed as number
of nauplii per brood, between the control and the supplemental groups is remarkable,
although the animals were of similar total length. Fecundity from week 2 to 4 in the GW
group was significantly lower as compared to the other three treatments (P<0.01). GW
fecundity peaked slightly in week 5 and then gradually declined or was stable until the end
of culture. In the three supplemental groups the fecundity showed a progressive increase
from week 2 to 4, with the SB group showing the highest peak. From week 5 onwards,
56
Chapter 3
fecundity decreased in these three groups. The group fed RB had intermediate values
between the PM and SB groups until week 8. From week 9 to 12, fecundity was similar in
all three groups.
Table 3. Survival, total length, individual weight and maturation percentage of Artemia
reared with different supplementations
GW
GW+ PM
GW+PM+RB
GW+PM+SB
Day 5
75.4±1.8a
75.8±1.0a
69.2±4.1a
70.7±1.3a
Day 11
52.1±1.8a
53.1±1.5a
54.0±2.8a
52.9±2.1a
Day 5
5.0±1.1a
4.8±0.9a
5.0±1.0a
4.9±1.1a
Day 14
8.8±1.0a
9.2±0.7b
9.4±0.6b
9.4±0.6b
Day 21
9.3±0.8a
9.5±0.6b
9.6±0.5b
9.7±0.5b
Day 5
3.2±0.1a
3.2±0.3a
3.1±0.2a
3.1±0.3a
Day 14
7.5±0.9a
8.4±0.8b
9.1±0.8bc
9.3±0.8c
Day 21
11.2±0.3a
11.5±0.3b
11.6±0.2bc
11.8±0.2c
Maturation (%)
83.6±6.8a
95.2±5.9b
98.6±2.0bc
99.7±0.6c
Survival (%)
Length (mm)
Weight (mg)
Fecundity (nauplii/female)
Different letters in a row indicate significant differences (P <0.05) among treatments.
90
GW
80
GW+PM
GW+PM+RB
GW+PM+ SB
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
Culture period (week)
Figure 1. Average fecundity (number nauplii female-1), the error bars stand for standard
deviation.
57
Chapter 3
In the first brood (week 2), a relatively high percentage of Artemia females reproduced
ovoviviparously (15.8-17.5%), after which most of them switched to oviparity in the
second brood (week 3: ovoviviparity in the range of 5.8-12.5%) (Figure 2). From week 4
onwards, ovoviviparity percentage showed a more or less steady increase until the end of
the culture period. The percentage of females producing nauplii from week 3-5 was
significantly higher in the control than in the three supplemented groups (P<0.05), and from
week 6 onwards the pattern was similar in all groups.
GW
GW+PM
GW+PM+RB
GW+PM+ SB
40
Ovoviviparity (%)
35
30
25
20
15
10
5
0
1
2
3
4
5
6
7
Culture period (week)
8
9
10
11
12
Figure 2. Average percentage of ovoviviparity (%)
Biomass yield (ton/ha)
3.0
b
2.5
b
b
2.0
a
1.5
1.0
0.5
0.0
GW
GW+PM
GW+PM+RB GW+PM+SB
Feeding treatment
Figure 3. Average yield of Artemia biomass (ton ww ha-1) reared with different
supplementations after 12 weeks of culture. The error bars stand for standard deviation.
Different letters indicates significant differences (P<0.05) among treatments.
58
Chapter 3
Table 4. Proximate analysis, expressed in percent dry weight of fresh Artemia biomass
sampled during different culture periods after application of different supplementation
treatments.
Protein
Lipid
Ash
Fibre
Carbohydrates
GW
56.25±1.04
13.49±0.52
15.26±1.09
0.37±0.13
14.63±0.86
PM
56.05±1.59
13.87±0.35
15.64±1.38
0.42±0.12
14.02±2.69
RB
55.90±1.09
13.59±0.46
14.89±0.75
0.39±0.13
15.23±1.56
SB
56.19±1.11
13.71±0.43
14.84±1.43
0.44±0.09
14.82±1.50
GW
54.90±2.31
10.69±1.03
20.34±0.68
0.50±0.07
13.57±2.81
PM
56.82±1.96
11.10±1.44
19.57±1.47
0.47±0.16
12.04±2.25
RB
57.76±1.94
11.39±1.03
18.71±2.14
0.54±0.07
11.60±4.21
SB
57.53±1.76
11.58±1.25
19.02±2.35
0.48±0.14
11.39±2.52
GW
53.67±2.32
10.45±1.36
23.72±2.62
0.59±0.17
11.57±2.15
PM
54.43±1.22
10.71±1.10
22.30±1.96
0.67±0.04
11.90±2.30
RB
54.69±2.53
11.94±1.56
22.05±3.54
0.61±0.11
10.71±1.81
SB
54.52±2.87
11.28±0.64
21.97±2.91
0.58±0.08
11.65±0.74
GW
51.00±2.74
9.95±0.85
25.46±4.14
0.65±0.14
12.93±2.12
PM
52.93±1.05
10.02±0.82
25.82±1.98
0.77±0.10
10.46±2.15
RB
53.29±2.65
10.52±0.54
23.23±2.45
0.65±0.07
12.31±2.50
SB
53.53±2.81
11.14±1.76
24.10±2.06
0.62±0.09
10.62±3.43
GW
49.67±1.88
9.43±1.32
26.46±2.87
0.78±0.08
13.66±1.49
PM
49.36±2.72
9.54±1.29
26.74±3.80
0.77±0.10
13.60±2.33
RB
50.24±2.32
10.14±0.39
22.61±2.67
0.71±0.09
16.29±4.16
SB
50.61±1.19
10.18±1.19
25.10±3.05
0.87±0.17
13.24±0.70
Day 5
Week 3
Week 6
Week 9
Week 12
Values are average and standard deviation of three replicates.
59
Chapter 3
3.5. Artemia biomass yield
The average Artemia biomass produced with different supplementations is shown in Figure
3. The supplemental diets gave an increase in biomass production (values in the range 1.792.44 ton ww ha-1 after 12 weeks of culture) compared to the control. (1.06 ton ww ha-1). In
addition, co-feeding of pig manure with either rice bran or soy bean gave the best results
among the four test diets, though it was not significantly higher than when using solely pig
manure.
3.6. Proximate composition of Artemia biomass
Comparison of the proximate composition of Artemia biomass reared on different
supplementations is given in Table 4. At the same culture period, the contents of protein,
lipid, ash, fiber and carbohydrates were not significantly different among treatments
(P>0.05). However, mean protein and lipid concentrations tended to decline with the
culture period, especially the last week of culture (week 12) showed the lowest values,
whereas the ash content increased. Carbohydrates and fiber remained similar or slightly
lower than the initial day 5 values.
4. Discussion
The results from this study indicated that different supplemental feeds had a significant
effect on the growth and total yields, but did not affect on the proximate composition of
Artemia biomass, which are crucial parameters for production purposes.
4.1. Survival and growth performance
The survival of Artemia was not affected by the treatment (P > 0.05). However, these
values (52-54% at day 11) are lower than data obtained in laboratory culture conditions;
Naegel (1999) obtained 72-79% survival after 11 days of culture, using an inert commercial
diet and Chaetoceros sp. as feed. Similar results were obtained by Teresita and Leticia
(2005) after 15 days of culture feeding with rice bran and Tetraselmis suecica. This may
due to the fact that the Artemia population is characterized by a very heterogeneous
distribution in the culture ponds; this distribution pattern is strongly affected by
environmental factors such as light, temperature and wind among others (Persoone and
Sorgeloos, 1980; Wear and Haslett, 1987; Camargo et al., 2002; Baert et al., 2002).
60
Chapter 3
Therefore, the large quantitative variation found when sampling the same pond might cause
a random error of these values.
Growth performance in terms of length and weight were similar after five days of
inoculation. At this period, all animals had received a nearly equal amount of natural food
present in the culture pond and from the fertilization pond. On days 14 and 21 total length
and individual weight in the groups fed the supplemental feeds were significantly higher
than in the control (P<0.05). On day 14, the animals reached the adult stage; therefore the
increase in total length from day 14 to 21 was negligible, while individual weight still
showed a moderate growth. This is in line with the visual observation in the ponds, where
the first riding couples in the three groups receiving the supplemental feeds, were detected
on day 7, while this phenomenon was found on day 10 in the control. Similar observations
were made by Tunsutapanich (1982) who found that riding couples could be observed 9
days after the inoculations of SFB Artemia in salt ponds in Thailand. Quynh and Lam
(1987) reported that populations of three Artemia strains (Macau, Great Salt Lake and
China) showed first riding couples at day 7, and the Chinese parthenogenetic females began
to develop ovaries after 10 days culture in salt ponds in Vietnam. Amat (1980) observed a
quicker maturation of SFB Artemia in a natural environment than under laboratory
conditions, although his value (22 days in nature) is still longer than our observation.
However, Wurtsbaugh and Gliwicz (2001) reported that with optimal temperatures and
nutritious food, juvenile Artemia reached reproductive size within 7 days in the laboratory.
According to Balasundaram and Kumaraguru (1987), Artemia fed mixed diets (rice bran,
yeast, algae from saltpans, and decomposed cabbage) yielded the best growth and also
attained faster maturation (9 days) than those fed on a monodiet (14-20 days of culture).
D’Agostino (1980) and Fábregas et al. (1996a; 1998) reported that the growth and the
reproduction of Artemia were governed by several factors such as the amount and quality of
available food, the salinity and the water temperature. On the other hand, Wurtsbaugh and
Gliwicz (2001), studying the Great Salt Lake, found that both the quantity and quality of
the food source, combined with temperature will likely influence growth rate and time to
maturation of Artemia. From the short maturation time observed in this study, we may
deduce that satisfactory trophic conditions prevailed at the inoculation time. However, the
maximum adult sizes observed were slightly smaller than those usually reported in
literature. Evjemo and Olsen (1999) reported that Artemia franciscana, grown under
laboratory conditions with maximal food concentration, reached sexual maturity at a length
61
Chapter 3
of 10.4 -11.6 mm after 16 days. In the present study, the mean length of adult Artemia was
less than 10 mm after 3 weeks of inoculation. Amat (1980) stated that Artemia living in a
natural environment become sexually mature earlier and usually have smaller sizes than
those cultured in the laboratory. Furthermore, the results showed that the maturation
response of these groups to food supplementation follows the same pattern as the
observations made for growth performance. Because in this period the environmental
conditions (i.e. temperature) were suitable for Artemia cultivation, food limitation can be
considered as a main factor affecting growth and maturation of the inoculated populations.
This is linked with the amount of “green water” in the fertilization pond (as a natural food
source for Artemia), that can be pumped in the culture ponds. This limit is set by the lowest
salinity acceptable in the pond (≥70 g L-1), and is especially a problem at the start of the
season when salinity in the reservoir and fertilizer pond is very low (20-25 g L-1). In
addition, the algae abundance present in the water can be partly affected by fertilization but
even so there are limits set by variables not under our control, such as starting concentration
and composition of the initial inoculum; light intensity; presence of unwanted grazers (i.e.
ciliates such as Fabrea salina), temperature. Therefore, this problem was observed in the
control group receiving only green water at the start of the season when light and
temperature are often low (cloudy weather). Food limitation might thus show up at the later
developmental stages, as illustrated by a sudden drop in turbidity from 28 cm to >60 cm
(visible bottom) at day 9 in the control ponds, while in the three other groups turbidity
ranged from 28 to 32 cm. Moreover, food availability in the culture ponds almost
completely depends on extra input: food levels can be increased by using organic fertilizer
(pig manure), pumping enriched water, stirring up bottom sediments and supplying extra
food such as rice bran or soy-bean powder.
According to Edwards (1982) and Moore (1986), food supplements applied directly in
aquaculture ponds can act through three ways: direct consumption; solubilization in the
water column and microbial/bacterial colonization and decomposition which serves as a
food for fish and shrimp. It is also important to note that organic fertilizer contains the
organic matter being digested by bacteria that Artemia can use as a food (Intriago and
Jones, 1993). Hence, Artemia receiving supplemental feeds grow on a highly nutritious
mixed algal, bacterial, protozoan and organic diet, and this may have contributed to the
very high growth and maturation rate (Wurtsbaugh and Gliwicz, 2001). This is illustrated
62
Chapter 3
by the increased growth and earlier maturation of Artemia with increasing food availability
in the present study.
4.2. Reproductive performance
The effect of food supplementation on female fecundity was similar as on growth
performance and maturation rate. Fecundity of the three supplemental groups followed a
similar pattern (Figure 1) as in the studies of Browne (1980, 1982) and Brands et al. (1992),
who found that the fecundity versus age curves were bell-shaped for several Artemia
strains. Amat (1980) also found an increase of the reproductive capacity within successive
broods, starting from ±50 offspring/brood/female for the first brood, and reaching very high
figures of 300-350/brood/female for SFB Artemia. Moreover, females had smaller brood
sizes when kept on a low food regime than at a high food regime (Brown, 1982; Lenz and
Dana, 1987). In the present study, the relatively high number of offspring recorded for the
first brood in the two groups supplemented with rice bran or soybean meal also supports the
hypothesis of initial good trophic conditions while fecundity in the control group was lower
than in the other three treatments during the experimental period. On the other hand, it
should be noted that fecundity was inversely proportional to ovoviviparity. Previous studies
have reported that an increased ovoviviparity at higher feeding rates results in a quickly
decreasing brood size due to increased intraspecific competition with the later generations.
Hence, both growth and reproduction were reduced in more crowded conditions, unless the
food availability was improved (Quynh and Lam, 1987, Brands et al., 1995; Baert et al.,
1997).
Considering the mode of reproduction, several studies found that Artemia females tend to
reproduce ovoviviparously at first and then switch to oviparity (Dana and Lenz, 1986;
Quynh and Lam, 1987). Our findings are in accordance with those of other studies where it
was shown that females with the first brood not only reproduce ovoviviparously but also
have a low fecundity (D’Agostino, 1980). In this study, adult Artemia were partially
harvested every three days, resulting in young females in later generations with smaller
size, having small brood sizes and tending to produce nauplii. Furthermore, ovoviviparity
has been usually found as the dominant reproduction mode of Artemia females inhabiting
saltworks with year-round favourable conditions (Wear et al., 1986; Wear and Haslett,
1987; Lenz and Dana, 1987; Lenz and Browne, 1991). Similar observations were made by
Camara (2001) who reported that most Artemia populations in the coastal saltworks in
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Chapter 3
Brazil are reproducing ovoviviparously in the relatively stable local environmental
conditions. In Vietnamese conditions, dominant ovoviviparity appeared to be in contrast
with those studies (Brands et al., 1995; Baert et al., 1997). In addition, switching of
reproductive mode in the natural environment can be expected to vary depending on the
environmental conditions (Lenz, 1987). Other factors such as temperature, salinity,
photoperiod and brood number also potentially contribute to a shift of mode of
reproduction (Berthélémy-Okazaki and Hedgecock, 1987), but temperature seems to play
an important role in our experiment, i.e. from week 4 onwards ovoviviparity increased with
temperature. Similar results were observed by Baert et al. (1997) and Anh and Hoa (2004).
Increasing ovoviviparous reproduction causes higher numbers of nauplii in the population,
reducing food availability in the culture ponds, possibly result in food shortage. To solve
this problem, about 10-20% of water was exchanged weekly from 7 weeks of inoculation
onwards. This also contributed to minimal increases in salinity, extra inputs of green water
(natural food) to the culture ponds, and may result in fast growth and good recruitment of
the Artemia population.
4.3. Different feeding supplement and their effects on Artemia biomass yields
The total yield of Artemia biomass with different food supplements reflects their effect
during the experimental period. The difference in biomass production among the treatments
with the three supplemental feeds was not significant (P>0.05), but they were significantly
higher than the control (P<0.05). Furthermore, biomass productivity in the treatments with
co-supplementation of PM with RB or SB yielded 2.1-2.3 and 1.2-1.4 times higher results
than the control (receiving only green water as a natural food) and supplementation with
solely PM, respectively (Figure 3). As mentioned earlier, these results demonstrate that
supplementation of inert food to the culture ponds enhances the production of Artemia
biomass.
Quynh and Lam (1987) and Brands et al. (1995) suggested that the total Artemia biomass or
cyst production reflects a combination of growth, maturation and reproduction of the
population. The result of our study was comparable with these authors: harvested biomass
in ponds in the Philippines was about 15-30 kg ha-1 day-1 (Jumalon et al., 1987), and in
Thailand 4.4 ton ha-1 year-1 (Tarnchalanukit and Wongrat, 1987). Zmora et al. (2002)
suggested that by a daily introduction of 3-5 million nauplii 1000 m-2 of pond, an average
of 5 kg 1000 m-2 day-1 (50 kg ha-1 day-1) of Artemia biomass was harvested over a period
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Chapter 3
of several months in Israel. In addition, they compared two ponds of 400 m2, which both
received 300 kg dried chicken manure. One of both ponds received in addition 2 kg of
micronized soy protein (70%) daily, the other did not. After 24 days of culture they
obtained 140 and 6 kg of Artemia biomass, respectively. They also concluded that using
soy protein powder allowed bridging over a period of shortage of natural food.
Johnson (1980) found that carbohydrates of fibrous nature (such as rice bran, soybean
meal) apparently ensure good growth either as a dietary ingredient or as a bacterial
substrate. The carbohydrates in the rice bran seem to have contributed to the growth of
Artemia since this species requires abundant carbohydrate during the first days of
development (Johnson, 1980; Teresita et al., 2003). Moreover, the success of Artemia
culture fed agricultural by-products is strictly dependent on the instantaneous establishment
of a microflora as a supplement to the nutrient-deficient diets (D’Agostino, 1980). Several
authors investigated the control of carbon/nitrogen (C/N) ratio by the addition of a
carbohydrate source (e.g. cassava meal, flour) to the culture systems. They found that (1)
the total ammonia nitrogen from the water and sediment were reduced significantly (2)
increased the heterotrophic bacterial growth which supply the microbial protein (single cell
protein or floccules) as a source of protein for fish or shrimp (3) reduced the demand for
feed protein (4) reduced nitrogen discharge making intensive shrimp farming more
ecologically sustainable and economically viable (Avnimelech, 1999; Hari et al., 2004;
2006; Verdegem et al., 2006). Additionally, net uptake of inorganic nitrogen by bacteria
only occurs when the C/N ratio of the organic matter is higher than 10 (Lancelot and Billen,
1985). Therefore, increasing the C/N ratio could increase bacterial production in the
systems (Burford et al., 2003). In our study, the C/N ratio of the three supplemental feeds
was highest in rice bran (23.1), followed by soy bean meal and pig manure (13.4 and 11.4).
These values could be considered a suitable range for microbial development in the culture
ponds that may provide more food for Artemia. According to Burford et al. (2003, 2004),
up to 40% of the bacteria were associated with flocculated particles which are composed of
a mixture of bacteria, senescent phytoplankton, protozoa and inorganic particles as a
potential food source and contribute substantially to the nutrition of shrimp Litopenaeus
vannamei.
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Chapter 3
4.4. Proximate composition of Artemia biomass
It is known that the nutritional value of Artemia is not constant, but varies both
geographically and temporally (Sorgeloos et al., 1998; Kara et al., 2004; Moraiti-Ioannidou
et al., 2007). Results of the present study showed that the proximate composition of
Artemia biomass received different supplementations was similar at the same culture
period. However, these values slightly changed through the culture time. Protein and lipid
level showed a gradual drop in week 6 to 12 (by the end of the dry season) while ash
content slightly increased in this period; fiber and carbohydrate showed the same pattern as
the ash level (Table 2). This is probably affected by the quantitative and qualitative
availability of feed for the Artemia in the culture pond. In the period from week 9 onwards
heavy rainfall occurred, and the supply of green water as natural food for Artemia was
limited. According to Wurtsbaugh and Gliwicz (2001), along with the depleted
phytoplankton food resource during the summer in the Great Salt Lake, USA, Artemia
growth slowed down and lipid indices decreased. Moreover, Naegel and Rodríguez (2002)
found that the proximate analysis of Artemia biomass in solar salt ponds in Mexico changed
slightly through the production season, except for crude fibre which changed markedly. Our
results are in agreement with these literature observations; proximate composition of adult
Artemia cultured in the solar salt ponds in Vietnam also showed slightly different values
over time. Proximate composition of a similar range was reported by Naegel and Rodríguez
(2002): ranges of content in protein (55.8-65.3%), lipid (6.8-9.8%), ash (12.3- 18.8%),
crude fiber (0.7-1.4%) and carbohydrate (11.1-17.8%) on dry weight basis were determined
in adults collected from the solar salt ponds in Mexico during different months.
On the other hand, proximate composition of Artemia culture under laboratory conditions
showed different values when fed different foods. Ronsivalli and Simpson (1987), growing
Artemia on either rice bran or whey powder as the sole diet for 15 days, found that final
content in protein was 50.13 and 10.13%, lipid 9.47 and 1.30%; ash 9.93 and 7.60% and
carbohydrates 24.10 and 75.86%, respectively. In addition, Naegel (1999) obtained protein
contents of 56.45, 42.87 and 41.16%, lipid contents of 2.95. 16.45% and 20.33% in Artemia
reared on Chaetoceros sp., Nestum (the powdered baby food) and enriched Nestum,
respectively. A similar result was obtained by Teresita and Leticia (2005) suggesting that
the proximate composition of Artemia reared on rice bran and Tetraselmis suecica was
closely similar to our result. Moreover, protein obtained in their study was 3.8 times higher
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Chapter 3
than that obtained for Artemia franciscana when fed upon rice bran only (Ronsivalli and
Simpson, 1987).
In conclusion, feed co-supplementation with pig manure and rice bran or soybean meal can
be applied in cultivation of Artemia in the salt works. However, rice bran seems to be more
practical than soybean meal due to its local availability and low price. As cost estimations
are not relevant for a small-scale experiment as the one described in this study, an
economic comparison of production costs (inputs) and Artemia biomass yields (outputs)
between different treatments should be performed in the future on a commercial scale.
Nevertheless, it can be stated that co-supplementation of food is more profitable than the
use of natural food solely, as the former requires the same costs for pond management and
labour as the latter, but results in biomass production twice as high.
The proximate composition of Artemia biomass in this study was comparable with the
researches cited above and can be considered as a suitable food source for aquatic species.
One of the main disadvantages for Artemia culture in the salt works is that environmental
conditions cannot be controlled. In this study, extreme conditions such as high temperature
and little wind at night often occurred in the second half of the dry season, causing high
mortality and retarded growth. On the contrary, heavy rainfall usually appeared at the end
of the dry season resulting in a sharp salinity drop, and high water turbidity because of clay
foam. Therefore, the supply of green water or food supplement to the culture ponds was
limited which may have caused a significant reduction in the production and quality of
Artemia biomass, thus hiding the effect of the feeding treatments in this period.
Acknowledgements
This study has been funded by the Vietnamese Government for the PhD study of Nguyen
Thi Ngoc Anh. Le Van Nhieu, Phan Thanh Phuoc and Giang Van Hay are acknowledged
for their assistance in the field works and Anita Dehaese and Geert Vandewiele with the
protein and lipid analyses.
67
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68
Chapter 3
Chapter 3
Section III
Effect of different ratios of N:P on primary productivity:
its combination with feeding strategies for Artemia
biomass production in salt ponds
Chapter 3
Chapter 3
Abstract
The effects of using 2 different nitrogen and phosphorus ratios (N:P=5 and N:P=10) on
primary productivity in “green water” and its combination with feeding strategies (rice bran
and pig manure) for the culture of Artemia biomass in salt ponds were evaluated in a
replicated factorial experiment over a period of 12 weeks. The levels of chlorophyll a were
similar and varied from 60 to 274 µg L-1. Multiple-regression analysis combining stepwise
selection of independent variables indicated that chlorophyll a had a positive relationship
with total nitrogen content and a negative correlation with the contents of N-NH4+ for both
fertilizer treatments. Moreover, four phyla of algae consisting of 67 species were found in
this study, in which Bacillariophyta (diatoms) were predominant (60-63%), followed by
Cyanophyta (15-16%), Dinophyta (13-15%) and Chlorophyta (8-10%).
There was no significant interaction (P>0.05) between the green water and supplemental
feeding treatments. Regardless of feed supplement, total biomass yield was slightly higher
(2.29 ton ha-1 crop-1) when Artemia was fed green water from the N:P=5 treatment as
compared to the N:P=10 treatment (1.97 ton ha-1 crop-1) although a statistical difference
could not be detected. Comparative cost-benefit analysis showed that the N:P=5 green
water and pig manure treatments (GW5+PM) attained a higher net profit and cost/benefit
ratio, and a lower cost of production than the other treatments. A budgeting analysis in this
study showed that higher profits could be obtained by using the N:P=5 ratio to produce
green water as natural food and pig manure as feed supplementation for production of
Artemia biomass in the Vietnamese salt ponds.
Key words: Artemia biomass, algae, fertilizer, N:P ratio, pig manure, rice bran
1. Introduction
Seasonal culture of Artemia in salt ponds has proved successful and widely applied in the
coastal areas of the Mekong Delta of Vietnam since the 1990s (Brands et al., 1995). So far
this region is an important supplier of high-quality Artemia cysts that are used in domestic
aquaculture as well as abroad and this achievement helps poor salt farmers to improve their
living standards (Son, 2005; Hoa et al., 2007). In practice, culture success relies on the
favourable growth of the Artemia population following inoculation as is influenced not only
by abiotic factors (temperature, salinity, etc.) but also by the availability of suitable food in
the culture ponds (Baert et al., 1996, Hoa et al., 2007). As Artemia is a non-selective filter
69
Chapter 3
feeder, they graze small food particles ranging in size from 1 to 50 μm and their main food
sources are microalgae, bacteria, organic detritus etc. (Van Stappen, 1996; Fernández,
2001; Dhont and Sorgeloos, 2002).
Algae are an important natural food source for Artemia cultured in the salt ponds. Some
algae groups and species are better suited in terms of size and digestibility as food for
Artemia than others (Brands et al., 1995; Baert et al., 1996; Hoa et al., 2007). In laboratory
experiments, several species of algae have been considered as an appropriate food for
Artemia such as Phaeodactylum tricornutum, Tetraselmis suecica (Fábregas et al., 1996a,
1996b; 1998), Isochrysis sp. (Evjemo and Olsen, 1999), Chaetoceros sp. (Naegel, 1999;
Toi et al., 2006); Dunaliella, Isochrysis sp. and Chaetoceros muelleri (Lora-Vilchis et al.,
2004). However, uncontrolled growth of phytoplankton communities commonly results in
water quality problems due to cyanobacterial dominance (Baert et al., 1996; Wetzel, 2001;
Smith, 2006). Bloom forming cyanobacteria are not readily utilized as a natural food source
by Artemia (Baert et al., 1996; Hoa et al., 2007).
When culturing Artemia in the salt ponds, farmers have their own “rule of thumb” to feed
their Artemia by adding organic manure and fertilizers into the fertilization pond, and then
pumping “green water” to culture ponds; sometimes manure and rice bran are applied as a
direct food for Artemia. However, even skilled salt farmers may face problems when trying
to stimulate appropriate algae (i.e. Dunaliella, Chaetoceros...) as a natural food source for
Artemia and may experience problems, such as the occurrence of larger algae (i.e. diatoms
with a dominance of Gyrosigma, Pleurosigma and others, or proliferation of filamentous
blue-green algae such as Lyngbya, Oscillatoria etc.). This blooming of unwanted algae
species causes a lot of problems for pond management, because fertilizers are inefficiently
used, and cysts may be trapped in the algal mat (the so-called lab-lab) created by
filamentous and benthic algae. Consequently, the Artemia population in the pond is
negatively affected, since brine shrimp, which can’t take up filamentous algae or digest a
thick cell wall, may starve under these conditions (Baert et al., 1996; 1997; Anh et al.,
1997a; Hoa et al., 2007).
Previous studies found that the two most important nutrients in aquaculture ponds are
nitrogen (N) and phosphorus (P) because these two nutrients are often present in short
supply and thus limit phytoplankton growth (Wetzel, 2001; Boyd et al., 2002). These two
nutrients are added to ponds under the form of fertilizers, manures, and feeds. Moreover,
70
Chapter 3
these two primary nutrients can regulate aquatic primary productivity in most lakes and
coastal marine ecosystems, although the actual response of primary producers to N and P
enrichment can be affected by factors such as light conditions, hydrology, and grazing
(Wetzel, 2001; Boyd et al., 2002). Furthermore, the N:P ratio is a factor regulating
phytoplankton community structure (Bulgakov and Levich 1999; Geider and Roche, 2002;
Smith et al., 2006). Phytoplankton species show a common response to the total
nitrogen:total phosphorus (TN:TP) ratio, regardless of whether they are of marine or
freshwater origin (Wetzel, 2001; Smith et al., 2006). In natural phytoplankton communities,
many species are present at any given time. Presumably they represent a wide spectrum of
individual requirements for N and P. Hence, over a rather broad range of N:P supply ratios
(roughly from 10:1 to 40:1), some species may be N-limited and others P-limited (Suttle
and Harrison, 1988; Bulgakov and Levich, 1999; Wetzel, 2001; Smith, 2006).
According to Baert et al. (1996) manipulation of the algal composition in the fertilization
pond is still more of an art than a science. Usually a high N:P ratio is recommended (N:P of
10) if the growth of green algae (Tetraselmis, Dunaliella) and diatoms (Chaetoceros,
Navicula, Nitzschia) is desirable. However, as phosphor dissolves badly in salt water and is
absorbed very quickly at the pond bottom, N:P ratios of 3 to 5 might be more appropriate.
Besides N:P ratios, temperature, salinity, light intensity and pumping rate (input of new
nutrients and CO2) also play an important role in algae development. High N:P ratios
mostly stimulate green algae compared to diatoms at lower salinity and higher light
intensities. Some green algae are poorly digested by Artemia (Nannochloropsis,
Chlamydomonas). Hence, management of the algae population also depends on the
composition of the local algae community; the most dominant algae in the intake water will
also be the most dominant ones after fertilization. In addition, an appropriate management
of primary production has been identified as an important factor determining the final yield
of Artemia. Manipulations to increase primary production (e.g. by fertilization or extra
pumping) or to increase the availability of organic matter in the culture ponds have been
found to be a main factor affecting total Artemia cyst (Baert et al., 1996, 1997) and biomass
yields (Anh et al., 1997a).
Our previous study (Section II in Chapter 3) has found that co-supplementation of pig
manure with rice bran or soybean meal in the culture ponds resulted in a significant
increase in Artemia biomass yields compared to control ponds receiving only green water
as a single food source. However, some problems may occur if large quantities of organic
71
Chapter 3
matter present in the culture pond cause water pollution, negatively affecting the Artemia
population. Adhikari (2003) reported that utilization of excessive amounts of raw organic
manure can result in excessive blooms of bacteria during aerobic breakdown of this
manure, and may also cause oxygen depletion in the culture ponds. Consequently, in order
to further optimize the culture technique of Artemia biomass in salt ponds and to enhance
Artemia biomass productivity, this study wanted to obtain more information on the optimal
management of the fertilization pond by applying different N:P ratios to stimulate a steady
algal bloom and to improve “green water” quality and quantity as natural food, used in
combination with different supplementary feeds in the culture ponds
2. Material and Methods
The combined experimental system consisted of two different compartments: a series of
ponds for Artemia production and the fertilization pond where the nutrient management
was conducted to stimulate algae production. “Green water” from this pond was then
supplied to the culture ponds via the supply canal as natural food for Artemia. The
experiment lasted 12 weeks from February to May 2007 at Vinh Hau village, Bac Lieu
province, southeast coast of the Mekong Delta, Vietnam.
2.1. Fertilization pond and culture system
Four newly dug ponds of 90 m² each (length: 36 m, width: 2.5 m, depth: 1.0 m, flat bottom)
were built. After pond construction, pond bottoms were limed with 10 kg CaCO3 m-2 and
dried for 7 days, and then pig manure was added at a rate of 1.2 and 0.6 ton dry weight
(DW) ha-1 initially and every four weeks consecutively. Three days later, the ponds were
filled with seawater from the reservoir to a water depth of 0.5 m and during the following
three days the water level was increased up to 0.8 m. Then inorganic fertilizers
(combination of urea and super phosphate with different ratios of N:P=5:1 and 10:1,
according to the treatment allocated) were applied in the beginning at an amount of 7 g m-3.
Next applications were done once or twice a week; between 2-5 g m-3 was added depending
on the observed turbidity in the fertilization ponds: as a rule, fertilizer was added when
water turbidity exceeded 20 cm. Water level in these ponds was maintained equally at 0.70.9 m depth during the experiment. In general, about 30-40% of the water volume in each
fertilization pond was left after pumping into the culture ponds, and was instantly filled up
to the original depth. To achieve high productivity in the fertilization pond, raking the pond
bottom was applied every two days. Fertilizers were dissolved in water before application.
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Chapter 3
The ratio of nitrogen to phosphorus was calculated based on the information supplied by
the producer, i.e. 16.5% P2O5 in super phosphate and 46.3% nitrogen available in urea.
Two blocks of six earthen ponds were constructed for culturing Artemia (each pond had a
central platform and a ditch around the perimeter of the platform) with an area of 300m2
each (20m x 15m); the peripheral ditch was 2 m wide and 0.4 m deep.
2.2. Experimental setup
In this study, combinations of different green water types (fertilizer treatments) and
supplementary feeds (feeding treatment) were tested for their effects on the production of
Artemia biomass reared in the salt ponds.
Different fertilizer treatments corresponded with a fertilization pond to which inorganic
fertilizers were added at different ratios of N:P =5:1 and 10:1 (two replicates). One block of
culture ponds received green water from the N:P=5 treatment (GW5) and another from the
N:P=10 treatment (GW10) once every two days during the culture period.
A two-factor experiment with three replicates for each treatment, randomly selected from
the 6 available ponds in each block, was conducted as follows:
-
GW5+RB: green water from N:P=5 treatment, supplemented with rice bran;
-
GW5+PM: green water from N:P=5 treatment, supplemented with pig manure;
-
GW10+RB: green water from N:P=10 treatment, supplemented with rice bran;
-
GW10+PM: green water from N:P=10 treatment, supplemented with pig manure;
For details on feed supplementation, see ‘Feeding procedures’ below.
2.3. Pond preparation
The preparation of culture ponds was done prior to inoculation. For details see Section II in
Chapter 3. Ponds were inoculated when water salinity exceeded 80 g L-1.
At the start of the culture period, the water level in the ponds was about 4-5 cm above the
platform and then gradually increased up to >40 cm during the culture period. In order to
increase the availability of the natural food for Artemia, culture ponds were fertilized with
urea and super phosphate at a rate of 1 and 0.2 g m-2 two days before inoculation to
stimulate phytoplankton bloom.
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Chapter 3
2.4. Artemia inoculation
Artemia cysts collected from the previous year were used for our experiment. Hatching
conditions were according to Van Stappen (1996): seawater of 35g L-1 salinity, constant
illumination and aeration, temperature 25-28 °C. Freshly-hatched nauplii were inoculated in
the late afternoon. Ponds were inoculated at a stocking density of 100 individuals L-1.
2.5. Pond management
Culture of Artemia in the salt ponds was controlled daily to ensure optimal culture
conditions for growth of the Artemia population. For details see Section II in Chapter III.
2.6. Source of fertilizers and manure
Super phosphate fertilizer containing 16.5% P2O5 and maximum 0.6 % moisture, was
supplied by Long Thanh factory, Go Dau Industrial zone, Dong Nai province, Vietnam.
Urea fertilizer containing 46.3% nitrogen, maximum 1% biuret and maximum 0.4%
moisture was supplied by Phu My Company, Ho Chi Minh city, Vietnam.
The pig manure was purchased from the Bac Lieu animal husbandry farm. Rice bran (2nd
grade rice meal) was bought from a local market.
2.7. Feeding procedures
Pond supplementation started from day 5 after inoculation. Pig manure was applied at 200300 kg DW ha-1 week-1 and rice bran was added at a rate of 20-30 kg ha-1 day-1. Feeding
was done once daily between 16:00-17:00 h to provide extra food for Artemia in the
evening during the first 5 weeks of culture, and in-between the interval of green water
supply beyond this period, until the end of the culture.
Pig manure was stored in a plastic bag, which was perforated and submerged under water
for 2-3 days and then daily moved around the Artemia pond to allow its gradually spreading
over the pond bottom. Rice bran (about 10-20% of particle sizes ≤50µm) was fermented
with alcohol yeast (approximately 2-3%) for 24 hours before application; rice bran was
diluted with water and then manually distributed over the pond surface.
2.8. Data collection
2.8.1 Fertilization pond
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Chapter 3
Water temperature and pH were measured at 7:00 and 14:00 h with a combined pHtemperature meter (Model HI 98127, HANNA Instruments, USA). Salinity was measured
at 7:00 h with a hand refractometer (Atago, Japan) and turbidity at 14:00 h with a Secchi
disk; all measurements were made on a daily basis. Water colour and weather conditions
were also noted every day.
Water samples were collected weekly between 9:00 and 10:00 h for determination of
ammonium (N-NH4+), nitrate (N-NO3-), total nitrogen (TN), soluble reactive phosphorous
(PO43-), total phosphorous (TP) and chlorophyll a. Six different sampling points were
identified in each pond at the start of experiment. Samples were collected using a 5 Lplastic beaker; these samples were pooled and mixed in a big bucket and two sub-samples
(0.6 L) were taken out and preserved in ice for transport to the laboratory for analysis using
standard methods according to the American Public Health Association (APHA, 1998).
Algal composition was evaluated using a plankton net (mesh size 25μm) that was dragged
horizontally all over the pond surface. Samples were fixed in 4% formaldehyde. Algal
species were identified microscopically at 400x magnification in a settling chamber by
using an electronic microscope and based on the studies of Shirota (1966), Jomas (1996)
and Tien (1996). The relative presence of each species was noted as follows: + = only rare
or few cells present; ++ = sub-dominant and +++ = dominant.
All water parameters were also determined before application of the fertilizers.
2.8.2. Artemia ponds
Water temperature, salinity and turbidity in the culture ponds were recorded at the same
time as for the fertilization ponds (see above).
Artemia survival and growth were determined at days 11 and 14 (only evaluating the
original population inoculated). Nine point samples per pond were collected using a square
plankton net (mesh size 100μm, surface area 0.25m2) that was towed vertically through the
water column at each sampling site in the early morning (Baert et al., 2002). Samples were
fixed in Lugol’s solution, counted, and the number of animals per litre was calculated.
These data were compared to the initial stocking density, which allowed the calculation of
the Artemia survival in the culture ponds.
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Chapter 3
Growth was expressed as length and weight of the sampled Artemia. Using a scoop net with
1 cm mesh size allowed to collect only the original population, and not its offspring (in this
experiment Artemia females started producing offspring after 10-11 days following
inoculation).
For determination of the total length of Artemia, 30 animals were randomly taken from the
samples collected for determination of survival (see above) after they had been pooled per
pond. Length was measured from the anterior part of the head to the end of the telson, using
a dissection microscope equipped with a micrometric ruler.
Wet weight of Artemia was determined by taking five samples (about one gram each) of
Artemia in each pond, removing extra water by tissue-paper, weighing (0.00g precision
balance) and counting the number of animals in each sample, which allowed to calculate
the mean individual wet weight of the Artemia.
Sampling for evaluation of the fecundity (expressed as number of nauplii per brood per
female) followed the same procedure as for growth parameters. Sixty females with full
brood sack were randomly taken from the pooled samples collected from five different
places in each pond. The brood sacks were immediately cut off and preserved in a 4%
formalin solution; then the offspring (nauplii) was counted under the binocular. The
number of females producing nauplii, observed among sixty specimens, was calculated as
percentage ovoviviparity. Fecundity and ovoviviparity were evaluated on a weekly basis.
The purpose of this study was to evaluate the production of Artemia biomass; therefore
only ovoviviparous reproduction was taken into account.
2.9. Supplemental feed
Nitrogen, carbon and phosphorus and dry weight of supplemental feed (pig manure and rice
bran) were analyzed at three different times. In particular, pig manure was analyzed before
application, and after 1 and 2 months of storage at room temperature.
2.10. Biomass harvesting
Harvesting of Artemia biomass was based on the study of biomass harvest strategies by
Anh and Hoa (2004). Adult Artemia was partially collected after three weeks of culture,
and harvest was conducted every three consecutive days at an amount of 75-150 kg wet
weight ha-1 per 3 days (2.25-4.50 kg pond-1). For details see Section II in Chapter 3.
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Chapter 3
2.11. Statistical analysis
The percentage values were normalized through a square root arcsine transformation before
statistical treatment. A two-factor ANOVA test (SPSS, version 13.0) was used to detect
significant interactions between green water and feed supplement. For all treatments, results
were analyzed statistically with one-way ANOVA analysis of variance to find the overall
effect of the treatment. Tukey’s HSD (Tukey’s Honestly Significant Difference) test was
used to identify significant differences between the experimental sample means at a
significance level of P<0.05. Linear or multiple regression was used to detect the
relationship between parameters in the fertilizer experiment. For the multiple-regression
model, the Stepwise Criteria method was applied to find out the best correlation of factors
and the dependent variables.
3. Results
3.1. Fertilization ponds
3.1.1. Water quality in the fertilization ponds
In the two fertilizer treatments the water parameters: salinity, temperature, pH and turbidity
were similar (Figure 1). Salinity varied between 30 and 46 g L-1, temperature and pH were
in the ranges of 22.5-29.5°C; 8.1-9.9 and 29.6-34.8°C; 8.4-11.9 in the morning and
afternoon, respectively. Higher salinity and temperature were observed between weeks 7
and 11 (March-April). Especially heavy rainfall caused a sharp decline in salinity in May
(week 12). At the starting day, the value for turbidity was 62 cm and tended to increase
after 1 week of fertilizer application, and varied between 10 and 30 cm from week 2
onwards.
The mean concentrations of N-NH4+, N-NO3- and TN ranged from 0.3-2.4, 0.2-1.9 and 1.211.6 mg L-1, respectively. These values were highest at the end of the experiment and
slightly higher in the NP10 treatment than in the NP5 treatment (Figure 2a-c).
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Chapter 3
On the contrary, the average P-PO43- and TP levels in the NP5 treatment fluctuated in the
ranges of 0.03-0.24 and 0.07-1.84 mg L-1, respectively and were higher compared to those
in the NP10 treatment which were in the range of 0.02-0.19 and 0.07-1.19 mg L-1,
respectively. In particular, the P-PO43- level showed the highest peak in week 7 (Figure 2de). Generally, the concentrations of N-NH4+, N-NO3-, TN and TP had a tendency to
38
7am
35
2pm
(a)
pH-2pm
(b)
11
32
29
26
23
10
9
8
7
20
70
60
50
40
30
20
10
0
1
2
0 1 2 3 4 5 6 7 8 9 10 11 12
3 4 5 6 7 8 9 10 11 12
Experimetal period (week)
Experimental period (week)
(d)
(c)
N:P=5
N:P=10
Salility (g/L)
0
Turbidity (cm)
pH-7am
12
pH value
Temperature (°C)
increase towards the end of the experiment.
46
43
40
37
34
31
28
25
N:P=5
N:P=10
0 1 2 3 4 5 6 7 8 9 10 11 12
Experimental period (week)
0 1 2 3 4 5 6 7 8 9 10 11 12
Experimental period (week)
Figure 1. Fluctuation of abiotic water parameters in the fertilization ponds.
(a) Temperature, (b) pH, (c) Salinity, (d) Turbidity.
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Chapter 3
N:P=10
1.5
1.0
0.5
0.0
1 2
3
4 5
6
7 8
N :P = 5
2.0
N :P = 10
0.5
0.0
0
(c)
N:P =1 0
(b)
1.0
9 10 11 12
N:P =5
14
12
10
8
6
4
2
0
2.5
1.5
PO4 (mg/L)
TN (mg/L)
0
1
2
3
4
1.0
N:P=5
0.8
N:P=10
5
6
7
8
9 10 11 12
(d)
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
7
8
0
(e)
N:P=5
1.8
1.5
1.2
0.9
0.6
0.3
0.0
9 10 11 12
N:P=10
N:P ratio
TP (mg/L)
(a)
N:P=5
2.5
2.0
N-NO3 (mg/L)
N-NH4 (mg/L)
3.0
0
1
2
3
4
5
6
7
8
1
2
3
4
5
24
21
18
15
12
9
6
7
(f)
8
9 10 11 12
N :P = 5
N :P = 10
6
3
0
9 10 11 12
0
Exp erimen tal p erio d (week)
1
2 3 4 5 6 7 8 9 10 11 12
Exp eriment al p eriod (w eek)
Figure 2. Fluctuation of nutrient parameters in the fertilization ponds.
(a) N-NH4+; (b) N-NO3-; (c) TN; (d) P-PO43-; (e) TP; (f) TN:TP ratio
The ratio of TN:TP was affected by the fertilizer treatment. The mean ratio of TN:TP was
16.8 at the start and ranged from 6.4 to 13.6 and from 7.4 to 20.6 for the NP5 and NP10
treatments, respectively (Figure 2f). Especially after two weeks of fertilizing the highest
value (20.6) was observed in the NP10 treatments. Comparing the corresponding values
between two treatments, the ratio of TN:TP in the NP10 treatment was significantly higher
than in the NP5 treatment (P<0.05).
During the sampling period, the Ch a level in the two fertilizer treatments followed a
similar pattern; i.e. low at the beginning (22 µg L-1) and varying from 78 to 274 µg L-1 for
the NP5 treatment and from 60 to 253 µg L-1 for the NP10 treatment. Peak values for both
fertilizer regimes were observed between weeks 3-4 and 8-9 (Figure 3a). In parallel, linear
regression showed that the contents of Ch a in both treatments was positively correlated
with temperature and pH (Figure 3b-c), whereas it showed an inverse relationship with
turbidity (Figure 3d).
79
Chapter 3
Furthermore, multiple-regression analysis combining stepwise selection of independent
variables to find a correlation between Ch a and nutrients (nitrogen and phosphorus) in the
fertilization ponds for both treatments resulted in two equations:
YNP5 = 22.377x1-51.012x2+26.444 (R=0.759, P=0.014) (1)
YNP10 = 21.569x1-85.180x2+52.877 (R=0.905, P=0.000) (2)
Where: YNP5 and YNP10 : concentration of chlorophyll a in NP5 and NP10 treatments
x1 and x2: concentration of TN and N-NH4+
From equations (1) and (2) we can conclude that the Ch a concentration was largely
affected by the nitrogen content present in the water column; Ch a had positive relationship
with total nitrogen content (TN) whereas it showed a negative correlation with the contents
of N-NH4+ for both fertilizer treatments. The excluded variables in the two models were NNO3- , P-PO43-, TP and ratio of TN:TP.
300
250
Chlorophyl a (µg/L)
Chlorophyl a (µg/L)
(b)
(a)
200
150
100
50
N:P=5
N:P=10
0
0
1
2
3
4
5
6
7
8
9
300
YNP5 = 50.265x- 1454.3
250
R = 0.7545
200
150
29
(c)
150
YNP10 = 60.036x- 481.62
R2 = 0.8383
50
9
10
11
12
31
32
33
Temperature (°C)
34
13
YNP5 = -6.7254x+ 296.44
200
2
R = 0.7427
150
100
YNP10 = -7.7759x+ 314.56
50
2
R = 0.7838
5
pH
35
(d)
250
0
0
8
30
300
Chlorophyll a (µg/L)
Chlorophyl a (mg/L)
200
100
2
R = 0.7119
50
0
10 11 12
YNP5 = 55.427x- 420.01
R2 = 0.7588
250
YNP10 = 50.077x- 1458
100
Experimental period (week)
300
2
10
15
20
25
30
35
40
Turbidity (cm)
Figure 3. Chlorophyll a levels in fertilization ponds. (a) Variation in Chl a levels with
time. (b) Correlation between Chl a and temperature; (c) Correlation between Chl a and pH
(d) Correlation between Chl a and turbidity.
80
Chapter 3
CYANOBACTERIA
CHLOROPHYTA
BACILLARIOPHYTA
DINOPHYTA
CYANOBACTERIA
(a)
10
12
CHLOROPHYTA
BACILLARIOPHYTA
DINOPHYTA
8
6
4
2
Number of algal species
Number of algal species
12
0
(b)
10
8
6
4
2
0
0
1
2
3
4
5
6
7
8
0
9 10 11 12
1
2
3
4 5 6 7 8
Weekly sampling
Weekly sampling
9 10 11 12
Figure 4. Occurrence of algal phyla in fertilization ponds as a function of time.
(a)
21
18
15
12
9
6
3
0
0
2
Total number of algal species
Total number ofalgal species
(a) N:P=5 treatment, (b) N:P=10 treatment
y =-0.9719x+16.754
R² =0.4005, P=0.020
4
6
8
10
12
(b)
21
18
15
12
9
6
3
0
0
2
4
y =-0.3866x+13.735
R² =0.0914, P=0.326
6
8
10
12
14
-1
Total nitrogen (mg L )
Figure 5. Correlation between total number of algal species and the total nitrogen in the
fertilization ponds. (a) N:P=5 treatment, (b) N:P=10 treatment
3.1.2. Algae compositions
Four phyla of algae, i.e. Cyanophyta, Bacillariophyta, Chlorophyta and Dinophyta, were
found in this study. The variation in the number of algae species for both fertilizer
treatments is summarized in Table 1. The results showed that the total number of algae
species was 67 in which the NP10 samples were more diversified (62 species) than in the
NP5 treatment (52 species). The percentage of species compared to the whole community
indicated that Bacillariophyta (diatoms) were predominant (60-63%), followed by
Cyanophyta (15-16%), Dinophyta (13-15%) and Chlorophyta (8-10%). Furthermore,
Cyanophyta and diatoms were observed over the whole sampling period, Chlorophyta only
occurred in the first period while Dinophyta were detected in the last period and both
groups appeared in a fairly low number of species (Figure 4). Generally, there were no
81
Chapter 3
apparent differences in the algal occurrences over time between the two treatments
although the number of blue-green algae species was slightly higher in the NP10 than in the
NP5 treatment.
(a)
y = 0.6664x + 3.7135
18
Total number of algal species
Total number of algal species
21
2
R = 0.3624; P=0.030
15
12
9
6
3
21
y = -0.13x + 12.361
18
R = 0.0199; P=0.643
(b)
2
15
12
9
6
3
0
0
3
6
9
12
15
18
3
21
6
9
12
15
18
21
TN:TP ratio
TN:TP ratio
Figure 6. Correlation between the total number of algal species and the ratios of TN:TP.in
the fertilization ponds. (a) N:P=5 treatment; (b) N:P=10 treatment.
Table 1. Number of algae species in the fertilization pond during 12 weeks of experiment
Phylum
Bacillariophyta
NP5+NP10
NP5
NP10
Species Frequency
number
(%)
Species Frequency
number
(%)
Species Frequency
number
(%)
42
62.69
33
63.46
37
59.68
Chlorophyta
6
8.96
4
7.69
6
9.68
Cyanophyta
10
14.93
9
17.31
10
16.13
9
13.34
6
11.54
9
14.52
Dinophyta
Total
67
52
82
62
Chapter 3
Table 2. Variation in algae composition of each sample during the experimental period
Sampling
date
Study
period
10/2/07
Total number of
species
NP5
NP10
Dominant (+++)/Sub-dominant (++) species
Week 0*
19
19
nd
17/2/07
Week 1
12
14
nd
24/2/07
Week 2
9
6
NP5: Tetraselmis cordiformmis ++ (Chlorophyta)
NP10: Tetraselmis sp. +++ (Chlorophyta)
NP10: Phormidium sp. ++ (Cyanophyta)
3/3/07
Week 3
7
9
NP5: Chaetoceros curvisetus +++ (Bacillariophyta)
NP10: Chaetoceros lauderi ++ (Bacillariophyta)
NP10: Tetraselmis sp. ++ (Chlorophyta)
10/3/07
Week 4
6
9
NP5: Chaetoceros diversus +++ (Bacillariophyta)
NP5: Nitzschia sigma ++ (Bacillariophyta)
NP10: Cymbella ventriccosa +++ (Bacillariophyta)
17/3/07
Week 5
8
11
NP5: Nitzschia sp. +++ (Bacillariophyta)
NP10: Gyrosigma attenuatum ++ (Bacillariophyta)
NP10: Nitzschia sigma var. intercedens ++
(Bacillariophyta)
24/3/07
Week 6
9
10
NP5: Nitzschia longissima var. reversa +++
(Bacillariophyta)
NP10: Nitzschia sigma ++ (Bacillariophyta)
31/3/07
Week 7
12
13
NP5: Nitzschia philippinarum +++ (Bacillariophyta)
NP10: Nitzschia sp. ++ (Bacillariophyta)
7/4/7
Week 8
9
7
NP5: Navicula gracilis ++ (Bacillariophyta)
NP5: Nitzschia philippinarum ++ (Bacillariophyta)
NP10: Navicula placentula fo rostrata ++ (Bacilla.)
NP10: Nitzschia sigma var Intercedens ++ (Bacilla.)
14/4/07
Week 9
5
7
NP5: Nitzschia philippinarum +++ (Bacillariophyta)
NP10: Nitzschia acicularis ++ (Bacillariophyta)
NP10: Pleurosigma fasciola ++ (Bacillariophyta)
21/4/07
Week 10
11
10
NP5: Nitzschia philippinarum ++ (Bacillariophyta)
28/4/07
Week 11
8
14
NP5: Navicula gracilis ++ (Bacillariophyta)
NP5: Nitzschia philippinarum ++ (Bacillariophyta)
NP10: Navicula sp. ++ (Bacillariophyta)
5/5/07
Week 12
9
11
NP5: Nitzschia philippinarum ++ (Bacillariophyta)
Week 0* sample collected before application of fertilizer; nd: not detected
83
Chapter 3
It was observed that the total number of algal species was negatively and positively
correlated with TN and the TN:TP ratio (Figure 5a and 6a), respectively. However, this
relationship was only observed in the NP5 treatment while these characteristics did not
show any correlation in the NP10 samples (Figure 5b-6b). Additionally, total phosphorus
did not affect the structure of the algal community for both fertilizer treatments. On the
other hand, sub-dominant and dominant species of algae in the fertilization ponds were
altered with time and fertilizing regime (Table 2). For example, in NP5 samples of week 2
Tetraselmis cordiformmis (Chlorophyta) was sub-dominant (++), whereas in NP10 samples
Tetraselmis sp. (Chlorophyta) was most abundant (+++) followed by Phormidium sp. (++)
(Cyanophyta). From this period onwards, different algal species belonging to
Bacillariophyta prevailed over other algal communities for both treatments except for
Tetraselmis sp. (Chlorophyta) being sub-dominant (++) in the NP10 samples of week 3.
Moreover, Chaetoceros sp. only dominated between weeks 3 and 4. Particularly, Nitzschia
(diatoms) was frequently found throughout the sampling period.
3.2. Artemia ponds
3.2.1. Chemical composition of the supplementation products
The chemical composition of the two supplementation products is presented in Table 3. The
carbon level in rice bran (RB) was higher than in pig manure (PM) while nitrogen content
in PM and RB was similar. However, the phosphorus level in PM was almost twice the
value for RB. In addition, the ratios of C:N and N:P were higher in RB (16.2 and 10.3),
than in PM (2.0 and 1.3), respectively.
Table 3. Chemical composition of the two supplementation products (% dry matter).
Values are average and standard deviation of three replicates.
Rice bran
Pig manure
Dry matter
88.58 ± 1.28
50.02 ± 13.94
Carbon
42.31 ± 2.08
30.61 ± 4.38
Nitrogen
2.63 ± 0.36
2.98 ± 0.39
Phosphorus
1.39 ± 0.22
2.40 ± 0.32
C :N ratio
16.24 ± 1.43
10.27 ± 0.28
N:P ratio
1.95 ± 0.53
1.25 ± 0.26
84
Chapter 3
3.2.2. Abiotic factors
There was almost no difference in abiotic factors among the treatments. Mean salinity was
in the range from 74 to 99 g L-1, water temperature increased from February to April with
observed ranges of 22-29°C and 31-36°C at 7.00 am and 2.00 pm, respectively (Figure 7).
However, extreme temperatures in the afternoon usually occurred between weeks 10 and 11
(from 16th until 26th of April) with absolute values of 37-39°C. Water depth increased from
5 to 50 cm. Turbidity at the day of inoculation was 22-23 cm (Figure 8) and fluctuated
39
100
36
95
33
90
30
85
27
80
24
75
Temperature_2 pm
21
70
Temperature_7 am
18
Salinity (ppt)
Temperature (°C)
within the range of 28-40 cm during the culture period.
65
Salinity
15
60
0
1
2
3
4
5
6
7
8
9
10
11
12
Culture period (week)
Figure 7. Fluctuation of water temperatures and salinity in the Artemia ponds during the
culture period.
42
39
36
Turbidity (cm)
33
30
NP5+RB
NP5+PM
NP10+RB
NP5+PM
27
24
21
18
15
0
1
2
3
4
5
6
7
8
9
10
11
12
Culture period (week)
Figure 8. Fluctuation of turbidity in the Artemia ponds during the culture period for the
various treatments
85
Chapter 3
3.2.3. Survival and growth
Survival and growth of Artemia cultured with different green water type and feed
supplements are shown in Table 4. Two-factor ANOVA test for the survival of Artemia
after 11 days of culture (the original population) reveals that there was no significant
interaction between green water and feed supplement (P=0.847). Average survival ranged
from 70.3% to 73.5% and no significant differences were found among treatments
(P>0.05).
Table 4. Mean survival at day 11, length and weight at day 14 and final yield of Artemia
biomass cultured with different green water source (fertilizing with different N:P ratio) and
supplementary feed. Values are average and standard deviation of three replicate ponds.
Values within a column with different letters were significantly different (P<0.05).
Survival
(%)
Length
(mm)
Weight
(mg)
Biomass yield
(ton ha-1 crop-1)
GW5
71.84±3.56a
9.34±0.71a
9.39±0.37a
2.29±0.48a
GW10
71.98±3.60a
9.22±0.75a
9.19±0.44a
1.97±0.36a
RB
73.54±3.01a
9.24±0.77a
9.29±0.32a
2.19±0.36a
PM
70.28±3.20a
9.31±0.70a
9.29±0.49a
2.07±0.53a
Green water (1)
0.946
0.114
0.065
0.267
Feed supplement (2)
0.617
0.359
0.981
0.671
0.847
0.003
0.030
0.499
GW5+RB
9.19±0.81a
9.27±0.38ab
GW5+PM
9.48±0.57b
9.50±0.33b
GW10+RB
9.29±0.72ab
9.31±0.26ab
GW10+PM
9.14±0.77a
9.08±0.55a
Main effect
Green water
Feed supplement
P value
Interaction:(1)x(2)
14 days after inoculation, there was significant interaction between green water and feed
supplement factors on total length and individual weight of Artemia (two-factor ANOVA,
P=0.003 and 0.030). The values of mean length in the GW5+PM group (9.48 mm) were
slightly higher than in the GW10+RB treatment (9.29 mm) and significantly larger
(P<0.05) as compared to those in the GW5+RB and GW10+PM groups (9.19 and 9.14
86
Chapter 3
mm). A similar pattern was observed for individual weight; these values ranged from 9.08
to 9.50 mg. However, a significant difference was observed only between the GW5+PM
and the GW10+PM samples (P<0.05). One-way ANOVA analysis of the green water or
supplementary feed as a main effect showed no significant differences in length and weight
values (P>0.05).
The interaction between green water and feed supplement was not significantly different
(two-factor ANOVA, P=0.499) for the total yield of Artemia biomass (Table 4). When
comparing between the two green water treatments, GW5 had a slightly higher value (2.29
ton WW ha-1 crop-1) than GW10 (1.97 ton WW ha-1 crop-1) although there was no statistical
difference between both values. Moreover, feed supplementation with RB or PM resulted in
similar yields of Artemia biomass (2.19 and 2.07 ton ha-1 crop-1), respectively.
3.2.4. Reproduction parameters
Table 5. Reproduction characteristics (percentage of ovoviviparity and brood size) at first
spawning and average values of the whole culture period of Artemia biomass cultured with
different green water source (fertilizing with different N:P ratio) and feed supplement.
First spawning
Ovoviviparity
(%)
Main effect
Whole culture period
Brood size Ovoviviparity
(%)
(No. nauplii
-1
female )
Brood size
(No. nauplii
female-1)
Green water
GW5
13.06±3.86
44.5±5.4b
19.17±10.59
47.5±3.7b
GW10
11.94±1.95
30.2±1.7a
18.36±10.04
41.3±3.5a
RB
12.78±3.60
36.4±6.5
18.43±9.92
44.7±4.0
PM
12.22±2.51
38.4±10.5
19.09±10.71
44.0±5.8
Green water (1)
0.585
0.000
0.228
0.011
Feed supplement (2)
0.783
0.344
0.320
0.731
0.899
0.438
0.076
GW5+RB
0.046
41.3±5.9b
GW5+PM
47.8±2.7b
GW10+RB
31.5±1.9a
GW10+PM
28.9±1.3a
Feed supplement
P value
Interaction:(1)x(2)
Values within a column with different letters were significantly different (P<0.05).
87
Chapter 3
By two-factor ANOVA analysis for percentage of ovoviviparity at the first spawning (the
original population inoculated) and the average value of the whole culture period, no
significant interaction between green water and feed supplement was found (P= 0.899 and
P=0.438). Again, values for the ovoviviparity percentage were similar, both for green water
and feed supplement and varied in the ranges 11.9-13.1% and 18.4-19.4%, respectively
(Table 5). Furthermore, Figure 9a illustrates the percentage of ovoviviparity versus the
culture period for all treatments. In the first brood (week 2), a relatively high percentage of
Artemia females reproduced ovoviviparously (11.7-13.3%), after which most of them
switched to oviparity in the second brood (week 3: ovoviviparity in the range of 4.4-6.1%).
From week 4 onwards, ovoviviparity proportion showed a gradual increase with the culture
period and reached the highest values in week 10 (33.3-40.6%), followed by a sharp decline
(21.7-26.7%) at the end of the culture period. In general, ovoviviparous reproduction in all
treatments displayed a similar trend.
45
NP5+RB
40
NP5+PM
%Ovoviviparity
35
(a)
NP10+RB
30
NP10+PM
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
Culture period (week)
Brood size (nauplii/female)
NP5+RB
NP5+PM
(b)
90
80
NP10+RB
70
NP10+PM
60
50
40
30
20
10
1
2
3
4
5
6
7
8
9
10
11
12
Culture period (week)
Figure 9. (a)Variations in ovoviviparity (%), (b) Variations in brood size of Artemia
females (number of nauplii female-1) during the culture period for the various treatments
(error bars stand for standard deviation).
88
Chapter 3
A two-factor ANOVA analysis indicated significant interaction (P=0.046) between green
water and feed supplement only for the size of the first brood; no significant interaction was
found for the average brood size of the whole culture period (P=0.076). Nevertheless,
analysis of green water effect on brood size revealed significant differences for both
parameters (P=0.000 and P=0.011). The GW5 had significantly higher values (44.5 and
47.5 nauplii brood-1 female-1) compared with the GW10 sample (30.2 and 41.3 nauplli
brood-1 female-1) for the size of the first brood and the mean brood size of the whole culture
period, respectively. In all treatments the fecundity showed a progressive increase from
week 2 to 4 (Figure 9b). From week 5 onwards, brood sizes pointed to a moderate decrease
in these groups. Generally, the NP5+PM and NP10+PM treatments showed higher and
lower values, respectively, compared to the other two treatments.
3.2.5. Feed conversion ratio
The data show that after 12 weeks of experiment, 1 hectare of Artemia biomass production
needs about 2213 kg DW pig manure, 102-111 kg urea and 31-57 kg super phosphate to
produce sufficient algae as a natural food for Artemia (Table 6).
Table 6. Total pig manure and fertilizers used in the fertilization ponds
Treatment
N:P=5
N:P=10
Total fertilizer applied for 0.18 hectare Artemia biomass experiment
Pig manure (kg DW treatment-1)
398
398
Urea (kg treatment-1)
18.4
20.0
Super phosphate (kg treatment-1)
10.3
5.6
2213
2213
102
111
57
31
Total fertilizer applied for 1 hectare Artemia production
Pig manure (kg DW ha-1 Artemia)
Urea (kg ha -1 Artemia)
Super phosphate (kg ha-1 Artemia)
The estimated conversion ratio of rice bran, pig manure and Chlorophyll a to Artemia
biomass is summarized in Table 7. The conversion ratios for rice bran were 0.62 and 0.65,
and for Ch a 0.80 and 0.98 mg Ch a kg-1 Artemia biomass for the GW5+RB and
GW10+RB treatments, respectively. For the GW5+PM and GW10+PM treatments the
corresponding conversion ratios of pig manure were 1.17 and 1.43, and 0.81 and 1.14 mg
89
Chapter 3
Ch a kg-1 Artemia biomass. These results indicate that the conversion ratios of RB or PM in
the GW5 groups were lower than in the GW10 treatments although a significant difference
was not detected (P>0.05).
Table 7. Estimates of conversion ratio of rice bran, pig manure and chlorophyll a to
Artemia biomass (300m2 pond-1).
Treatment
Total feed
applied in
Artemia pond
GW5+RB
Rice bran
(DW)
(kg pond-1)
Pig manure
(DW)
Chl a
(mg pond-1)
GW5+PM
GW10+PM
40.66
40.66
-
-
1355.27
1355.27
-
-
(kg pond-1)
-
-
76.50
76.50
(kg ha-1)
-
-
2550.00
2550.00
52.71
60.72
52.71
60.72
1.76
2.02
1.76
2.02
kg WW pond-1
67.5±12.3
63.7±11.4
69.6±19.3
54.6±9.8
kg ha-1 crop-1
2251±410
2123±380
2321±643
1821±327
0.62±0.12
0.65±0.13
-
-
-
-
1.17±0.38
1.43±0.26
0.80±0.16
0.98±0.19
0.81±0.27
1.14±0.21
(kg ha-1)
(g ha-1)
Total Artemia
biomass yield
GW10+RB
Conversion ratio (kg rice bran kg-1 biomass)
Conversion ratio (kg pig manure kg-1 biomass)
Conversion ratio (mg Ch a kg-1 biomass)
3.2.6. Production costs
Total production cost for twelve weeks of Artemia biomass culture is summarized in Table
8. In this study, the costs of pond construction, labour, cysts for inoculation, pump, fuel and
other consumables were assumed to be the same for all treatments and only expenditures of
feed (pig manure, fertilizer and rice bran) were different according to treatments allocated.
Production costs were similar among treatments, varying between 1226 and 1341 US$ ha-1
in 2007 (Table 8). Feed for Artemia ranked first (28-34%) in the category of expenses and
manual labour ranked second (24-25%), followed by fuel (15-17%) and pond construction
(10-11%); feed and manual labour accounted for more than 50% of the total production
cost. Additionally, the expenditure for the production of 1 kg of Artemia biomass was
lowest (0.56 US$) and highest (0.69 US$) in the NP5+PM and NP10+PM treatments,
respectively. However, significant differences were not observed among treatments
90
Chapter 3
(P>0.05). Similar estimations for the income, net profit and benefit-cost ratio, ranged from
2276-2901, 1049-1676 US$ ha-1 and 0.85-1.37, respectively.
Table 8. Production costs (US$) for 1 ha of Artemia biomass production in the salt ponds
Description
NP5+RB
NP5+PM
NP10+RB
NP10+PM
140.6
140.6
140.6
140.6
300
300
300
300
75
75
75
75
Pump
62.5
62.5
62.5
62.5
Fuel
206.3
206.3
206.3
206.3
Pig manure
96.8
305.2
96.8
305.2
Fertilizer
35.9
35.9
37.6
37.6
Rice bran
321.9
0
321.9
0
Materials
100
100
100
100
1339
1226
1341
1227
0.61±0.12a
0.56±0.19a
0.65±0.13a
0.69±0.13a
Income ha-1
2814±512
2,901±804
2,654±475
2276±408
Net profit ha-1
1,475±512
1,676±804
1,313±475
1,049±408
Cost benefit ratio
1.10±0.38
1.37±0.36
0.98±0.35
0.85±0.33
Pond construction
Labour
Seed
Total cost ha-1
Cost kg-1 Artemia biomass
Costs of pig manure and fertilizers (urea and super phosphate) used in the fertilization
ponds were included in the production costs.
The expense in this experiment was based on prices in 2007, 1 US$ ≅ 16,000 VND
4. Discussion
4.1. Fertilization ponds
4.1.1. Water parameters in the fertilization ponds
The results showed that abiotic factors such as salinity, water temperature, pH and turbidity
showed a similar pattern in the two fertilizer treatments (NP5 and NP10). Salinity and
temperature increased during the dry season (February-April) and declined in the rainy
season (May). Moreover, in the present experiment, salinity in the fertilization pond was
influenced by the water intake from the reservoir, and temperature was affected by salinity
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Chapter 3
and water depth. Temperature and salinity are important factors affecting the algal growth
and structure of algal assemblages (Baert et al., 1996; Letland et al., 2001; Smith, 2006).
Moreover, algae have definite temperature optima and tolerance ranges, which interact with
other parameters in a seasonal succession (Wetzel, 2001). According to Renaud et al.
(2002), in the tropics microalgae in outdoor cultures must grow within a high diurnal
temperature range of 25-35°C throughout the year. The observed daily temperature
variation during the experimental period was in the range of tolerance for algae
development (Tuyen, 2003).
It was observed in this study that pH and turbidity were related to the Ch a level (Fig.3c-d).
On the other hand, high fluctuation of pH and turbidity could mainly be influenced by
water exchange i.e. about 40-60% of the water volume in these ponds was replaced every
two days by seawater from the reservoir. If these two parameters were measured at the day
when new seawater was supplied, the Ch a level and pH were low and turbidity was high.
However, a significant relationship was not detected between pH and turbidity.
Ammonium (N-NH4+) and phosphorus (P-PO43-) are important parameters to assess the
potential primary production in fertilization ponds (Baert et al., 1996; Boyd et al., 2002;
Smith, 2006). According to Wetzel (2001) the growth of an algal population under
adequate light and temperature conditions is often limited by a single nutrient. This
limitation can rapidly shift from nutrient to nutrient as their availabilities change on a
diurnal, daily and seasonal basis.
In our experiment the nitrogen and phosphorus concentrations for both fertilizer treatments
showed great variations with time and followed a similar trend (Figure 2). Besides water
exchange as mentioned before, other key factors such as fertilizing strategies and type of
fertilizer (organic or inorganic), source of water intake, weather, etc. may strongly affect
these parameters. In practice, higher nitrogen and phosphorus concentrations coincided
with the application of fertilizers. A moderate peak of nitrogen and phosphorus levels in the
first week of fertilizer application was due to the high initial dose of manure and fertilizer
application i.e. 1.2 ton pig manure (DW) ha-1 was added at the beginning of the experiment,
then 7 g m-3 of fertilizer was applied at day 3, followed by 5 g m-3 at day 6. However, the
highest phosphorus peak occurred at week 7, maybe caused by the addition to the
fertilization ponds of pig manure (which has a high phosphorus level: 2.4% DW (see Table
3) two day prior to sampling. On the other hand, a decline of N-NH4+, N-NO3- and P-PO4392
Chapter 3
concentrations was probably due to the rapid utilization of nutrients by phytoplankton in
that period. According to several studies, concentrations of soluble reactive phosphorus and
dissolved inorganic nitrogen, which are nutrients primarily assimilated by algae, show great
temporal variations (Boyd et al., 2002; Figueredo and Giani, 2005; Rahman et al., 2008a).
Furthermore, Khoi et al. (2006a) recognized that algae growth resulted in an exhaustion of
ammonium in all culture media of Chaetoceros calcitrans.
The Redfield ratio of N:P=16:1 (Redfield et al., 1963) has widely been used to evaluate the
nutrient limitation status of many marine ecosystems for which measurements of particulate
nutrients, dissolved inorganic nutrients or total nutrient concentrations are available (Geider
and Roche, 2002). In aquatic systems where N:P ratios exceed this value, the conditions
shift to P limitation. In contrast, N becomes a limiting nutrient for aquatic plants as the N:P
ratio in the water column is below 16.
At the beginning of this experiment the ratio of TN:TP (16.8) was close to the Redfield
ratio value. Nonetheless, in most cases, these values were lower than the Redfield values,
except for the value in the NP10 treatment at week 2, which was higher. The input of pig
manure with low ratio of N:P (1.3) may have contributed to lower the TN:TP ratios in the
water column during the sampling period. Generally, the ratios TN:TP in the NP10
treatment were higher than those in the NP5 treatment (Figure 2f). Geider and Roche
(2002) found that under optimal nutrient-replete growth conditions for phytoplankton, the
cellular N:P ratio is somewhat more constrained, ranging from 5 to 19 mol N: mol P, with
most observations below the Redfield ratio of 16. The lowest values of N:P are associated
with nitrate- and phosphate-replete conditions. The highest values of N:P are observed in
oligotrophic waters.
Chlorophyll a data provide a good estimate for phytoplankton dynamics in the pond; it is
about 1-2% of the total algal dry weight. Comparison of the Ch a level between the two
treatments shows a similar pattern during the experimental period. The availability of
nutrients either from the decomposition of accumulated organic matter from pig manure or
from fertilizer addition caused repeated algal peaks in both treatments. On the other hand,
Ch a of two fertilizer treatments increased to a moderate peak, then declined at the moment
of new water intake since algal biomass was partially removed (40-60% of pond volume)
from the system every two days, at a moment when algae can be in the exponential phase
(the cell density increases as a function of time, Coutteau, 1996). The study of Smith (2006)
93
Chapter 3
compared the N and P dependence of Ch a in 92 coastal ecosystems and demonstrated a
strong positive response of marine phytoplankton growth to N and P enrichment. Moreover,
the content of Chl a in this experiment was also considerably affected by the algal
abundance in the reservoir which supplied water to the fertilization ponds.
With regard to abiotic factors, linear regression indicated a significant positive correlation
between Ch a and temperature (R²=0.52-0.62, P=0.00) or pH (R²=0.76-0.84, P=0.00) while
a negative relationship (R²= 0.61-0.74, P=0.00) with turbidity was identified for the N:P=5
and N:P=10 treatments, respectively (Figure 3b-d). Ch a concentration increased with water
temperature and its reduction was associated with the reduction in temperature by the end
of the experiment. Also, high pH value and low turbidity resulted in high Ch a
concentration. Additionally, multiple-regression analysis indicated that Ch a had potential
collinearity with nitrogen concentration (TN and N-NH4+) while phosphorus concentration
(TP and P-PO43-) was not included in the models. The two models for both NP5 and NP10
treatments were highly similar.
Khoi et al. (2006a) suggested that maintaining dissolved reactive phosphorus
concentrations above 0.06 mg P L-1 in the water column can enhance the growth of algae.
Our data showed that P-PO43-levels were in the range of 0.2-0.6 P-PO43- mg L-1, and the
ratios of TN:TP were lower than the Redfield ratio. Nitrogen may have been a limiting
factor for algal production in this study. Previous researches also proved that in most
coastal marine ecosystems, primary production tends to be nitrogen-limited (Howarth,
1988). Tomasky et al. (1999) conducted a nutrient enrichment in Waquoit Bay, USA, and
found that nitrogen limitation generally increased with increasing salinity, apart from short
periods of phosphorus limitation. In our experiment, salinity in the fertilization ponds
varied between 33 and 48 g L-1, which could contribute to nitrogen limitation for algal
growth. However, Smith (2006) recommended that changes in nutrients, chlorophyll, and
particulate carbon and nitrogen levels over time indicated that most of the brackish water
sites showed phosphorus limitation. Guildford and Hecky (2000) revealed a significant
correlation between Chl a and TP for marine sites but parallel evidence that marine
phytoplankton biomass was controlled by TN was inconclusive. In contrast, Hoyer et al.
(2002) found significant correlations between Ch a and both TN and TP in Florida’s coastal
waters. Similar findings were observed by Smith et al. (2006), who showed that average
concentrations of Ch a in estuarine and coastal marine systems were strongly dependent on
94
Chapter 3
the mean concentrations of TN and TP in the water column. As biologically available N
and P become non-limiting, other factors such as light and CO2 may become the
determinant for algal development (Wetzel, 2001; Smith et al., 2006).
Generally, nutrient availability affects photosynthesis, which in turn increases the
phytoplankton availability in the water column (Souza et al., 2003; Rahman et al., 2008b).
However, this approach does not always work, especially in the presence of filter feeders,
which control algal growth, and algal biomass no longer relates to nutrient concentrations
(Baert et al., 1996; Wetzel, 2001; Smith, 2006). Also Smith et al. (2006) reported that by
management of nutrient loading they understood well the ways in which the local
biological expression of nutrient enrichment can be modified by site-specific factors,
including food web structure.
Lin and Yi (2002) suggested that to maintain a level of phytoplankton production at 80300 mg chlorophyll a m-3, and Secchi disc depth of 20-40 cm, the total P and N
concentrations in the water column should be kept at a range of 0.2-0.5 mg P L-1 and 1-3
mg N L-1, with a N:P ration of 5-10:1, respectively.
4.1.2. Algae composition
During the sampling period, the total number of algal species was higher in the NP10
treatment (62) than in the NP5 sample (52). Furthermore, Bacillariophyta (diatoms)
dominated and occupied a large percentage in the algal community (60-63%), followed by
Cyanophyta (15-16%), Dinophyta (13-15%) and Chlorophyta (8-10%). Our data are in
accordance with previous studies in Vietnam; i.e. Phu et al. (2006) who investigated
phytoplankton composition in 2 crops of intensive shrimp-with-tilapia ponds in Soc Trang
province (salinity range 10-18 g L-1) and found that the total number of algal species was
97 and 127 for crop 1 and 2, respectively, with the following composition: Bacillariophyta:
42.3 and 62.4 %, Chlorophyta: 20.6 and 5.6%, Euglenophyta: 20,6 and 41%, Cyanophyta:
15.5 and 15.3% and Pyrrophyta: 9.3 and 12.8% for crop 1 and 2, respectively. Similar
results were obtained by Oanh et al. (2008) who studied the relationship between algae and
nutrient factors (nitrogen and phosphorus) in intensive shrimp ponds, and who reported that
diatoms were the dominant group with about 65% of 131 species occurring. This result is
also supported by Ut et al. (2008), who surveyed the water quality in the Artemia culture
area of the Vinh Chau district, Soc Trang province, and who found a domination of diatoms
95
Chapter 3
with 61% of the 125 algal species recorded in total. Blue-green algae, however, were
dominating quantitatively. There are 537 microalgal species in the natural marine waters in
Vietnam (Nguyen and Ha, 2006). The above authors all confirmed that the species
composition of the algal community in this region is typical for the brackish water
ecosystem in the Mekong delta, Vietnam.
The total number of algal species observed in this experiment was much lower than in the
previous studies cited above, which could be due to various reasons i.e. high salinity (33-48
g L-1), restricted ecosystem (fertilization pond) and short duration of the experiment (12
weeks) whereas other investigations generally refer to an observation period of at least one
year or more and in different habitats. Wetzel (2001) found that in saline lakes,
phytoplanktonic species diversity is inversely related to salinity, and that green algae and a
few species of cyanobacteria are dominant; some diatoms are ubiquitous even in very saline
lakes (>100 g salinity L-1). Additionally, earlier researchers confirmed that many factors are
ultimately responsible for structuring phytoplankton communities, including predation,
environmental fluctuations, and competitive interactions. Hence, distribution of natural
algal communities shows seasonal fluctuations (Wetzel, 2001; Descamps-Julien and
Gonzalez, 2005; Smith, 2006; Smith et al., 2006; Winder et al., 2008).
The linear regression analysis indicated that an increased TN level resulted in low diversity
of algal species (R=0.63 and P=0.02) and the concentration of TP did not exhibit any
correlation with algal diversity for both fertilizer treatments in this study. In contrast, the
studies from Phu et al. (2006) and Oanh et al. (2008) demonstrated that both TN and TP
concentrations were inversely correlated with the number of algal species in intensive
shrimp ponds and in coastal brackish water in Vietnam (Ut et al., 2008). Furthermore, the
higher the TN:TP ratio, the higher the number of algal species observed (R=0.60, P=0.03).
In our study, the relationship between TN and algal diversity was detected in the N:P5
treatment while it did not match in the NP10 treatment; this could be explained by the
interference of other factors such as water exchange, fertilizer, local algae species
composition, presence of filter feeders, etc. We observed that the frequency of dominant
and sub-dominant species changed with time. Following two weeks of fertilizing,
Tetraselmis cordiformis (Chlorophyta) was sub-dominant (++) in the NP5 samples and
Tetraselmis sp. (Chlorophyta) was most abundant (+++), followed by Phormidium sp. (++)
(Cyanophyta), in the NP10 treatment. From this period onwards, different diatom species
became more dominant than other algal communities in both treatments. Oanh et al. (2008)
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Chapter 3
found that the concentrations of TN and TP were positively correlated with algal abundance
whereas they showed negative relationship with the number of algal species; when the N:P
ratio was in the range of 10-18, diatoms dominated whereas a N:P ratio between 6 and 10
resulted in the highest abundance of blue-green algae, as is observed in intensive shrimp
ponds. Our results are not in line with the above observations, as the TN:TP ratio in our
NP5 treatment was usually lower than 10 but diatoms predominated during the
experimental period (Figure 2f).
In our experiment, within the dominating diatoms, Nitzschia sp. was present in most
samples while Chaetoceros sp., Cymbella, Gyrosigma and Navicula appeared at certain
times. A similar observation was reported by Brands (1992), who found that the most
common algal species encountered in the fertilization pond in Vinh Chau salt fields,
Vietnam belong to the genus Nitzschia. According to Tuyen (2003), most diatom species
are euryhaline.
Generally, when the fertilization pond was managed by applying N:P ratios of =5 and 10 to
stimulate algal bloom, the results showed that the concentration of chlorophyll a was
similar and that the algal composition was not distinctly different between the two
treatments. Therefore, further evaluation by using “green water” from these ponds as a
natural food source for Artemia could provide some interesting additional information.
4.2. Artemia ponds
4.2.1. Effect of green water and supplementary feeds on survival, growth and total yield of
Artemia biomass
The survival of Artemia 11 days after inoculation was not influenced by the treatment
(P > 0.05). The values for survival in the present experiment were higher (73-74%) than in
our previous study (52-54%; see Section II of Chapter 3). This result is comparable to data
obtained in laboratory experiments. Teresita and Letica (2005) achieved 79% survival after
15 days of culture, feeding rice bran and Tetraselmis suecica. Similar results were obtained
by Toi et al. (2006) who obtained 75-85% survival after 10 days of culture, using
Chaetoceros sp. and Nitzschia sp. as feed.
Our results showed that a combination of different green water types and supplementary
feeds for stocking Artemia in the salt ponds had a significant effect on growth performance
97
Chapter 3
(length and weight) after a culture period of 14 days. The range of mean length (9.2-9.5
mm) and individual weight (9.1-9.5 mg) in the present experiment were comparable with
the results obtained in our previous research work (see Section II of Chapter 3).
The total yield of Artemia biomass reflects the different green water types and food
supplements received during twelve weeks of culture. However, statistical analysis on
single-factor effect or on the interaction of factors made clear that the differences in
biomass production among the treatments were not significant (P>0.05) for both tests,
although the mean value in the GW5 treatment was higher than in the GW10 treatment.
It was observed that the mean yield in the GW+PM treatment was 2.1 ton ww ha-1 and the
average value of all treatments was 2.2 ton ww ha-1. These results are better or comparable
to our earlier study (see Section II of Chapter 3) where a mean yield of 1.8 and 2.3 ton ww
ha-1 was achieved for the supplementation with pig manure (GW+PM) and for the cosupplement of PM with rice bran or soybean meal, respectively.
4.2.2. Effect of green water and supplementary feeds on reproduction of Artemia
population
The mode of reproduction in the Artemia population in the first brood and over the whole
culture period was not affected by the feeding treatments (Table 5). Nevertheless, this
characteristic showed a variation in time, with the lowest percentage of ovoviviparity
occurring in week 3 (Figure 9); from week 4 onwards, this parameter displayed a more or
less gradual increase until week 10 and then declined by the end of the culture period.
Previous studies reported that switching of the reproductive mode of Artemia in the natural
environment can be expected to vary depending on the environmental conditions such as
temperature, salinity, and photoperiod (Lenz, 1987; Berthélémy-Okazaki and Hedgecock,
1987). Temperature seems to play an important role in the present experiment, which is
confirmed by observations of various authors (Baert et al., 1997; Anh and Hoa, 2004). A
quite low brood size in the first brood, and highest values betweens weeks 3 and 5 were
observed as a general pattern in all treatments; the brood size, then gradually declined until
the end of the culture period (Figure 10). This result is in accordance with previous studies
focusing on the production of Artemia cyst (Brands et al., 1995; Baert, et al., 1997) and of
Artemia biomass (Anh and Hoa, 2004).
98
Chapter 3
Concerning to the fecundity of Artemia female, the mean brood size at first spawning and
the mean brood of the whole culture period in the GW5 treatment were significantly larger
than in the GW10 (P<0.05). These results could be associated to the difference in algal
composition present in the green water as a natural food for Artemia.
With reference to data obtained from laboratory experiments, Thinh et al. (1999) using 13
microalgae isolated from the Australian sea, found that Chaetoceros sp. is the best food for
Artemia (98% survival after 7 days of culture). Similar results were obtained by Naegel
(1999) and Lora-Vilchis et al. (2004). Furthermore, Toi et al. (2006) reported that
Chaetoceros sp. and Nitzschia sp. isolated from the Vietnamese coastal area are a suitable
food for Artemia (70-85% and 53-75% survival, 6-7 cm and 4-5 cm in length after 10 days
of culture) while Oscillatoria sp. proved an unsuitable food (3-11% survival and 1.5-1.7 cm
in length after 5 days, followed by complete mortality). Additionally, Wurtsbaugh and
Gliwicz (2001) stated that growth, survival and reproduction of Artemia franciscana in the
Great Salt Lake were markedly influenced by the nutritional quality of the locally occurring
microalgae. In accordance with these literature data, in our study the GW5 treatment
contained more suitable algal species (Chaetoceros sp. and Nitzschia sp.) as compared to
GW10, which is the most probable explanation of the higher fecundity and total Artemia
biomass yield of animals fed GW5 than those fed GW10 although there was no statistical
difference in biomass productivity.
According to Moore (1986) and Boyd et al. (2002) organic fertilizers act mainly through
the heterotrophic food chain by supplying organic matter and detritus to the pond
ecosystem; the manure serves principally as a substrate for the growth of bacteria and
protozoa, which in turn serve as a protein-rich food for other pond animals, including the
cultured fish or shrimp. Moreover, organic fertilizer contains the organic matter being
digested by bacteria that Artemia can use as a food (Intriago and Jones, 1993). Other
studies demonstrated that Artemia receiving supplemental feeds grow on a mixed algal,
bacterial, protozoan and organic diet (Wurtsbaugh and Gliwicz, 2001). Similar results were
obtained by Burford et al. (2003, 2004), who noted that up to 40% of the bacteria were
associated with flocculated particles which are composed of a mixture of bacteria,
senescent phytoplankton, protozoa and inorganic particles. These floccules are a potential
food source and contribute substantially to the nutrition of shrimp Litopenaeus vannamei.
Supplemental feeds applied to the culture pond provide extra direct food, and its dissolved
nutrients may stimulate algal growth (Boyd et al., 2002; Adhikari, 2003; Rahman et al.,
99
Chapter 3
2008b). Also, net uptake of inorganic nitrogen by bacteria only occurs when the C/N ratio
of the organic matter is higher than 10 (Lancelot and Billen, 1985). Hence, increasing the
C/N ratio could increase bacterial production in the systems (Burford et al., 2003). In our
study, the C/N ratios of the two supplemental feeds were 16.2 and 10.3 for rice bran and pig
manure, respectively. These values may possibly be considered a suitable range for
microbial development in the culture ponds that may provide more food for Artemia. Thus,
when rice bran and pig manure are decomposed, the Artemia population can benefit from
their breakdown products in the culture pond. Consequently, the effect of different green
water types and supplementary feeds on the total yield of Artemia biomass could have been
masked in the present experiment. As our experiment has been carried out in salt ponds,
other factors may have an effect on the success of the Artemia culture such as pond soil
profile (Khoi et al., 2006b; 2008) or weather conditions.
4.2.3. Food conversion ratio
The conversion ratio of rice bran, pig manure and chlorophyll a to Artemia biomass in the
GW5 treatments was somewhat lower than in the GW10 groups. However, no statistical
difference between the two treatments was found (P>0.05). As mentioned earlier, the effect
of food supplementation on the Artemia population is not only realized through its effect on
the primary production as the Artemia population is also taking direct benefit from the
supplementation products that provide extra direct food for the filter-feeding Artemia.
Furthermore, food supplements decomposing into organic matter are a substrate for
bacteria, and both are also a suitable food source for Artemia. Hence, the food conversion
ratio (FCR) in Artemia cultured under field conditions can be lower than in Artemia
cultured under laboratory conditions which rely on a single input. Vanhaecke and
Sorgeloos (1989) obtained FCR values between 2.40 and 5.82 for different geographical
strains of Artemia reared at temperatures between 20 and 32.5°C using Dunaliella
tertiolecta as food for 9 days. Naegel (1999) achieved a FCR of 1.64 for the Nestum-fed
animals and of 1.17 for the animals fed with enriched Nestum (powdered baby food).
However, Zmora and Shpigel (2006) when using an outdoor closed system for intensive
Artemia biomass production with microalgae in the initial days of culture, followed by a
mixture of torula yeast and soy protein, obtained FCR values ranging from 0.17 to 0.25
which is much lower than in the present experiments.
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Chapter 3
4.2.4. Economic aspects
Brands et al. (1995) reported that manual labour and fertilizers used in the culture of
Artemia cysts account for close to 50% of total production costs. Similar findings were
found by Anh et al. (1997b) who made a survey of the situation of Artemia cyst production
in Soc Trang and Bac Lieu provinces, Vietnam. Our results are in line with these previous
research works, where labour and feed together were about 50-55% of total expenses.
Moreover, total expenditure in the present experiment was close to the cost for cyst
production from the farmers who spent between 1,000 and 1,200 US$ ha-1 in 2007
(personal communication from some farmers near the experimental site in Bac Lieu).
According to survey data of Hoa et al. (2007) on Artemia cyst production in Vinh Chau,
most farmers obtained an average benefit-cost ratio (BCR) of 0.63 and 0.36 for the years
2000 and 2003, respectively, and this ratio changed as a function of the cyst yield and
market price. In addition, in the years 2007 and 2008 many farmers realized a net profit of
1,000-2,000 US$ ha-1 and a BCR of 0.8-1.5 (unpublished data), which is comparable with
the estimations for our experiment.
The results showed that the income, net profit and benefit-cost ratio in the GW5 treatment
were higher than those in the GW10 treatment but statistical difference was not detected.
This could be due to the large variation in the total yield within a treatment. Because the
economic aspect is related to biomass productivity, using the ratio of N:P=5 to produce
natural food for Artemia thus seems to be quite more cost-effective compared to the ratio of
N:P=10.
Conclusions
This study showed that when using ratios of N:P=5 and 10 in the fertilization pond, the
differences in chlorophyll a concentration and algal composition are small. Bacillariophyta
(diatoms) were the dominant group over the sampling period. This is beneficial to the
growth of Artemia, especially the presence of diatom species of relatively small size such as
Nitzschia longissima, N. longissima var reversa and N. acicularis. The ratios of TN: TP
were lower than the Redfield value, indicating that nitrogen is a limiting nutrient for algal
growth in this experiment.
Moreover, using the GW5 treatments (at N:P=5) combined with rice bran or pig manure to
feed Artemia growth, fecundity and total yields were enhanced compared with the GW10
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Chapter 3
treatments (N:P=10). However, statistical difference was not found for biomass yield.
Neither were survival and ovoviviparous reproduction affected by the treatments.
Additionally, when either rice bran or pig manure was used as supplementation product,
similar results were obtained.
As for the economic aspects, the GW5 gave a slightly higher income, net profit and
economic return than the GW10 treatments. Thus, it could be suggested that application of
the ratio of N:P=5 in the fertilization ponds to produce algae as natural food source for
Artemia is preferable to a ratio of N:P=10.
102
CHAPTER
Drying Artemia biomass
Chapter 4
Chapter 4
Section I
Total lipid and fatty acid contents of Artemia biomass
dried using different drying techniques
Chapter 4
Chapter 4
Abstract
Frozen Artemia biomass were dehydrated by outdoor sun drying and three indoor drying
techniques which consisted of convective hot air drying (HA), intermittent microwave
combined with convective hot air drying (MWHA) and oven drying at temperatures of 50,
60 and 70°C. The aim of this study is to evaluate the effect of different drying techniques at
different temperatures on the contents of total lipids and fatty acids of Artemia biomass.
The results showed that among three indoor drying techniques, the shortest drying times
(57-74 min) were observed in MWHA followed by HA (380-480 min) and oven drying
(480-1320 min), respectively, while sun drying showed the longest dehydration times (9002160 min) compared to other drying methods. In addition, drying time decreased with
increasing temperature. Generally, for the three indoor drying methods the contents of total
lipids and fatty acids of dried Artemia were not significantly different from the control in
most cases. On the contrary, sun drying resulted in a high loss of these substances
compared to the original material. Moreover, at the same drying temperature, the longer
drying time caused a higher loss of nutrients in the dried products as shown by the values of
the total lipid and fatty aicds in the HA and oven-dried samples, which was slightly lower
than in MWHA-dried Artemia. However, between the three indoor drying methods
significant differences were not observed (P>0.05). In general, the intermittent MWHA
drying is a promising technique, which could produce high quality dried products in short
drying times. However, it may not be suitable for large-scale application because of high
capital investment and operating costs.
Keywords: Artemia biomass, total lipids, fatty acid, drying methods
1. Introduction
Lipids and fatty acids play an important role in the nutrition of crustaceans and fish. They
mainly function as a source of energy and for the maintenance of the functional integrity of
biomembranes (D’Abramo, 1998; Tocher, 2003). Furthermore, the essential fatty acid
content of artificial diets has been found to have a strong impact on survival, growth,
reproduction and stress tolerance of shrimp and fish species (Sargent et al., 1999; Lee,
2001; Watanabe and Vassallo-Agius, 2003; Takeuchi and Murakami, 2007).
Fresh Artemia biomass has been considered a good source of proteins, lipids and highly
unsaturated fatty acids (Sorgeloos, 1980a; Léger et al., 1986; Lim et al., 2001). However,
Artemia biomass contains a high water content (approximately 90% water) and is rich in
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Chapter 4
proteolytic enzymes; the soft Artemia body is thus subject to decomposition soon after
being collected and hence appropriate preservation methods are needed to keep a good
quality of biomass (Léger et al., 1986; Baert et al., 1996). According to several authors
(Brennand, 1994; Maskan, 2001; George et al., 2004), drying is an appropriate method of
product preservation. If the quality of dried Artemia biomass can be maintained, it may
have certain advantages over fresh or frozen products due to the low cost of transportation,
reduced space needed for storage and longer shelf-life. Previous studies have demonstrated
that dried Artemia biomass can be used as an ingredient in post-larval shrimp feeds (Abelin
et al., 1989; Naegel and Rodríguez-Astudillo, 2004). Moreover, freeze-dried meal of adult
Artemia has also been used as a partial or sole ingredient of shrimp broodstock diets
increasing diet ingestion and stimulating ovarian maturation in commercial scale trials
(Wouters et al., 2001; 2002).
Several technologies have been described for drying plant and animal products, such as
freeze drying, vacuum drying, microwave drying, hot air drying, conventional sun drying
etc., or combinations of some of these methods; they are employed depending upon the
desired quality and flavour of the dried products, the initial moisture content and the
chemical composition of products (Sumnu, 2001; George et al., 2004; Chua and Chou,
2005). Drying time and temperature can be considered the most important operating
parameters affecting dried product quality, which is usually evaluated on the basis of
nutrient retention and sensory characteristics (Maskan, 2001; George et al., 2004; Gowen et
al., 2006; Ayanwale et al., 2007). Hence, different drying methods would have a direct
impact on nutrient availability. Lipid and fatty acid profiles of Artemia as feed are
important for the survival, growth and reproduction of shrimp and fish species (Léger et al.,
1986; Naessens et al., 1997; Sorgeloos et al., 2001; Wouters et al., 2001; 2002; Martins et
al., 2006). However, the drying process may cause the loss of these substances through
oxidative deterioration. Therefore, the main objective of our study was to compare the
contents of total lipids and fatty acids of Artemia biomass, dried using different drying
methods and at different temperatures, aiming to assess the effect of the drying method on
the dietary lipids in aquafeeds.
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2. Material and Methods
2.1. Drying experiment
The drying techniques and drying equipment were provided by the Department of Farm
Machinery and Post-harvest Technology, College of Technology, Can Tho University,
Vietnam. Four drying methods were tested: outdoor sun drying and three indoor drying
methods: convective hot air drying, combination of intermittent microwave and convective
hot air drying, and oven drying.
For the convective hot air drying and combined microwave-convective hot air drying, the
drying system was specially designed, and consisted of a hot air-microwave oven, equipped
with an adjustable temperature and velocity convective mode, and an adjustable power
continuous or intermittent output microwave mode. In this experiment, the equipment was
operated using the intermittent mode. For all convective drying treatments, the air velocity
was set at 1.5 m s-1. The settings of the respective drying techniques were as follows:
-
Convective hot air drying: convective air temperature was set at 50, 60 and 70°C.
-
Intermittent microwave combined with convective hot air drying: convective air
temperature was set at 50, 60 and 70°C, microwave power was set at the medium
high level and the intermittent time was 2 min with a cycle time of 1 min ‘on’ and 2
min ‘off’ to prevent the samples from becoming charred or burnt.
-
Oven drying: temperature was set at 50, 60 and 70°C.
-
Outdoor sun drying: samples were exposed directly to sunlight from 8:00 to 17:00h.
At night, these samples were kept in airtight nylon bag; sun drying was continued
the next day until the desired moisture content was obtained.
Each drying trial was repeated three times and the final moisture content for all drying
techniques was ≤13%. For abbreviations of drying techniques: see Table 1.
2.2. Sample preparation
Fresh Artemia biomass was obtained from the experimental ponds in Bac Lieu province,
Vietnam. The freshly-harvested biomass was placed in a nylon bag in a layer of about 2,5
cm and transported in a plastic box with ice to the laboratory of Can Tho University and
stored at -15°C until use. All Artemia biomass utilized in this experiment was from the
same batch.
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Chapter 4
Prior to each drying experiment Artemia biomass was taken out of storage, thawed and
washed with tap water to eliminate impurities. Excess water in Artemia samples was
removed with tissue paper and Artemia samples of 100 g were spread on a plastic net in a
layer with a thickness of about 4 mm.
For sun drying, 0.3% of antioxidant (butylated hydroxytoluene, BHT) was added to the
sample before drying.
2.3. Sample analysis
Moisture content, total lipid and fatty acid contents of the Artemia samples were
determined before and after drying.
Dried Artemia samples were ground into fine particles and stored at -80°C until further
analysis, and frozen Artemia biomass was used as control. Moisture was determined by
drying in an oven at 110°C until constant weight. Total lipids were extracted according to
the method described by Folch et al. (1957) as modified by Ways and Hanahan (1964).
Fatty acid composition was analytically verified by flame ionization detection (FID) after
injecting the sample into a Chrompack CP9001 gas chromatograph according to the
procedure described by Coutteau and Sorgeloos (1995). Integration and calculations were
done with the software program Maestro (Chrompack).
2.4. Statistical analysis
The contents of total lipid and fatty acid composition of Artemia samples subjected to
different drying methods and temperatures were compared with the frozen Artemia by oneway ANOVA. The Tukey HSD post-hoc analysis was used to detect differences between
means. Significant difference was considered at P<0.05 (SPSS for Windows, Version 13.0).
The percentage values were normalized through a square root arcsine transformation before
statistical treatment.
3. Results
3.1. Drying time
The moisture content and drying time of Artemia biomass at temperatures of 50, 60 and
70°C by different drying methods are shown in Table 1. All drying treatments resulted in
similar moisture content. Regardless of other drying parameters, drying temperatures at 50,
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Chapter 4
60 and 70°C, employing the intermittent microwave combined with convective hot air
drying (MWHA) significantly shortened drying time (57-74 min) compared with
convective hot air drying (HA) alone (380-460 min), oven drying (480-1320 min) and open
sun drying (1380 min). Moreover, for the same drying temperature, drying time in HA
drying was faster than in oven drying. Increased temperature resulted in a shorter drying
time. Generally, drying time was fastest in the MWHA drying which was 10, 14 and 21
times faster than HA, oven and sun drying, respectively.
Table 1. Comparative evaluation of the effect of different drying techniques on drying time
Initial moisture
content (%)
Final moisture
content (%)
Drying time
(min)
Convective hot air (HA50)
86.7 ± 0.5
12.2 ± 1.2
960
Microwave+HA (MWHA50)
88.4 ± 0.7
12.6 ± 0.8
74
Oven (Oven50)
87.6 ± 0.7
12.3 ±1.4
1320
Convective hot air (HA60)
86.7 ± 1.0
12.6 ± 1.1
560
Microwave+HA (MWHA60)
87.5 ± 0.8
12.4 ± 0.6
66
Oven (oven60)
86.7 ± 0.6
12.6 ± 0.9
870
Convective hot air (HA70)
87.5 ± 0.6
12.4 ± 0.7
380
Microwave+HA (MWHA70)
88.1 ± 0.9
12.2 ± 0.5
57
Oven (Oven70)
86.9 ± 1.0
12.5 ± 0.6
480
Run 1 (30-39°C)
88.1
11.9
900
Run 2 (26-34°C)
87.6
12.8
1980
Run 3 (28-37°C)
87.8
11.8
2160
87.8 ± 0.3
12.3 ± 0.6
1380
Drying technique
Drying at 50°C
Drying at 60°C
Drying at 70°C
Open sun drying*
Mean value of three runs
* Average air temperatures were 35.5, 30.0 and 31.6°C for run 1, 2 and 3, respectively; the
values in the brackets indicated the min and max values of air temperature during the
drying process.
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Chapter 4
3.2. Total lipids and fatty acid composition of dried Artemia
Data on total lipid and fatty acid composition of the frozen (control) and dried Artemia
biomass are presented in Table 2. For the three indoor drying methods (HA, MWHA and
oven drying) total lipids of dried Artemia were similar for the different drying temperatures
50, 60 and 70°C ranged from 10.29 to 10.93%, but the HA70, Oven50 and Oven70 samples
showed a statistically lower value than the frozen sample (11.35%).
In addition, total lipid content of sun-dried biomass (9.82%) was significantly lower than
the control and HA50, HA60, MWHA and Oven60 samples (P<0.05). There was no
significant effect of three indoor drying methods on the contents of eicosapentaenoic acid
(EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n6), except for DHA values in Oven60 and Oven70 samples, which were significantly lower
than in the control. In sun-dried biomass, the EPA content was significantly reduced as
compared with the control, HA50 and MWHA50. Additionally, no significant difference in
DHA was observed between the sun-dried sample and the Oven60 and Oven70 samples
(P>0.05) but the DHA value of the sun-dried sample significantly differed from the other
drying treatments (P<0.05) and the control. Although the values of ARA and total saturated
fatty acids (SFA) in sun-dried samples were lower than the control and other drying
methods, statistical differences were not detected.
For the three indoor drying methods, total mono-unsaturated fatty acid (MUFA) levels were
similar in all drying temperatures, with levels for HA70, Oven50 and Oven70 being
significantly lower than in frozen biomass. The sun-dried sample showed a significant
reduction in total MUFA content compared to the control and other drying methods
(P<0.05). Furthermore, total polyunsaturated fatty acid (PUFA) levels and total n-3 PUFA
tended to decrease slightly with increasing temperatures, with the HA70 and Oven70
samples similar to the sun-dried product and significantly lower than the control. Total n-6
PUFA contents showed the same trend as total n-3 PUFA in the three indoor drying
methods. However, significant differences were only found between the sun-dried sample
and the control (P<0.05). The ratios of n3/n-6 were nearly equal in all samples, ranging
from 1.8 to 2.2. In general, there were no significant differences in total lipid and fatty acid
levels of dried Artemia between the drying temperatures of 50, 60 and 70°C although
slightly lower values were observed at higher drying temperatures, and contents of total
lipids and fatty acids of the indoor-dried Artemia samples differed less from the control
than that of the sun-dried sample.
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Chapter 4
Table 2. Total lipids (% of DW) and fatty acid composition (mg g-1 DW) of frozen (control) and Artemia biomass dried using different drying techniques.
Drying techniques
Convective hot air drying
Microwave + HA drying
Oven drying
Sun
drying
Control*
50°C
60°C
70°C
50°C
60°C
70°C
50°C
60°C
70°C
11.35c
10.88bc
10.70bc
10.26ab
10.93bc
10.67bc
10.53bc
10.44ab
10.61bc
10.29ab
9.82a
20:5n-3
11.81b
11.27b
10.62ab
10.62ab
11.44b
10.88 ab
10.69 ab
10.84ab
10.63ab
10.53ab
9.30a
22:6n-3
0.51c
0.42bc
0.36 ab
0.31ab
0.40bc
0.39 bc
0.43 bc
0.39bc
0.36ab
0.29ab
0.19a
20:4n-6
3.59a
3.31a
3.37a
3.15a
3.41a
3.26a
3.30 a
3.32a
3.23a
3.22a
2.59a
ΣSFA
29.17a
29.27a
29.69a
28.49a
30.36a
30.02a
28.79 a
27.22 a
28.96a
27.47a
26.96a
ΣMUFA
45.18c
40.77bc
41.25bc
40.56b
42.54bc
42.88bc
41.69 bc
40.51b
40.73bc
39.94b
35.02a
ΣPUFA
24.10c
22.11bc
21.75 bc
20.54ab
22.33bc
21.62bc
21.05 bc
20.76 bc
20.98bc
19.91ab
17.22a
1
Σn-3 UFA
16.17c
14.82bc
13.97bc
13.54ab
15.10bc
14.52bc
14.27bc
14.24bc
13.96bc
13.35ab
11.84a
2
Σn-6 UFA
7.93b
7.29ab
7.78ab
7.01ab
7.23ab
7.11ab
6.78ab
6.52ab
7.02ab
6.56ab
5.39a
Ratio n-3/n-6
2.0a
2.0a
1.8a
1.9a
2.1a
2.0a
2.1a
2.2a
2.0a
2.0a
2.2a
Total lipids
Fatty acids
1
Σ (n-6) ≥18:2n-6, 2Σ (n-3) ≥18:3n-3, EPA, eicosapentaenoic acid (20:5 n-3); DHA, docosahexaenoic acid, (22:6 n-3); ARA, arachidonic acid
(20:4n-6); SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
*Control indicates the frozen Artemia sample was also analyzed for comparing with the dried Artemia biomass.
Data represent averages of triple and duplicate analyses for total lipids and fatty acids, respectively. Values in the same row that do not share the
same letter are statistically significant different (P<0.05).
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Chapter 4
4. Discussion
4.1. Effect of different drying techniques and temperatures on drying time
Our study indicated that among three indoor drying methods, the combined microwave and
conductive hot air drying (MWHA) resulted in faster drying as compared to the convective
hot air (HA) and oven drying. These observations are in agreement with other researchers
who found that combining microwave energy with convective drying can lead to
considerable reductions in drying times for mushrooms (Funebo and Ohlsson, 1998),
bananas (Maskan, 2000), garlic cloves (Sharma and Prasad, 2001), apples (Bilbao-Sainz et
al., 2005), cooked chickpeas and soybeans (Gowen et al., 2007) compared to the convective
drying alone. According to Nindo et al. (2003), drying asparagus by the combination of
microwave and spouted bed drying resulted in the fastest drying rate compared to freeze
drying, tray drying and spouted bed drying. Similar results were observed by Chua and
Chou (2005), i.e. that intermittent microwave drying can significantly reduce drying time in
comparison with convective or intermittent infrared drying, without the samples being
charred; these authors found that using a suitable combination of convective-microwave
drying, drying time can be shortened by as much as 42 and 31% for potato and carrot
samples, respectively.
Previous investigations have reported that high-moisture fruits, vegetables and other
products are very responsive to microwave application, and absorb microwave energy
quickly and efficiently (Feng and Tang, 1998; Sumnu, 2001; Feng et al., 2002), whereas
hot air drying of food has a low energy efficiency and needs a lengthy drying time. Because
of the low thermal conductivity of food materials, heat transfer to the inner sections of food
during conventional heating is limited (Feng and Tang, 1998; Maskan, 2001; McMinn et
al., 2005). In microwave drying, the microwaves can easily penetrate into the inert dry
layers to be absorbed directly by the moisture; the quick energy absorption causes rapid
evaporation of water resulting in shorter drying time. Therefore, it is a rapid, more uniform
and more energy-efficient technique as compared with conventional hot air drying
(Maskan, 2000; Wang et al., 2004; Altan and Maskan, 2005; Wang and Xi, 2005; Wang et
al., 2007). Schiffmann (1995) explains the efficiency of combined microwave and
convective hot air drying by the fact that convective hot-air is relatively efficient in
removing free water at or near the surface, whereas the unique action of microwave
provides an efficient way of removing internal free and less free water. An appropriate
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Chapter 4
microwave power level, used in sequence with hot-air drying prevents the samples from
charring.
It has been observed that increasing drying temperatures from 50 to 70°C resulted in a
shorter drying time of Artemia biomass for all drying methods used. According to Krokida
et al. (2003), the temperature of drying is the most important factor determining the drying
rate for all vegetables examined in their study. Faster drying is due to high evaporation that
can drive the moisture migrating to the surface in minutes. Our results were in accordance
with similar studies conducted on taro flour (Njintang and Mbofung, 2003); figs (Babalis
and Belessiotis, 2004); apricot (Karabulut et al., 2007) and water chestnut (Singh et al.,
2008). On the other hand, in our study oven drying was found to be slower than convective
HA drying, probably because the oven lacked a built-in fan for air circulation, resulting in a
lower energy-efficiency for oven drying as compared to convective HA drying (Brennand,
1994). A similar result was reported by Belghit et al. (2000) who investigated the
characteristics of the drying curve of verbena and found that drying air velocity seemed to
have a more important effect than temperature. Moreover, in our study the drying time was
longer for sun drying due to the fluctuating temperature during the drying period, which is
strongly affected by the weather conditions. Therefore, in case of sun drying, the drying
period may be interrupted during rainy or cloudy days (low temperature and high
humidity), causing the most extended drying time compared to other drying methods
(Togrul and Pehlivan, 2004; Özcan et al., 2005).
4.2. Effect of different drying techniques and temperatures on the contents of total lipids
and fatty acids in dried Artemia biomass
Our results showed that the total lipid content of dried Artemia in all drying methods was
lower than in frozen Artemia (control). These results confirm the findings of Liou and
Simpson (1989), who reported that the lipid levels of Artemia nauplii dried by vacuum and
hot air drying were lower than in newly-hatched Artemia. In our study, however, significant
losses of total lipids were observed in HA70, Oven50 and Oven70 and sun-dried samples
when compared to the control (P<0.05). This indicates that loss of lipids after drying may
be not only affected by drying temperatures but also by drying time. For example, oven
drying at 50°C and sun drying at temperatures between 26-39°C took much longer (1320
min and 1380 min, respectively) than MWHA and HA drying (74 and 960 min,
respectively). According to several authors (Maskan, 2001; George et al., 2004; Gowen et
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Chapter 4
al., 2006; Ayanwale et al., 2007), drying time and temperature can be considered the most
important operating parameters affecting the quality of the dried product. On the other
hand, some food products require several hours and others may take more than a day; and
prolonging drying time (by using lower temperatures) or interrupting drying time may
result in spoilage of dried products (Hughes and Willenberg, 1994) whereas high
temperature during drying leads to the partial destruction of the nutrients (Sampaio et al.,
2006). This was also observed in our study, where Artemia samples dried at higher
temperature resulted in slightly lower content of total lipids, but not significant difference
(P>0.05). Possibly the 10°C increment of drying temperature might be insufficient to cause
statistical differences. Similar results were obtained by Paleari et al. (2003), who reported
that a decrease of fat content during processing has been shown in cured and dried products
from different animal species. For sun drying, the prolonged direct incidence of sunlight
may accelerate lipid oxidation, as illustrated by the lipid content in our sun-dried sample
being significantly lower than the control and MWHA samples (P<0.01).
The fatty acid levels in the Artemia biomass, dried indoors with various techniques at
temperatures of 50, 60 and 70°C, all were very similar. When comparing with the frozen
Artemia significant differences were only found in the contents of some of the fatty acids,
with the actual values showing only minor differences (Table 2). In general, the saturated
fatty acids (SFA) were almost equal or slightly higher, whereas the unsaturated fatty acids
(UFA) decreased when compared to the frozen sample. This can be observed in nearly all
individual FA as well as in the different sums and ratios. These results are confirmed by
Sampels et al. (2004), who reported that dried reindeer meat showed higher values of total
SFA whereas total UFA were lower compared with fresh meat. According to Cosgrove et
al. (1987) and Mottram (1998), unsaturated fatty acids undergo oxidation more easily than
SFA. Nonetheless, Liou and Simpson (1989) found that no statistical differences were
recorded in the total percentage of saturated, monoenoic, dienoic, unsaturated n-3 and n-6
fatty acids between fresh Artemia and Artemia dried by freezing, vacuum or hot air drying.
Furthermore, several researchers reported that the fatty acid profiles of raw, baked, broiled,
gilled and microwave cooked red snapper, pompano, mackerel, sardines and sea bass fillets
were not significantly different (Gall et al., 1983; Maeda et al., 1985; Yanar et al., 2007).
However, in the present experiment, total MUFA, PUFA and total n-3 PUFA including
EPA and DHA contents of dried samples were more affected by the drying process and
temperature than total n-6 PUFA and ARA values. Pigott and Tucker (1990) reported that a
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Chapter 4
major loss of n-3 fatty acids in fish oil was observed at high temperature and that highly
unsaturated fatty acids are highly susceptible to oxidative rancidity, with the development
of off-flavours. As mentioned above, drying temperature and drying time caused the same
effect as on total lipid content. According to Bórquez et al. (1997), as drying time increases,
losses of n-3 fatty acids in fish protein concentrates increased in fluidized bed-drying and
drying temperature had little effect (between 60 and 80°C) on n-3 fatty acid losses under
drying conditions. A higher drying temperature in fluidized bed-drying with a shorter
drying time would yield a higher quality fish protein concentrate (i.e. with minimal
rancidity). Similar results were obtained by Bórquez (2003) who found that the loss of n-3
fatty acids of fish particles increased with drying time in impingement drying, and that the
drying medium temperature is the most important variable, influencing both processing
time and product quality. In addition, Mudgett and Westphal (1989) found that proteins,
lipids and other components can also absorb microwave energy, but are relatively less
responsive. Although n-3 and n-6 PUFA levels in conventionally cooked rainbow trout
fillets were lower than in microwave-cooked fillets, the difference was not statistically
significant (Unusan, 2007). These results were in agreement with the present experiment,
where at the same drying temperature contents of total lipids and FA compositions in
MWHA-dried samples were slightly higher than that of HA and oven drying, but where
significant differences were not detected. Moreover, all individual fatty acid levels of
Artemia dried using the three different indoor techniques revealed the same effect as with
the total lipids, with no significant differences at different drying temperatures.
Temperatures in the range 50-70°C may thus be considered acceptable for drying Artemia.
Overall, the fatty acid contents in the sun-dried product was significantly lower than in the
frozen sample (P<0.01) except for the total SFA. When compared with the three indoor
drying methods, significant differences were only found for levels of DHA, MUFA, PUFA
and n-3 PUFA. Similar results have been reported in the literature for the losses of other
nutrients and sensory values in sun drying of various fruits and vegetables, e.g. Nyambaka
and Ryley (2004) found that sun dried cowpea leaves were not comparable to fresh material
in terms of aroma, texture and appearance; the color of sun-dried tomato slices is
significantly darker than fresh ones and vacuum assisted solar-dried samples due to direct
exposure and longer drying time (Rajkumar et al., 2007); higher losses of β-carotene,
ascorbic acid and chlorophyll in open sun drying as compared to cabinet drying have been
reported for leafy vegetables (Negi and Roy, 2000). According to Karabulut et al. (2007),
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Chapter 4
the β-carotene content of sun-dried apricots was not comparable with hot air dried apricots
probably due to differences in drying conditions between hot air drying and sun drying,
such as sun light, air velocity, drying temperature and drying time; Özcan et al. (2005)
found that the mineral content of oven-dried herbs was higher than in sun-dried herbs.
Alghren et al. (1994) considered the n-3/n-6 ratio as the most important indicator of fish
lipid quality, which also reflects the quality of fish as a food. The ratios of n-3/n-6 PUFA in
all dried Artemia samples were in the range 1.8-2.2 and the control value was 2.0. This
indicates that these drying methods did not affect this ratio. Our results are in accordance
with Sampels et al. (2004) who reported that the n-6/n-3 ratio was not affected by the
drying method. A similar result was detected by Yanar et al. (2007) who found that baking,
grilling and microwave cooking did not change the ratio of n-3/n-6 fatty acids compared
with the raw fillets of sea bass. In this study, within the temperature range of 50-70°C, a
longer drying time caused lower values of total lipids and fatty acids in dried Artemia
samples.
Conclusions
Data obtained from our study showed that the drying time was shortest for intermittent
MWHA drying and longest for sun drying. In addition, drying was faster in convective hot
air than in oven drying and drying time was reduced significantly when drying temperature
increased.
Overall, conductive HA drying, intermittent MWHA drying and oven drying at
temperatures of 50, 60 and 70°C are adequate techniques to dry Artemia biomass without
significant loss in total lipids and fatty acids. Especially intermittent MWHA drying is a
promising method, which could produce high quality dried products in short drying times.
However, it may not be suitable for large-scale application because of high initial capital
investment and operating costs. Conversely, sun drying resulted in significant reductions of
total lipid and fatty acid contents in dried Artemia but was much less energy consuming.
There is thus a need to find alternative drying methods both with respect to the economic
aspect and product quality. According to Chua and Chou (2003), both sun and solar drying
are cheap methods because they benefit from solar heat in which solar drying is a
modification of sun drying, i.e. the sun's rays are collected inside a specially designed unit
resulting in higher temperature with adequate ventilation for removal of moist air. It is
likely that the use of a solar dryer can result in shorter drying time as well a higher quality
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Chapter 4
product than in sun drying due to the judicious control of the radiative heat. In the coastal
areas in Vietnam several hundreds of hectares of Artemia cyst production area are in
operation during the dry season (Brands et al., 1995; Hoa et al., 2007). Therefore, future
research should aim to develop appropriate techniques for solar drying and on its effect on
nutritional quality of dried Artemia biomass. Such a solution could help the farmers to
salvage large amounts of live Artemia biomass, which is a by-product of their cyst-oriented
Artemia ponds and convert it into feed or as ingredient in formulated feeds for shrimp, fish,
livestock and poultry. This integrated production could contribute to the profitability of
Artemia farmers’ operations and thus have a positive impact on their socio-economic status
in this area.
115
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116
Chapter 4
Chapter 4
Section II
Effect of solar drying on lipid and fatty acid composition
of dried Artemia biomass
Chapter 4
Chapter 4
Abstract
Since sun-drying of Artemia biomass affects product quality, the potential use of a solar
drier for Artemia biomass has been evaluated. The drying time and quality of dried Artemia
biomass in terms of lipid and fatty acid composition using a solar drier versus sun drying
was compared under various weather conditions in Bac Lieu (latitude of 9o38'9"N,
longitude of 105o51'45"E), South of Vietnam. When drying Artemia biomass on sunny days
or days with sunny intervals, the drying time was substantially reduced with 45% and 25%,
respectively, compared to open sun drying. However, on cloudy/rainy days the reduction
was only 7%. Solar-dried Artemia contained higher levels of lipids and fatty acids than
open sun-dried Artemia. Especially when drying was performed on sunny days the quality
of the dried product was relatively comparable to fresh Artemia, whereas in biomass dried
on cloudy/rainy days these values were similar or inferior to the control under both drying
conditions. Additionally, for lipid classes the amounts of total neutral lipids of dried
Artemia significantly increased while total polar lipids significantly decreased when
compared to the non-dried sample (P<0.01). In particular, at the same drying weather
condition, the content of free fatty acids in sun-dried samples showed greater increase than
in solar-dried samples indicating the open sun drying underwent more lipolysis (lipid
degradation) as compared with the solar-dried product. Generally, all analyzed parameters
of solar-dried Artemia differed less from fresh Artemia than those of sun-dried samples.
This preliminary work has proved that the use of a solar drier for drying Artemia biomass is
a cheap and promising method, which could produce a better quality of dried Artemia that
can be used as feed ingredient in formulated feeds for aquaculture species.
Keywords: Artemia biomass, total lipids, lipid class, fatty acid, solar drying, sun drying
Introduction
Artemia biomass is rich in protein, lipid, attractants, pigments and other active substances
that make it an attractive direct feed for ornamental fish (Lim et al., 2001) or an excellent
ingredient for aquafeeds, e.g. as maturation trigger in shrimp broodstock (Naessens et al.,
1997; Wouters et al., 2001) and for young juveniles of marine fish, shrimp and crab (Léger
et al., 1986; Dhert et al., 1993; Sorgeloos et al., 2001; Ut et al., 2007a). Fresh Artemia
biomass contains a very high level of moisture (approximately 90%) and is rich in
proteolytic enzymes; consequently it decomposes quickly and is difficult to store and
transport, and is subject to quick quality deterioration (Baert et al., 1996).
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Chapter 4
In the coastal areas of Soc Trang and Bac Lieu provinces (South of Vietnam), large
amounts of Artemia biomass, a by-product from commercial cyst-oriented Artemia
production, can be collected at average rates of 0.2-0.3 ton ha-1 when the culture season
ends (Brands et al., 1995; Anh et al., 1997a). According to College of Aquaculture and
Fisheries, Can Tho University, the production area in the 2008 culture season was about
400 ha, and thus more than 100 tons of Artemia biomass could be obtained from this
region. However, so far this product has not been utilized in local aquaculture. Therefore, a
proper processing technique of Artemia biomass, allowing to store this product sufficiently
long without significant quality loss, may contribute to the production of high-quality feed
ingredients for aquafeeds that could be of direct application in local and regional
aquaculture.
Drying is a common technique for preservation of human and animal food products. The
major advantage of drying food products is the reduction of moisture content to a level that
allows an extended shelf life of the dried products and a substantial reduction in terms of
mass and volume, which facilitates packaging and storage, and thus reduces transportation
costs (Brennand, 1994; Chua and Chou, 2003). A previous laboratory experiment had
demonstrated that drying Artemia using a combined microwave and convective hot air
method resulted in the shortest drying time and better quality than convective hot air and
oven drying methods (Section I of chapter 4). However, this method may not be suitable for
large scale application in rural areas in Vietnam because of its high operating cost. Hence,
research should focus on the development of cost-effective methods with low investment to
dry Artemia biomass for direct use or for further processing. Sun drying is a traditional
practice where fresh product is left to dry in the open air and sun while solar drying is a
modification of sun drying in which the sun's rays are collected inside a specially designed
unit with adequate ventilation for removal of moist air (Hughes and Willenberg, 1994).
According to Chua and Chou (2003), both sun drying and solar drying are cheap methods
because they rely on solar energy. Furthermore, the utilization of a solar dryer can lead to a
superior product quality than sun drying, as the radiative heat can be ‘better controlled’.
Other researchers have also been using solar-energy drying as an alternative to the
traditional open sun drying in developing countries (FAO, 1992; Sodha and Chandra,
1994). Construction details and operational principles of solar dryers for different
biological materials have been reviewed by Ekechukwu and Norton (1999) and Farkas
(2004), who confirmed that using solar energy is a promising solution for meeting the
118
Chapter 4
technical, economical, and environmental demands raised by the drying process. Also,
Purohit et al. (2006) emphasized the importance of developing low cost solar drying
systems, preferably using local materials and skills.
Vietnam is located in a tropical monsoon climate zone and near the equatorial region,
especially in the central and south of the country, has a natural advantage of abundant solar
radiation. Total sunshine is between 1600-2720 h year-1, mean daily sunshine is from four
to eight hours and total solar radiation varies from 346.8 to 2153.5 kWh m-2 year-1 with
average total daily radiation is 5 kWh m-2 day-1 (http://ecchanoi.gov.vn/vi/?id=news=208).
According to the Vietnam Institute of Agricultural Engineering (VIAE), in the rural areas
of Vietnam sun drying has been the most common method for drying of products, but it
often results in poor quality, such as a high moisture content and/or possible contamination
with dirt, insects, moulds etc. (if the product has been dried too slowly). In large farm and
grain processing plants, artificial driers heated with oil and coal stoves (furnaces) are also
used but with high cost and environmental pollution. In 1998, VIAE installed a solar drier
(type SD-25) at household level in some provinces in the north for drying fish and shrimp.
They reported that using the solar dryer reduces drying time by 65% when compared to sun
drying, resulting in higher product quality and reduction of costs and of pollution
(http://xttm.agroviet.gov.vn).
For commercial production of agricultural products the use of a forced convection solar
dryer provides a better control of the drying air conditions (Ekechukwu and Norton, 1999;
Farkas, 2004), whereas the natural convection solar drier does not require any other energy
during operation (Chua and Chou, 2003). For this reason, the natural convection solar
dryer, especially when applied to thin layers, may become a more suitable method for the
rural sector and other areas where electricity is scarce and irregularly available
(Pangavhane et al., 2002). Therefore, this study was undertaken to evaluate a natural
convection solar dryer with simple design and low investment for drying Artemia biomass,
and to compare its performance in terms of drying time and product quality versus open sun
drying under the Vietnamese climatic conditions. Our results may help Artemia-farmers in
utilizing locally available materials in the construction of a low-cost drier for Artemia
biomass, and to valorize this by-product in the production of formulated feeds for target
species, which opens perspectives for increased profits and an improved living standard for
the local salt farmers.
119
Chapter 4
Previous studies have proven that qualitative and quantitative aspects of the lipid contents
of Artemia as direct feed or as ingredient in artificial diets are important for the survival,
growth and reproduction of shrimp and fish species (Léger et al., 1986; Coutteau and
Mourente 1997; Sorgeloos et al., 2001; Wouters et al., 2002; Martins et al., 2006). Hence,
in assessing the quality of dried Artemia, special consideration was given to the lipid
quality, taking into account their susceptibility to deterioration as a consequence of the
drying conditions as reported by several authors (Liou and Simpson, 1989; Pigott and
Tucker, 1990; Bórquez et al., 1997; Toldrá, 2006; Ayanwale et al., 2007).
2. Material and Methods
2.1. Description of the experimental drier
A small-scale experimental solar drier was constructed by copying the design from FAO
(1992) for drying Artemia biomass under Vietnamese climate conditions.
The solar drier (Figure 1) measured 2 m x 1m x 1m in size and has four legs of 20 cm
height. The frame was made of bamboo with an angle of inclination of about 20° frontward,
which created air circulation within the system. Transparent nylon covered the entire drier’s
side and the chimney except bottom side. The drying chamber (0.9m x1.2 m) contained the
3 cm wire-mesh that served to hold the Artemia biomass trays. A solar collector unit (1m x
0.8m) was integrated in the floor of the drier; with a 3 mm thick tin absorber plate, painted
black to enhance absorption. A chimney of 20 cm diameter and 60 cm height was fixed in
the middle of the upper part of the drier to increase the airflow through the drying chamber
and through the material to be dried. The opening (0.2 m x 1m) at the front of the drier
allowed for the entry of air, which, once heated, rose by natural convection and left through
the outlet (chimney). The drier was positioned facing the east during drying.
Open sun drying experiments were carried out by placing the Artemia samples under direct
sunlight on a wire-meshed surface elevated approximately 1.1 m above the ground to allow
for air circulation (Figure 1).
120
Chapter 4
S
Air outlet
N
Air inlet
Solar collector
Figure 1. Left: Photograph of experimental solar drier. Right: Open sun drying.
2.2. Sample preparation
Live Artemia biomass (adult Artemia) was collected from commercial Artemia cystoriented ponds in Vinh Hau village, Bac Lieu province, Vietnam. The harvested Artemia
biomass was washed with freshwater, excess water was removed by tissue paper and 0.5%
of antioxidant (butylated hydroxytoluene, BHT) was added to the sample before drying.
Then 500g Artemia sample was spread on the plastic net (mesh size 1.2 mm) in a thin layer
of 4 mm. The Artemia thin layer samples were dried using both solar and open sun drying
methods.
2.3. Experimental set-up
The drying test was carried out by the end of the Artemia culture season (from 4th May until
15th June 2007, the transition period from dry to rainy season in Vietnam) under the
climatic conditions where Artemia culture takes place in Bac Lieu province. The study was
done in this period in order to evaluate the performance of the solar drier at various weather
conditions (sunny, cloudy or rainy days) randomly occurring during the Artemia drying
experiment.
Each run, four trays of 500 g each of Artemia biomass, spread in a thin layer (thickness
approximately 4 mm, were installed in the drying chamber of the solar drier. Open sun
drying was conducted simultaneously under the same weather conditions. Drying was
performed between 7.00 and 17.00 h. Every evening, the Artemia samples were removed
and stored in an air-tight plastic bag.
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Chapter 4
Weight loss was determined at regular intervals of two hours: the Artemia samples were
taken out, quickly weighed on an electronic balance with 0.1 g accuracy and returned to the
drier. From these data, the average values of moisture content as a function of time were
determined and used to calculate the drying curves. Drying was continued the following
days until the sample reached the desired moisture level.
2.4. Climatic data
The dry bulb and wet bulb temperatures at inlet and outlet of the solar drier were measured
at regular intervals of 30 min. The relative humidity of air was calculated from recorded
wet and dry bulb temperatures using a psychrometric chart. At the same time, the
temperature of the product was determined at four different locations for both solar and sun
drying (by inserting a thermometer into the product). In sun drying, the ambient
temperature and relative humidity were also recorded throughout the drying period. Solar
radiation data and other meteorological data were obtained from the Meteorological Station
in Bac Lieu (it is about 6 km from the drying site).
2.5. Chemical analysis
Since fresh Artemia biomass utilized in this experiment originated from different batches,
each sample (control) was also analyzed for sake of comparison with the corresponding
dried Artemia samples.
The initial and final moisture content of the Artemia samples were determined in triplicate
by oven-drying at 110°C until constant weight (AOAC, 1995). Total lipid was determined
according to Folch et al. (1957) as modified by Ways and Hanahan (1964), and fatty acid
methyl ester (FAME) composition was analytically verified by gas chromatography
following the methodology of Coutteau and Sorgeloos (1995). Lipid classes were analyzed
using high-performance-thin-layer chromatography (HPTLC) (Olsen and Henderson,
1989).
2.6. Statistical analysis
Data for all measured parameters were analysed using SPSS for Windows, Version 13.0.
Total lipid, lipid classes and fatty acid levels of fresh Artemia (control) and dried Artemia
samples exposed to different drying weather conditions were compared by one-way
ANOVA. The Tukey HSD post-hoc analysis was used to detect differences between means.
Significant differences were considered at P<0.05. All percentage values were normalized
through a square root arcsin transformation prior to statistical treatment.
122
Chapter 4
RH- Sun drying
100
(b)
50
15:00
14:00
13:00
12:00
11:00
10:00
9:00
8:00
7:00
40
17:00
25
16:00
50
15:00
30
14:00
60
13:00
35
12:00
70
11:00
40
10:00
80
9:00
45
8:00
90
Outlet T°- Solar drying
Air T°- Sun drying
100
Outlet RH- Solar drying
RH- Sun drying
90
80
40
70
35
60
30
50
25
40
7:00
8:00
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
17:00
7:00
8:00
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
17:00
7:00
8:00
9:00
10:00
11:00
45
50
(c)
Outlet T°- Solar drying
Air T°- Sun drying
Outlet RH- Solar drying
RH- Sun drying
100
90
80
40
70
35
60
30
50
25
40
7:00
8:00
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
17:00
7:00
8:00
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
17:00
7:00
8:00
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
17:00
Temperature (°C)
45
Relative humidity (%)
Outlet RH- Solar drying
Relative humidity (%)
Air T°- Sun drying
50
55
Temperature (°C)
Outlet T°- Solar drying
Relative humidity (%)
(a)
7:00
Temperature (°C)
55
Time of day
Figure 2. Hourly variations in mean temperature and relative humidity during drying.
The arrows indicate starting a new day of drying. (a) predominantly sunny weather
conditions, (b) weather with sunny intervals, (c) predominantly cloudy/rainy weather
conditions.
123
Chapter 4
3. Results
Over a period of 42 days twelve runs of Artemia drying tests were performed under various
weather conditions, four of which with predominantly sunny weather, six with ample sunny
intervals, and two runs with variable weather of sunshine, clouds and rain.
3.1. Weather conditions during the experiment
During the drying experiment between May and June, 2007 in Bac Lieu (latitude of
9o38'9"N, longitude of 105o51'45"E), total sunshine varied from 6 to 10h per day (12 h of
daylight). The total daily variations of solar radiation, wind velocity, air relative humidity
and air temperature ranged from 250 to 980 Wm-2, 1 to 8 ms-1, 60 to 85% and 24 to 35 oC,
respectively (Bac Lieu Meteorological Station). These data show that abundantly available
solar energy can be used for the drying of the agricultural products.
3.2. Temperature and relative humidity of drying experiment
The hourly fluctuations of the mean temperature and relative humidity (RH) under various
weather conditions for solar and sun drying are shown in Figures 2. The ambient
temperature rose considerably between 10:00 and 14:00 h, whereas RH reached the lowest
value during this period.
Solar drying
Product temperature (°C)
50
Sun drying
45
40
35
30
25
Sunny weather
Sunny interval
Cloudy/rainy
Figure 3. Mean product temperatures (Artemia biomass) dried under different weather
conditions.
Generally, the drying temperature and RH varied continuously during the drying period of
7:00 to 17:00 h due to the variation of insolation. Air temperature and RH in sun drying and
124
Chapter 4
at inlet of solar drier were the same and averaged 35.5, 33.4 and 30.7°C and 72, 75 and
80% when weather was predominantly sunny, with sunny intervals and variable weather of
sunshine, clouds and rain, respectively. (Values of temperature and RH at the inlet of solar
drier were the same as data obtained in sun drying so Figure 2 compared data at the outlet
of solar drier with air temperature and RH in sun drying). Temperatures at the outlet of the
solar drier were higher than the ambient and inlet temperatures, whereas the RH at the
outlet was lower than RH observed for sun drying or at the inlet, and these differences were
more pronounced at noon on sunny days (e.g. 53°C vs. 38°C and 39% vs. 65%,
respectively). However, these differences were minor on rainy/cloudy days (Figure 2c). The
mean product temperature inside the solar drier was significantly higher as compared to that
in the open sun drying when weather was sunny or with sunny intervals (Figure 3). Overall,
the product temperature was higher than the air temperature due to the absorption of solar
radiation by the Artemia biomass and this difference was more pronounced in solar than in
open sun drying. However, the product temperature was highly influenced by the weather
parameters such as change in ambient air temperature, relative humidity and air velocity
inside the drier due to insolation.
3.3. Drying time
The drying curves of Artemia biomass in the solar dryer and open sun drying under various
weather conditions are presented in Figure 4.
In all cases the solar dryer required a shorter drying time than the open sun drying.
Depending on weather conditions, the time needed to dry Artemia biomass in a 4 mm thick
layer from an initial moisture content of 86-89% to a final moisture content of 10-12% was
10, 18 and 28 h in the solar dryer and 18, 24 and 30 h in open sun drying when the weather
was sunny, with sunny intervals and with variable weather of sunshine, clouds and rain,
respectively.
125
Moisture content (%)
Chapter 4
Sun drying
90
80
70
60
50
40
30
20
10
0
(a)
0
2
Solar drying
4
6
8
10
12
14
16
18
Moisture content (%)
Drying time (hour)
Sun drying
90
80
70
60
50
40
30
20
10
0
(b)
0
2
4
Solar drying
6
8
10
12
14
16
18
20
22
24
Drying time (hour)
Moisture content (%)
90
80
Sun drying
(c)
Solar drying
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Drying time (hour)
Figure 4. Drying curves for solar dried and sun dried Artemia biomass under various
weather conditions. (a) predominantly sunny weather conditions, (b) weather with sunny
intervals, (c) predominantly cloudy/rainy weather conditions.
126
Chapter 4
3.4. Total lipids
Moisture content (%), total lipids (% of DW) and lipid class (% of total lipid) of fresh
Artemia (control) and Artemia dried using solar and sun drying at various weather
conditions are shown in Table 1.
In biomass dried during sunny weather or weather with sunny intervals, the contents of
total lipids in the solar-dried samples (10.50 and 8.85%, respectively) were lower than the
corresponding values in the fresh samples (11.03 and 9.26%), but statistical differences
were not detected (P>0.05).The corresponding values, however, in sun-dried samples (9.28
and 8.31%) showed a significant decrease as compared to the fresh and solar sample
(P<0.05). Moreover, on the cloudy/rainy days values of both dried samples were similar
and statistically different from the fresh samples.
3.5. Lipid classes
The major lipid classes in dried Artemia samples were noticeably affected by both drying
methods and weather conditions (Table 1). The amounts of total neutral lipids (NL)
significantly increased whereas total polar lipids (PL) significantly decreased when
compared to the fresh sample (P<0.01). In addition, these values showed statistical
differences between solar and open sun drying methods (P<0.05). For the neutral lipids, the
levels of cholesterol esters, free fatty acids (FFA) and cholesterol+diacylglycerol (DAG)
were significantly higher while triglycerides were lower in solar and sun-dried Artemia than
in the fresh Artemia (P<0.05). Particularly, under the same drying weather condition the
FFA content in sun-dried samples exhibited a greater increase than in solar-dried samples.
For polar lipids, the content of phosphatidic acids + phosphatidylethanolamine (PA+PE),
phosphatidylserine + phosphatidyl-inositol (PS+PI) and phosphatidylcholine (PC) were
lower in the dried samples, whereas sphingomyelines and lyso-phosphatidylcholine (LysoPC) remained constant or slightly declined. Moreover, linear regression (Figure 5)
indicated that the level of total polar lipids of dried Artemia was inversely correlated with
drying time (y = -0.6171x + 24.483, R² = 0.95) while the content of free fatty acids
extremely increased with drying time (y =1.8212x + 8.0145, R² = 0.98). Generally, when
Artemia was dried in the solar drier, lipid classes changed less than in open sun drying.
127
Chapter 4
Table 1 Moisture content (%), total lipids (% of DW) and lipid classes (% of total lipids) of Artemia biomass dried using solar and sun drying
under various weather conditions. Data represent average of triplicate analyses. Values within the same row of each group that do not share the
same letter are significantly different (P<0.05).
Moisture content (%)
Total lipids (% DW)
Total neutral lipids (%)
Total polar lipids (%)
Neutral lipids (%)
Cholesterolesters
Triglycerides
Free fatty acids
Cholesterol+DAG
Unknown NL
Polar lipids (%)
PA+PE
PS+PI
Phosphatidylcholine
Sphingomyelines
Lyso-PC
Unknown PL
Sunny day
Fresh
88.61
11.03b
73.72a
26.28c
Sun
10.40
9.28a
92.40c
7.60a
Sunny interval day
Fresh
Solar
86.32
10.60
9.26b
8.85b
71.49a
84.19b
28.51c
15.81b
Solar
10.24
10.50b
83.48b
16.52b
0.64a
56.48c
3.78a
7.79a
5.73
4.46b
1.05b
14.70c
0.92a
3.27b
1.87
Sun
10.64
8.31a
88.32c
11.68a
Cloudy/rainy day
Fresh
Solar
88.74
11.59
10.47b
8.67a
68.38a
88.85b
31.62c
11.15b
Sun
11.66
8.42a
92.89c
7.11a
3.70b
37.02b
26.62b
10.06b
6.08
3.90b
30.74a
38.68c
12.73b
6.35
0.53a
55.30c
3.08a
8.40a
4.18
3.15b
31.32b
34.29b
10.29b
5.14
3.58b
22.06a
46.36c
13.10b
3.22
0.47a
50.95b
2.31a
8.26a
6.41
4.44a
35.93a
33.26b
11.27b
3.95
4.65a
30.91a
39.09c
12.28b
5.96
3.72b
0.80b
5.57b
0.91a
3.06b
2.47
1.28a
0.34a
1.68a
0.92a
1.80a
1.57
4.72c
0.93c
17.94c
0.95a
2.53b
1.44
2.44b
0.67b
5.76b
0.97a
2.36b
3.60
1.79a
0.46a
2.44a
0.94a
1.78a
4.27
6.91b
0.74b
19.37b
0.81a
2.70b
1.10
1.57a
0.41a
3.27a
0.76a
1.17a
3.97
1.58a
0.28a
2.36a
0.65a
1.43a
0.82
NL= Neutral lipids, PL=polar lipids, PA= phosphatidic acids, PE= phosphatidyl-ethanolamine, PS= phosphatidylserine, PI=phosphatidylinositol,
DAG= Diacylglycerol, Lyso-PC= Lyso-phosphatidylcholine
128
Chapter 4
Table 2. Fatty acid composition (mg g DW-1) of Artemia biomass dried using solar and sun
drying under various weather conditions.
Fatty
acids
Sunny days
Sunny interval days
Cloudy/rainy days
Fresh
Solar
Sun
Fresh
Solar
Sun
Fresh
Solar
Sun
18:2n-6
1.95a
1.84a
1.76a
2.71a
2.30a
2.11a
3.92b
2.81a
2.69a
18:3n-3
0.82c
0.65b
0.53a
1.11c
0.84 b
0.54a
0.74b
0.35a
0.33a
20:5n-3
7.38b
6.48b
5.50a
6.53c
5.63b
4.69a
7.74b
5.47a
5.11a
22:6n-3
0.61c
0.43b
0.28a
0.43b
0.27a
0.22a
0.53b
0.22a
0.20a
20:4n-6
3.25a
3.00a
2.75a
3.20b
2.85ab
2.60a
3.70b
2.85a
2.60a
ΣSFA
19.20a
19.28a
17.39a
22.25a
19.12a
19.69a
20.62a
21.59a
21.57a
ΣMUFA
31.09b
27.43b
23.43a
36.05c
28.54b
23.99a
32.77b
22.57a
21.91a
ΣPUFA
16.41b
13.76b
11.72a
16.09c
13.35b
11.32a
18.54b
13.05a
12.28a
10.25b
8.47b
6.76a
9.22c
7.49b
5.98a
10.0b
6.61a
6.27a
Σn-6
PUFA
6.16b
5.42ab
4.96a
6.87b
5.86ab
5.35a
8.45b
6.45a
6.01a
n-3/n-6
1.71a
1.62a
1.43a
1.34 a
1.31a
1.13a
1.22a
1.00a
1.00a
1
Σn-3
PUFA
2
Data represent average of duplicate analyses. Values in the same row of each group with
the same letter are not significantly different (P>0.05).
LNA, linolenic acid (18:3n-3); EPA, eicosapentaenoic acid (20:5 n-3); DHA,
docosahexaenoic acid (22:6 n-3); LIA, linoleic acid (18:2n-6); ARA, arachidonic acid
(20:4n-6); SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA,
polyunsaturated fatty acids.
1
Σ (n-6) PUFA ≥18:2n-6;
2
Σ (n-3) PUFA ≥18:3n-3
129
Total PL & FFA (% of total lipid)
Chapter 4
y = 1.8212x + 8.0145
70
2
R = 0.9768
60
Free facty acids
50
40
30
y = -0.6171x + 24.483
20
2
R = 0.95
10
Total polar lipids
0
0
5
10
15
20
25
30
35
Drying time (hour)
Figure 5. Correlation between total polar lipid/free fatty acid contents of Artemia biomass
and drying time.
y = -0.3563x + 30.688
R2 = 0.9663
Total MUFA & PUFA (mg/gDW)
35
30
25
20
Total MUFA
15
Total PUFA
y = -0.1902x + 16.353
R2 = 0.957
10
5
0
0
5
10
15
20
25
30
35
Drying time (hour)
Figure 6. Correlation between total MUFA/PUFA contents of Artemia biomass and drying
time.
130
Chapter 4
Total n-3 and n-6 (mg/gDW)
12
y = -0.1456x + 10.039
R2 = 0.9032
10
8
6
Total n-3 PUFA
Total n-6 PUFA
4
y = -0.0587x + 6.1733
R2 = 0.8336
2
0
0
5
10
15
20
25
30
35
Drying time (hour)
Figure 7. Correlation between total n-3 PUFA/n-6 PUFA contents of Artemia biomass and
drying time.
3.5. Fatty acid composition
The fatty acid content, measured in both fresh and dried Artemia, is presented in Table 2. In
Artemia biomass dried on cloudy/rainy days all analyzed parameters in solar-dried and sundried samples were similar (P>0.05). Both drying methods exhibited significantly lower
values than fresh biomass (P<0.05), except for total saturated fatty acids (ΣSFA) which
were somewhat higher. Drying of Artemia biomass on sunny days or days with sunny
intervals, however, had a different effect on the fatty acid composition of the dried product
depending on the drying method. The amount of ΣSFA in the dried samples was slightly
lower or almost equal to fresh Artemia in both drying methods conducted under various
weather conditions. This was also the case for linoleic acid (LIA, 18:2n-6) and arachidonic
acid (ARA, 20:4n-6) levels (P>0.05). Nonetheless, in comparison to the fresh sample a
significant decrease of ARA was noted in sun-dried samples on days with sunny intervals.
When dried under these weather conditions there was a higher loss of linolenic acid (LNA,
18:3n-3) and docosahexaenoic acid (DHA, 22:6n-3) in sun-dried samples compared to
solar-dried samples, and both were significantly different from the corresponding value in
fresh Artemia. Although the contents of eicosapentaenoic acid (EPA, 20:5n-3), total monounsaturated fatty acids (ΣMUFA), total polyunsaturated fatty acids (ΣPUFA) and the Σn-3
PUFA in the solar-dried samples on sunny days were lower than in the control, significant
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Chapter 4
differences were not detected; the sun-dried samples showed a significant reduction as
compared to the fresh and solar-dried samples. On the other hand, when dried on days with
sunny intervals, the three products exhibited statistical differences, with again a greater
drop of these parameters in case of sun drying than in solar drying. Additionally, the
content of Σn-6 PUFA in the solar-dried sample showed an intermediate value between the
fresh and the sun-dried sample, but only the sun-dried sample exhibited a significant
decrease compared to the control (P<0.05). Besides, when Artemia were dried on
predominantly cloudy/rainy days, the levels fatty acids in both products were similar and
significantly lower than the fresh samples except for ΣSFA. Regression analysis showed
that ΣMUFA and ΣPUFA were reduced with drying time (Figure 6) and the same tendency
was found for Σn-3 and Σn-6 PUFA but the n-3 PUFA showed higher loss than in the n-6
PUFA (Figure 7). However, the ratios of n-3/n-6 PUFA were not affected by drying
methods nor by weather conditions (P>0.05).
4. Discussion
4.1. Effect of drying parameters on drying time
When comparing solar drying to open sun drying, the drying time in the former was
substantially reduced with about 45% and 25% when drying was performed on sunny days
or on days with sunny intervals, respectively. If dried on cloudy/rainy days, the reduction
was only 7%. This reduction is in part due to the higher temperature and/or lower relative
humidity in the solar dryer. Previous studies reported that the drying rate of various
products in a solar drier is much higher than in open sun drying. This is due to the product
temperature inside the solar drying chamber which is significantly higher than in open sun
drying, as a consequence of i) better absorption of solar energy by the product as most of
the solar energy entering the drying chamber is trapped inside the drier facilitating
absorption, ii) energy release by the solar collector (black surface) to the product by heat
conduction, iii) easy removal of moisture through the chimney by natural air circulation
(Ekechukwu and Norton, 1999; Chua and Chou, 2003; Farkas, 2004). Moreover,
Pangavhane et al. (2002) reported that in a natural convection solar drier, the drying time of
grapes is also reduced by 43% compared to open sun drying. When using solar tray drying
of grapes, the range of decrease in drying time compared to sun-drying was 22-36%
depending on pre-treatment (Farkas, 2004). Similar results, in accordance with this study,
have been reported by Bala and Mondol (2001) for fish, by Bala et al. (2003) for pineapple,
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Chapter 4
by Rajkumar et al. (2007) for tomato slices and by Hossain and Bala (2007) for chilli. On
the other hand, Sacilik et al. (2006) found that the drying characteristics of tomato slices in
a solar tunnel and in open sun drying were highly influenced by weather parameters such as
change in air temperature, relative humidity, and wind velocity due to insolation. Joshi et
al. (2004) concluded that due to differences in drying period for different drying materials,
different efficiency values have been found for the same solar dryer. The efficiency of a
solar drying system is affected by the properties of the drying materials e.g. moisture
content, size, shape and geometry as well as ambient conditions e.g. solar radiation and
temperature, relative humidity, velocity and atmospheric pressure of ambient air. Besides,
the air humidity is a critical factor controlling the drying rate as air with lower relative
humidity has a higher absorbing capacity (Mastekbayeva et al., 1998). However, Krokida et
al. (2003), investigating drying kinetics of various vegetables, revealed that the drying
temperature is the main factor affecting drying rate for all the examined materials, more
important than air velocity and air humidity.
The results in the present study are in accordance with previous researchers cited above.
However, our solar drier appears to be more efficient as compared to open-sun drying on
sunny days as compared to cloudy/rainy days; maybe this can be attributed to the small size
of the dryer and especially the solar collector which serves as thermal heat store and which
may cause a rapid loss of heat in case of cloudy weather.
4.2. Effect of solar and sun drying methods under various weather conditions on total lipids
and lipid quality of dried Artemia
The results of the present study indicated that total lipids, lipid classes and fatty acids of
dried Artemia were significantly affected by both drying methods and by weather
conditions.
4.2.1. Total lipids
Generally, the total lipid content of dried Artemia was lower than in fresh Artemia
(control), which is in agreement with the results obtained by Liou and Simpson (1989), who
reported that the lipid levels of Artemia nauplii dried by vacuum and by hot air were lower
than in newly-hatched Artemia. A similar observation was reported by Paleari et al. (2003),
who found a decrease of fat content during processing in cured and dried products from
different animal species. In the present investigation, however, significant losses of total
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Chapter 4
lipids were observed in solar-dried samples on cloudy/rainy days and in all sun-dried
samples when compared to the fresh sample (P<0.05). Loss of lipids in outdoor-dried
Artemia was distinctly affected by weather conditions such as ambient temperatures,
relative humidity etc. related to insolation, which in turn leads to the variations in drying
time as mentioned earlier. For example, on sunny days and days with sunny intervals open
sun drying at average temperatures of 35.4 and 33.7°C took much longer (18 and 24 h,
respectively) than solar drying at temperatures of 44.4 and 39.9°C (10 and 18 h,
respectively). According to previous studies (Maskan, 2001; George et al., 2004; Gowen et
al., 2006; Ayanwale et al., 2007), drying time and temperature can be considered the
principal operating parameters affecting the quality of the dried product. On the other hand,
some food products require several hours whereas others may take more than a day; and
prolonging drying time (by using lower temperatures) or interrupting drying time may
result in spoilage of dried products (Hughes and Willenberg, 1994). Moreover, Altan and
Maskan (2005) confirmed that it is important to control air temperature and circulation
during the drying process. If the temperature is too low or the humidity too high (resulting
in poor circulation of moist air) the food will dry more slowly resulting in a poorly dried
product. This is in line with our results, as both drying methods conducted on the
cloudy/rainy days at lower average temperatures of 33.4 and 30.6°C resulted in a more
extended drying time (28-30 h) than on the days with sunny weather or sunny intervals.
Additionally, the prolonged direct incidence of sunlight in case of sun drying may
accelerate lipid oxidation (Pigott and Tucker 1990; Mottram, 1998). A shorter drying time
may thus result in higher values of total lipids in dried samples, as observed in this study.
4.2.2. Lipid classes
It was observed that Artemia dried using solar and sun drying methods induced
considerable changes in lipid class composition (Table 1). A significant decrease of total
polar lipids (PL), cholesterol esters and triglycerides (TG) and a significant increase of free
fatty acids (FFA) were obtained in both solar and open sun drying conducted under various
weather conditions, which could be related to lipolysis. Toldrá and Flores (1998) reported
that lipolysis is one of the main processes of lipid degradation in fresh meat during
processing; high temperatures and prolonged drying and ripening favour lipid oxidation in
the processed products. Moreover, lipolysis indicates an enzymatic release of FFA from
both TG and phospholipids, and is thought to increase lipid oxidation, since FFA are very
sensitive to oxidation (Gray and Pearson, 1984; Motilva et al., 1993; Coutron-Gambotti and
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Chapter 4
Gandemer, 1999). According to Chizzolini et al. (1998) most volatile molecules identified
in dry-salted hams are lipid related. Therefore, lipolysis and lipid oxidation have been
considered to be key processes in processing procedures (drying, smoking, curing etc.).
They also claimed that there was a close relationship between the decrease in phospholipid
content and the increase in FFA during processing of dry-salted hams. Moreover,
technological parameters such as the time/temperature cycles of the different stages of
possessing greatly affect FFA content and lipolytic enzyme activity. Longer stages and
higher temperatures result in a higher FFA content of the hams (Gandemer, 2002). Similar
finding was reported by Toldrá (2006). Our results are in accordance with these
observations: the longer the drying time, the higher the increase of the FFA content that
was found in both studied drying methods. For example, the values of FFA in solar-dried
Artemia were increased 7, 11 and 14 times vs. 10, 15 and 17 times in sun-dried samples
when Artemia biomass was dried at sunny weather, weather with sunny intervals and on
cloudy/rainy days, respectively. Other studies proved that lipolysis causes an increase in
FFA and diacylglycerols and a correlated decrease in triacylglycerols (Garcia-Regueiro and
Diaz, 1989; Moltilva et al., 1993). They are also congruent with the results of Sampels et al.
(2004) who found that FFA displayed a threefold increase during drying, suggesting that
lipolysis occurs before or during drying, whereas polar lipids and TAG decreased in the
dried reindeer meat and were significantly different from both smoked and fresh meat. The
great increase in FFA observed in this work also agrees with the results reported for Iberian
dry-cured ham (Antequera et al., 1992), another type of ham (Vestegaard et al., 2000) and
for dry cured loin (Muriel et al., 2007). Furthermore, the percentage of polar lipids was
reduced with drying time as a result of reduction in PA+PE, PS+PI and phosphatidylcholine
(PC) in this experiment that is in line with the finding of Sharma et al. (1982) who found
that the most pronounced loss of polar lipids was found in phosphatidylethanolamine (PE)
followed by PC during refrigerated or frozen storage of cooked chicken meat. The overall
lipolysis that takes place in dry-cured hams during ripening affects polar lipids more
intensely than neutral lipids (Andres et al., 2005). Additionally, Balogun (1998) reported
that the lipids underwent lipolysis and autoxidation during sun drying of two freshwater
species of clupeids (Pellonula afzeliusi and Sierrathrisa leonensis) from Kainji Lake,
Nigeria. In the present study, differences in temperature and drying time between the two
studied drying methods under various weather conditions caused significant variations of
lipid classes in dried Artemia samples. Under the same drying weather conditions, the
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Chapter 4
higher FFA and lower polar lipid levels indicated more lipolysis in the sun-dried samples
than in the solar-dried samples.
4.2.3. Fatty acid composition
Overall, the fatty acid (FA) composition of dried Artemia was notably affected by both
drying methods and weather conditions as the reduction of these FA contents showed a
different tendency in both solar and open sun drying under various weather conditions. For
example, when drying on sunny days, most FA profiles in the solar-dried sample were
relatively comparable to fresh Artemia, while for drying on cloudy/rainy days these values
were similar to the sun-dried sample, and for drying on days with sunny intervals they
displayed intermediate values (Table 2). The data show that the levels of ΣSFA were more
or less the same in all dried Artemia samples, whereas the unsaturated FA (UFA) decreased
as compared to the fresh Artemia. This can be seen in all individual FA as well as in the
different sums and ratios. These results are in accordance with the study of Sampels et al.
(2004), who reported that dried reindeer meat showed slightly higher values of total SFA
whereas total UFA were lower compared with fresh meat. Similar results were reported for
dry-cured hams from hairless Mexican pigs (Delgado et al., 2002) and for the cured and
ripened goat thigh (Paleari et al., 2008). According to Cosgrove et al. (1987) and Mottram
(1998), UFA undergoes oxidation more easily than SFA. In this study, ΣMUFA, ΣPUFA
and Σn-3 PUFA including LNA, EPA and DHA contents of dried Artemia samples were
more affected by the drying methods and weather conditions than Σn-6 PUFA, LIA and
ARA values. Pigott and Tucker (1990) found that fish lipids, with their wealth of highly
unsaturated fatty acids (HUFA), are highly susceptible to oxidative rancidity. Especially
loss of n-3 fatty acids (FA) could be significant and the decrease is dependent on the time
during which the reaction can take place: longer dehydration times promote loss of UFA or
n-3 FA. Susceptibility of n-3 FA to oxidation seems to be particularly localized in HUFA
residues (Tapaneyasin et al., 2005). Furthermore, Bórquez et al. (1997) suggested that as
drying time increases, losses of n-3 fatty acids in fish protein concentrates increased during
fluidized bed-drying. Similar results were obtained by Bórquez (2003) who found that the
loss of n-3 fatty acids of fish particles increased with drying time in impingement drying,
and that the appropriate drying temperature is the most important variable, influencing both
processing time and product quality. However, Paleari et al. (2003) observed that in dried
meat both n-3 and n-6 PUFA decreased significantly, which indicates higher oxidation in
the dried meat compared with the fresh and smoked meat. Drying methods and their
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Chapter 4
conditions can also significantly affect the quality of dried fish (Rahman et al., 2002). Our
results are in agreement with these findings; drying time caused the same effect on FA
profiles as on total lipid contents of dried Artemia that was mentioned before. Hence, it is
evident that the UFA has a tendency to decrease with prolonged drying time, which
enhanced the oxidation of these substances (Figure 6-7). Additionally, Alghren et al. (1994)
considered the n-3/n-6 ratio as the most important indicator of fish lipid quality, which also
reflects the quality of fish as a food. The ratios of n-3/n-6 PUFA in all dried Artemia
samples were in the ranges of 1.4-1.6, 1.1-1.3 and 1.0 and the corresponding control values
were 1.7, 1.3 and 1.2 (Table 2). This indicates that both solar and sun drying methods did
not affect this ratio. Our results are in accordance with the finding of Sampels et al. (2004)
who reported that the ratio n-6/n-3 in reindeer meat was not influenced by the drying
method. A similar result was detected by Yanar et al. (2007) who found that baking,
grilling and microwave cooking did not change the ratio of n-3/n-6 fatty acids compared
with the raw fillets of sea bass. Paleari et al. (2003) also proved that the ratio n-6/n-3 of
smoked and dried meat was not affected by processing methods.
In the present investigation, for all cases the fatty acid contents in the sun-dried products
were significantly lower than in the fresh sample (P<0.01) except for the total SFA. When
compared with the solar drying methods, significant differences were observed in most
cases for drying on sunny days and days with sunny intervals, except for ΣSFA, LIA and
ARA while when drying on cloudy/rainy days all FA parameters between the two drying
methods were similar, which could be partly ascribed to the negligible difference in drying
time (28 vs. 30 h for solar and sun drying, respectively).The drying time of a product would
depend on the characteristics of the product as both too high and too low drying rates may
spoil the product. On the other hand, for highly perishable products it may be necessary to
dry them in a shorter time (Hughes and Willenberg, 1994; George et al., 2004)
Their explanation is corroborated by the results obtained in this study which indicated that
the decrease in UFA proportion was more pronounced in the dried samples with longer
drying time. On the contrary, the higher retention of FA of solar-dried samples in the
current study might be explained by the shorter exposure time and the protection from UVradiation during solar drying on sunny days.
Similar losses of other nutrients and sensory values have been reported in literature when
sun drying various products. Bala et al. (2001, 2003) reported that fish and pineapple dried
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Chapter 4
in a solar tunnel drier was of higher quality than the sun-dried product. Sablani et al. (2003)
found that the protein and fat of dried sardines were slightly higher in a convective solar
cabinet drier (56.82% and 9.42%) than in an open rack drier (49.12% and 8.52%).
Moreover, the quality of prawn is significantly lower in case of open sun drying, due to
over-drying, insufficient drying, contamination by foreign materials, insects and microorganisms, and discolouring by UV-radiation. Hence, open sun-dried prawn products
usually do not fulfil nutritional quality standards (Tiwari et al., 2006). Moreover, higher
losses of β-carotene, ascorbic acid and chlorophyll in open sun drying as compared to solar
cabinet drying have been reported for leafy vegetables (Negi et al., 2000).
In summary, all analyzed parameters of solar-dried Artemia differed less from fresh
Artemia than in sun-dried samples. Moreover, both for solar and open sun drying a longer
drying time resulted in lower values of total lipids, polar lipids and fatty acids.
In coastal areas in Vietnam several hundreds of hectares of salt ponds are in operation for
Artemia cyst production; they produce large amounts of live Artemia biomass as a byproduct which is not utilized (Brands et al., 1995; Hoa et al., 2007). The solar drier
described in this study can be easily and cheaply constructed using locally available
materials and is technically suitable for drying of Artemia or other products at household
scale. This may help the Artemia producers to valorise Artemia biomass to be used as a
feed or ingredient in the formulated feeds for shrimp, fish, livestock and poultry. This
integrated production may contribute to the profitability of Artemia farmers’ operations and
therefore have a positive impact on their socio-economic status in this area.
Conclusions
The study showed that the use of our solar drier prototype led to considerable reduction in
drying time in comparison with open sun drying when conducted on sunny days and days
with sunny intervals. In particular when sunny weather is predominant, the quality of solardried Artemia was better than the sun-dried sample. Nonetheless, there was a significant
reduction in lipid quality in solar-dried Artemia. The drying time and product temperatures
were highly influenced mainly by the weather parameters: temperature and solar exposure.
We also observed that total lipid and fatty acid levels were higher in solar-dried Artemia
than in open sun-dried Artemia. Additionally, for lipid classes, the sun-dried sample
showed a higher increase of free fatty acid content and higher reduction in polar lipids
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Chapter 4
which exhibited more lipid degradation as compared to the solar-dried sample. Therefore,
solar drying is a cheap and promising method, which could produce better quality dried
product within a shorter drying time.
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Chapter 4
140
CHAPTER
Application of Artemia biomass for some target
aquaculture species
Chapter 5
Chapter 5
Section I
Formulated feeds containing fresh or dried Artemia
biomass as live food supplement for larval rearing of
black tiger shrimp, Penaeus monodon
Chapter 5
Chapter 5
Abstract
In this study, supplementation of microalgae and Artemia nauplii with practical formulated
feeds containing fresh or dried Artemia biomass for larval rearing of black tiger shrimp
Penaeus monodon was assessed. Five feeding treatments were carried out in a recirculating
seawater system with fifteen 30-L composite tanks. Shrimp nauplii were stocked at a
density of 150 L-1. Each dietary treatment was performed in triplicate in a completely
randomized design for 23 days. In the control treatment, live feed was supplemented with
commercial formulated feeds (Inve Aquaculture NV, Belgium). In two other treatments,
live feed was supplemented with a pelleted feed based on either fresh or dried Artemia. In
the remaining two treatments live feed was supplemented with a combination of 50%
commercial feed and 50% fresh or dried Artemia feeds. The formulated feeds were applied
from the zoea 2 stage onwards. Under the experimental conditions, the time of
metamorphosis of the shrimp larvae in the different stages was the same in all treatments
and survival of the shrimp in all stages showed no statistical differences (P>0.05).
However, growth performance (length and dry weight) and stress tolerance were
significantly lower in PL supplemented with fresh or dried Artemia diets as compared to the
PL receiving only commercial feeds or the ones fed a combination of both. Overall,
performance of PL in the combination treatments (commercial feed and Artemia diets) were
better or equal compared to those fed commercial feed alone as seen by the better growth
rate and higher resistance to formalin stress. The results of the present study indicate that
feed containing fresh or dried Artemia can partially supplement live feeds for larval rearing
of P. monodon. However, the formulation of practical diets should be further improved to
meet the nutritional requirements of P. monodon larvae and postlarvae. This may support
the development of less expensive feeds for black tiger shrimp production, which may
increase profitability.
Key words: Formulated feeds, Artemia biomass, live feed supplement, Penaeus monodon,
larval rearing.
1. Introduction
Shrimp continues to be the largest single commodity in value terms, accounting for 17% of
the total value of internationally-traded fishery products in 2006. The black tiger shrimp
Penaeus monodon is the most prominent farmed crustacean product in the global market,
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and has driven a significant expansion in aquaculture in many developing countries in Asia
(FAO, 2008).
In Vietnam, Penaeus monodon is a highly valuable species and its culture has been
practised for more than 15 years (Quynh, 1995). As a consequence, the demand for
postlarvae (PL) for growout culture is high and most hatcheries have concentrated
production on this species. However, success in larviculture of penaeid shrimp is highly
dependent on the availability of well-balanced, nutritionally complete and cost-effective
feeds (Wickins and Lee, 2002), and the cost of feed is a major component of the cost of
larviculture of shrimp (Lawrence and Lee, 1997). During the natural planktonic existence
of penaeid larvae, live phytoplankton and zooplankton are the most important components
of the diet. In practical culture, best results are achieved with live feed such as microalgae
and Artemia nauplii. These are however very costly (Liao, 1992; Wickins and Lee, 2002;
Robinson et al., 2005). Especially the high price of Artemia cysts has increased the shrimp
production cost, and cheaper alternative diets with comparable nutritional quality are
needed to maintain the cost competitiveness of shrimp in the local market. Consequently,
this has triggered research on development of artificial microdiets to reduce dependence on
live feeds (Teshima et al., 2000; Wouters and Sorgeloos, 2005). Several research efforts
have been directed towards the development of microparticulate and microencapsulated
larval feeds during the past ten years (Kumlu et al., 1994; Jones, 1998) and today a wide
range of proprietary non-living larvae diets exist (Holme et al., 2006; Martin et al., 2006).
Microencapsulated penaeid larval feeds such as LANSY-Shrimp and FRIPPAK PL FEEDS
manufactured by INVE Aquaculture NV (Belgium) seem to be among the most successful
with about 60% of the world shrimp hatcheries using these products as supplements to live
feeds (Fegan, 1992; Wikenfeld, 1992). Also in Vietnam they are among the most widely
used supplemental feeds (Thanh et al., 2002).
Artemia juveniles and adults are known to have a better nutritional value than cysts. They
have higher protein content and are richer in essential amino acids (Sorgeloos et al., 1998;
2001; Lim et al., 2001, Dhont and Sorgeloos, 2002). The use of Artemia biomass for
nursery feeding of shrimp and marine fish was found to increase cost-effectiveness of
culture as expenses for cysts can be reduced (Dhert et al., 1993; Sorgeloos et al., 2001).
Live biomass has also been demonstrated to be an excellent maturation diet for penaeid
shrimp (Naessens et al., 1997, Wouters et al., 1999) and is also very popular as live food for
ornamental fish in the tropical aquarium industry (Lim et al., 2001). Although the fresh-live
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Chapter 5
form of biomass has the highest nutritive value, harvested Artemia biomass can also be
used directly as a feed either frozen or dried (Léger et al., 1986; Brands et al., 1995; Dhont
and Sorgeloos, 2002). From this it is clear that among the many available animal
ingredients, Artemia biomass has good potential as ingredient for shrimp larval diets. The
main source of Artemia biomass in southern Vietnam is however in remote areas, which
makes transport of live biomass very expensive. A technique for processing or drying
Artemia biomass on site could reduce these costs and open new possibilities to fully exploit
the potential of the Artemia biomass available in these regions (Brands et al., 1995; Hoa et
al., 2007). Some researchers demonstrated that dried Artemia biomass meal could be used
as an ingredient in post larval shrimp feeds (Abelin et al., 1989; Naegel and RodríguezAstudillo, 2004).
The objective of this study was to evaluate the effect of formulated feeds containing fresh
or dried Artemia biomass as live food supplement on the survival, growth and stress
resistance of Penaeus monodon larvae in order to develop a local application of Artemia
biomass in the region.
2. Material and methods
2.1. Experimental diets
Commercial feeds used for penaeid shrimp larval rearing (LANSY-Shrimp ZM, FRIPPAK
Fresh #1CAR and FRIPPAK Ultra PL+150; INVE Aquaculture NV, Belgium) were
purchased from a retail trader in Can Tho city.
Fresh Artemia biomass was obtained from the experimental Artemia biomass ponds in Vinh
Hau station of Can Tho University in Bac Lieu province, Vietnam. Dried Artemia meal was
obtained by using a combined microwave and hot air drying system at a drying temperature
of 50°C. Other ingredients such as soybean meal (soybean protein concentrate), squid oil,
gelatin, wheat flour... were purchased from commercial suppliers. The dietary ingredients
were analyzed for their chemical composition (Table 1) prior to the formulation of the diets.
Experimental diets were formulated to be approximately isonitrogenous and isolipidic with
the commercial feeds: LANSY-Shrimp ZM, FRIPPAK Fresh #1CAR and FRIPPAK Ultra
PL+150, respectively. This way 6 test diets containing either fresh or dried Artemia
biomass as the main protein source with protein levels of 50.6%, 53.4% and 45.8% and
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Chapter 5
corresponding pellet sizes of 63, 125 and 150 µm, in function of the developmental stage of
the shrimp were formulated (Table 2). The “SOLVER” program in Microsoft Excel was
used to formulate the experimental feeds. The pellets were produced using a pelletmachine, oven-dried at 60°C, ground and sieved to the desired particle sizes and stored at
4°C for later use.
Table 1. Proximate composition (% dry matter) of the ingredients used in the experimental
diets
Fresh Artemia
Dried Artemia
meal
Soy protein
concentrate
Wheat flour
Dry matter
11.75
89.97
90.14
89.23
Crude protein
55.62
54.88
82.22
11.04
Crude lipid
12.41
11.57
0.93
1.52
Ash
19.17
21.45
6.09
1.85
0.62
1.10
2.30
1.25
Ingredients
Crude fibre
Table 2. Composition (% of dry matter) of the experimental diets
Diet
Ingredients
Grade 1
Grade 2
Grade 3
FA
DA
FA
DA
FA
DA
0
65.03
0
67.10
0
59.05
Frozen Artemia
64.54
0
66.50
0
58.50
0
Soybean meal
15.51
15.64
16.93
16.91
12.42
13.02
Wheat flour
12.83
12.14
10.05
9.43
17.47
17.93
Lecithin
1.00
1.00
1.00
1.00
1.00
1.00
Squid oil
0.58
0.62
0.30
0.32
1.75
1.56
Vitamin premix
2.00
2.00
2.00
2.00
2.00
2.00
Gelatin
3.00
3.00
3.00
3.00
3.00
3.00
Cellulose
0.57
0.56
0.21
0.23
3.86
2.44
Dried Artemia
FA: frozen Artemia, DA: dried Artemia meal
(1) Vitamin premix (VEMEVIT) was produced by Rhone-Poulenc. In 1 kg of mixture, there are Vit. A:
2.000.000 UI; Vit. D3: 400.000 UI; Vit. E: 12.000 mg, Vit. K: 480 mg; Vit. B1: 800 mg; Vit. B2:
800 mg; Vit. B6: 500 mg, Nicotinic acid: 5.000 mg, Calcium: 2.000 mg, Vit. B12: 2.000 mg, Foli
acid: 160 mg; Microvit. H: 2000:1.000 mg; Vit.C: 100.000 mg; Fe++: 1.000 ppm, Zn++: 3.000 ppm;
Mn++: 2.000 ppm, Cu++: 100 ppm, Iodine: 20 ppm, Co++: 10 ppm and some others.
(2)
Gross energy was calculated based on protein = 5.56; lipid = 9.54 and NFE = 4.20 Kcal g-1
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Chapter 5
2.2. Chemical analysis
Proximate analysis (moisture, crude protein, total lipid, fibre and ash) of the ingredients and
experimental diets were carried out according to the standard methods of AOAC (1995).
Nitrogen-free extract (NFE) was estimated on a dry weight basis by subtracting the
percentages of crude protein, lipids, crude fibre and ash from 100%.
Table 3a. Proximate composition (% of dry matter) of the experimental diets
Diet: Grade 1
Diet: Grade 2
Diet: Grade 3
(particle size: 63µm)
(particle size:125µm) (particle size:125µm)
FA
DA
FA
DA
FA
DA
Dry matter
91.25
90.57
90.78
91.16
90.82
91.13
Crude Protein
50.65
50.54
53.37
53.44
45.86
45.68
9.64
9.70
9.69
9.65
9.97
9.77
NFE
17.19
17.44
13.93
13.66
20.62
20.78
Ash
19.38
18.65
19.74
19.95
19.53
19.62
Crude Fibre
3.14
3.67
3.26
3.29
4.02
4.14
Energy (Kcal g-1)
4.49
4.50
4.52
4.51
4.40
4.38
Crude Lipid
FA: fresh Artemia, DA: dried Artemia meal
Table 3b. Proximate composition of the commercial feeds (information provided by
manufacturer)
Moisture
LANSY-Shrimp ZM
FRIPPAK Fresh #1CAR
FRIPPAK Ultra PL+150
(particle size: 63µm)
(particle size:125µm)
(particle size:125µm)
Max. 8
Max. 10
Max.9
Crude protein
Min. 48
Min. 52
Min. 42
Crude lipid
Min. 13
Min. 14.5
Min. 7
Max. 2.5
Max. 3.0
Max. 2.5
Fibre
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Chapter 5
2.3. Experimental design
A feeding trial was conducted for 23 days in the shrimp hatchery of the College of
Aquaculture and Fisheries, Can Tho University, Vietnam. The test was set up as a
completely randomized design with 3 replicates per treatment in a recirculating system. The
methods for larval rearing of P. monodon in this experiment overall followed the methods
described by Thanh et al. (2002).
The live feed was in the different treatments supplemented with formulated feeds as
follows:
-
Commercial feed (Inve Aquaculture NV) used as control (CF)
-
Formulated feed based on fresh Artemia biomass (FA)
-
Formulated feed based on dried Artemia meal (DA)
-
50% CF + 50% FA
-
50% CF+ 50% DA
Healthy and pathogen-free (PCR-negative for WSSV) nauplii of P. monodon from one
single batch were purchased from a commercial shrimp hatchery in Can Tho city, Vietnam
for the experiment.
At the starting of the experiment, the composite 30-L tanks were filled with 35 g L-1 treated
seawater to 60% of their capacity and were then progressively filled up each day. Before
stocking, nauplii were disinfected with 150 ppm formalin solution for 10 min and
transferred to the culture tanks at a density of 150 nauplii L-1. Each tank was provided with
continuous aeration. Uneaten feed and fecal matter were siphoned out before the first
feeding in the morning. A reciculation system was applied to the culture units from day 8 of
stocking onwards (when shrimp larvae were no longer fed algae).
2.4. Feeding protocol
Nauplii stages were not fed. Once the nauplii metamorphosed into zoea, feeding with the
diatom Chaetoceros gracillis commenced, and lasted to PL1. From the zoea 2 to the mysis
stage, formulated feed was added along with the diatoms. From mysis stage onwards,
larvae were fed Artemia nauplii in addition to the formulated feeds. For the formulated
feeds, initial particle size was 63 and it was increased to 125-150 µm as the animals grew in
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Chapter 5
size. Feeding was done every 3 hours based on the standard feeding schedule presented in
Table 4 and 5. Live feed rations were equal for all treatments.
Table 4. Standard feeding schedule for 1m3 culture of larvae and postlarvae of Penaeus
monodon. Feed amounts are presented both as concentration and as percentage of diet dry
weight (Thanh et al., 2002).
Formulated feed (g m-3 day-1)
Stages
Z1
Z2
Z3
M1
M2
M3
PL1
PL2
PL3
PL4
PL5
PL6
PL7
PL8
PL9
PL10
PL11
PL12
PL13
PL14
PL15
Chaetoceros
(1000 cell ml -1)
Artemia nauplii
(Ind. ml -1 day-1)
Grade 1
Grade 2
Grade 3
Total
(Amount)
(Amount)
(Amount)
(Amount)
(Amount)
(%DW)
(%DW)
67
100.00
84
129
125
67
40
20
84.55
81.90
64.62
44.34
28.60
12.19
(%DW)
1.4
2.6
0.6
1.0
1.5
2.0
3.0
3.0
3.5
4.0
4.5
4.5
5.0
5.0
5.5
6.0
6.5
7.0
7.5
8.0
15.45
18.10
28.33
40.62
47.03
60.12
5.0
5.6
6.0
9.0
9.0
9.0
8.0
6.0
5.0
7.05
15.04
24.37
27.70
29.31
23.72
26.62
24.91
26.41
25.68
26.33
25.06
25.68
26.22
26.70
27.64
28.50
29.31
6.0
11.0
12.0
19.0
21.0
27.0
29.0
31.0
33.0
35.0
37.0
38.0
39.0
40.0
70.69
76.28
73.38
75.09
73.59
74.32
73.67
74.94
74.32
73.78
73.30
72.36
71.50
70.69
Table 5. Feeding frequency of live and formulated feeds for larval and postlarval shrimp
over a 24- hour period (Thanh et al., 2002)
Time of day
Stage
6:00
9:00
12:00
15:00
18:00
21:00
24:00
3:00
Z1
A
A
A
A
A
A
A
A
Z2
A, FF
A
A
A
A, FF
A
A
A
Z3
A, FF
A
A
A
A, FF
A
A
A
M1
A, Art
A, FF
A, FF
A, FF
A, Art
A, FF
A, FF
A, FF
M2
A, Art
FF
FF
FF
A, Art
FF
FF
FF
M3
FF, Art
FF
FF
FF
FF, Art
FF
FF
FF
PL1-PL15
FF, Art
FF
FF
FF
FF, Art
FF
FF
FF
A: Algae, Art: Artemia nauplii, FF: Formulated feed
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Chapter 5
2.5. Data collection
Daily water temperature and pH were measured at 8:00 and 14:00 using a thermo-pH meter
(YSI 60 Model pH meter). Total ammonia nitrogen (NH3/NH4+), NO2-N and NO3-N were
monitored every two days using a spectrophotometer according to APHA (1998).
2.6. Performance parameters
Development stage of the shrimp was determined on a daily basis for all treatments. 30
animals were selected randomly, and their larval development stage was determined using a
dissection microscope.
Survival was determined at Zoae 3 (Z 3), Mysis 3 (M 3) and Postlarval 15 (PL 15) stages.
For Zoea 3 a Mysis 3 stages a 1000 ml-beaker was used to collect larvae randomly at three
different places in each culture tank, and then all animals per sample were counted. At the
end of the experimental period, all shrimps in each tank were counted. These data were
compared to the initial stocking density, which allowed the calculation of the survival of
shrimp in the culture tanks. At the same time, 30 animals in each tank were randomly
collected to determine the total length. Total length of animals was measured from the tip
of the rostrum to the tip of the telson by means of a dissection microscope, equipped with a
camera lucida. For dry weight estimation, 100 animals were taken randomly from each
rearing tank, washed with tape water, and then dried at 60°C in an oven for 24 hours. Dried
animals were group weighed on a balance with a precision of 0.0001 g and individual
weight was determined based on the total number of animals.
The quality of the postlarvae was assessed by studying the resistance of the postlarvae to
formalin shock, following the methods of Samocha et al. (1998) and modified by Thanh et
al. (2002). Thirty postlarvae from each tank were exposed to 150-ppm formalin solution in
a 1 L-plastic basin for 60 minutes. The same temperature and salinity as in the culture
medium was maintained and gentle aeration was supplied. Dead animals were monitored at
10 minute intervals.
The Cumulative Mortality Index (CMI) was calculated by summing the mortality counts
noted at each time interval. CMI= Nx1 + Nx2 + Nx3 + ... Nx6
Where N is the number of dead individuals at time x1, x2, x3...x6. The higher the numeric
value of the index, the less the postlarvae are resistant to stress and vice versa.
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Chapter 5
2.7. Statistical analysis
Data of survival were normalized through a square root arcsine transformation before
statistical treatment. Results were analyzed with one-way ANOVA (SPSS, version 13.0)
analysis of variance to find the overall effect of the treatment. Tukey’s HSD test was used
to identify significant differences between the dietary treatment means at a significance
level of p<0.05.
3. Results
3.1. Water quality
Water temperature fluctuated between 28.2 and 30.2 oC and pH values were between 7.6
and 7.8. Variations in TAN, NO2-N and NO3-N concentrations during the culture period
were in the ranges of 0.01-0.92, 0.01-0.26 and 0.80-17.58 mg L-1, respectively. Generally,
the water quality was not much different among treatments.
3.2. Shrimp performance
The different supplemental feeds did not affect larval development of the P. monodon
larvae as the time of metamorphosis in the different stages was the same in all treatments
(Table 6). The survival of the shrimp declined with developmental stage and was in the
range of 93-94%, 88-89% and 61-68% for zoea 3, Mysis 3 and PL 15 stages, respectively.
There were no significant differences among treatments (Figure 1).
Table 6. Time of metamorphosis (hours) in shrimp larvae fed different formulated
supplemental feeds.
Stages
CF
FA
DA
CF+FA
CF+DA
Nauplius
48
48
48
48
48
Zoea
96
96
96
96
96
Mysis
72
72
72
72
72
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Chapter 5
CF
FA
DA
CF+FA
CF+DA
100
Survival (%)
80
60
40
20
0
ZOEA3
MYSIS3
Developmental stages
PL15
Figure 1. Survival of shrimp fed different formulated supplemental feeds.
Statistical analysis did not reveal any effect of the dietary treatments on total length of the
animals (Table 7) up to the Mysis 3 stage. At PL15 stage, larvae in the groups
supplemented with commercial feeds alone (CF) or with a combination of commercial feed
and Artemia based diets (CF+FA and CF+DA) showed a significantly longer length than
the groups supplemented with only fresh (FA) or dried Artemia (DA) based diets (P<0.05).
When comparing the effect of the different supplemental feeds on dry weight of PL15,
again no significant differences were found between the CF and combination treatments.
However, the CF group had a significantly higher value than the group fed dried Artemia
supplemental feed (P<0.05).
Table 7. Total length (mm) and dry weight (mg) of shrimp fed different formulated
supplemental feeds.
Diet
CF
FA
DA
CF+FA
CF+DA
Zoea3
3.44±0.17a
3.46±0.17a
3.45±0.11a
3.46±0.11a
3.45±0.11a
Mysis3
4.64±0.10a
4.63±0.09a
4.62±0.10a
4.66±0.09a
4.65±0.09a
13.12±0.64a
13.02±0.82a 13.78±0.70bc 13.57±0.81bc
Total length
Postlarvae15 13.32±0.70b
Dry weight
of PL15
12.97±0.49bc 11.48±1.48ab 11.09±1.49a 13.38±1.00c
13.01±1.12c
Values in the same row sharing the same letter are not significantly different (P>0.05).
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Chapter 5
Cumulative mortality index
5
4
3
2
1
0
CF
FA
DA
CF+FA
CF+DA
Supplemental feeds
Figure 2. Cumulative mortality index (CMI) of P. monodon postlarva (PL 15) fed different
supplemental feeds exposed to 150 ppm formalin solution for 60 min.
The cumulative mortality index of the shrimp subjected to formalin stress was shown in
Figure 2. The results indicated that the stress index in the PL fed only the supplemental
feed containing fresh or dried Artemia biomass was significantly higher than in the animals
receiving commercial feed (control) or a combination of both (P<0.05). Furthermore, visual
observation indicated that mortalities in the former two treatments also occurred earlier
than in the other three treatments. This means that PL in these treatments were less tolerant
to formalin stress than in the other groups. Although PL in the CF+FA treatment had a
slightly lowest stress index as compared to the CF+DA group, this was not significantly
different (P>0.05). Shrimp in the FA group overall showed a better performance than those
fed the dried Artemia based supplemental diet (DA group), however this difference was not
statistically significant (P>0.05).
4. Discussion
4.1. Water patameters
Water quality is more critical during larval culture than at any other stage in the life cycle
of crustaceans (Winckins and Lee, 2002). Therefore, in this experiment water quality
parameters in the rearing tanks were kept stable and within the limits recommended for
shrimp larval culture. This should minimize the stress to the animals so that only dietary
effects on shrimp performance can be expected. The levels of TAN (NH3/NH4+), NO2-N
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Chapter 5
and NO3-N recorded in this study were within the tolerance range for penaeid larvae (Chen
et al., 1990; Liao, 1992; Winckins and Lee, 2002).
4.2. Gowth and survival
Appropriate larval nutrition ensures proper growth, survival and development in a given
species. Effective and nutritionally complete microparticulate diets are essential to produce
consistent quality postlarvae with high survival (Fedroza-Islas et al., 2004; Robinson et al.,
2005). Our results showed that shrimp in all treatments had the same time of
metamorphosis. Although survival at PL15 stage fed the diets containing fresh or dried
Artemia biomass were slightly lower than those fed the commercial feed or a combination
of both commercial and Artemia based diets, significant differences were not found among
the dietary treatments. This indicates that there were no negative effects in utilization of
nutrients by larvae induced by Artemia diets. Moreover these experimental diets were
readily accepted and consumed by the larvae as visually observed during the experiment.
These results confirm the studies of Abelin et al. (1989) and Naegel and RodríguezAstudillo (2004) that Artemia biomass meal can be successfully included in practical
penaeid shrimp diets.
Postlarval size (length, dry or wet weight) is a direct indicator of growth, and thus several
nutritional studies have successfully used this criterion to evaluate diet quality (Coutteau et
al., 1996; Wouters et al., 1997). Increased growth and reduced variability in size during
post larval stages has in turn been related to further growth to juvenile stages (Castille et al.,
1993). The feeds containing fresh or dried Artemia biomass had a similar protein content as
the commercial feed and thus similar performance may be expected if the requirements of
the larvae are met. However, a lower growth performance (length and dry weight) was
observed in the groups supplemented with only Artemia biomass based diets. This could be
due to differences in the quality of the proteins or lipids. Millamena et al. (1996) found that
the requirement of P. monodon postlarvae for methionine was 0.89% of the diet or 2.4% of
protein. In a diet containing 0.41% cystine, the total sulfur amino acid requirement
(methionine + cystine) would be 1.3% of the diet or 3.5% of protein. Although Artemia
biomass is considered a good animal protein with regard to amino acid profile, it may have
insufficient methionine, cystein and threonine (Sorgeloos, 1980b; Léger et al., 1986;
Evjemo, 2001) for meeting the essential amino acid requirement of P. monodon PL,
resulting in slower growth. The deficit of nutritional components in dried Artemia biomass
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Chapter 5
may be even more pronounced due to loss of nutrients caused by the drying process.
Garcia-Ortega et al. (2000) found that heat treatment of encapsulated cysts increased
protein denaturation and decreased protein solubility. Similarly it was found that total
unsaturated fatty acids in dried reindeer meat were lower compared with fresh meat
(Sampels et al., 2004). Furthermore, it has been documented by several authors that the two
most important essential fatty acids, 20:5 (n-3) (eicosapentaenoic acid or EPA) and 22:6
(n-3) (docosahexaenoic acid or DHA) are either absent or only present in low levels in
Artemia biomass (Sorgeloos, 1980b; Dhert et al., 1993; Evjemo, 2001; Lim et al., 2001). In
practice, Artemia biomass used in the present experiment showed a low level of DHA (see
Section I chapter 4). Therefore, it seems likely that fatty acid imbalances in the Artemia
biomass diets is at least partially responsible for the inferior performance of the larvae in
these treatments as compared to the control and combination groups.
Besides evaluating the effect of different diets on growth performance, stress tests are
usually applied to access quality whenever subjective criteria fail to differentiate batches,
thus providing the farmer with an additional tool for PL quality assessment prior to
stocking. Moreover, the test can be a valuable tool for rapid decision making for PL
treatment or price setting, and the stress test can be used as a more flexible tool for
diagnosing the larval quality and to formulate appropriate diets. Many different criteria
have been suggested for the evaluation of PL quality such as salinity stress tests (Dhert et
al., 1992a; Immanuel et al., 2004), ammonia stress tests (Cavalli et al., 2001) and formalin
stress tests (Samocha et al., 1998; Thanh et al., 2002). According to Thanh et al. (2002), the
method of measuring resistance to formalin shock has proven to be a good tool for quality
evaluation of penaeid postlarvae and can easily be conducted in hatcheries. It is the most
popular technique applied in Vietnam because it is a simple, cheap method and it only
requires a short time to deliver results. Using these techniques, a significantly improved
physiological condition of P. monodon was shown when feeding suitable diets (Immanuel
et al., 2004). In our studies, the effect of the dietary treatments on formalin stress resistance
displayed the same pattern as for growth performance, where PL supplemented with a
combination of commercial feed and Artemia based feed performed best. In contrast, the
animals fed the dried Artemia based diet supplement displayed a lower formalin tolerance
probably because the nutritional value of this diet did not satisfy the requirements of the
shrimp PL as mentioned above. These results are in agreement with the studies of Dhert et
al. (1992a); Cavalli et al. (2001) and Immanuel et al. (2004), who found that animals fed
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Chapter 5
nutritionally complete diets show a higher tolerance to stress conditions. In the current
experiment, the overall performance in terms of survival, growth rate and stress tolerance of
P. monodon postlarvae fed a combination of commercial feed and Artemia based diets as
live feed supplement was better than either diet alone.
From the above, it is clear that there still is significant potential to improve the
experimental Artemia diets through improvements in the essential nutrient content of the
diets. For example, it could be interesting to supplement the diets with essential amino
acids such as methionin which may have been present at levels below the larval
requirements (Millamena et al., 1996). It was also reported that the amino acid pattern of
the protozoeal, juvenile and adult stage of P. monodon showed increasing arginine and
decreasing phenylalanine levels with growth stage (Peñaflorida, 1989). Other result showed
that the essential amino acid index (EAAI) could be used in screening potential protein
sources. However, when formulating diets, EAAI should be supported with feeding trials
and digestibility tests to determine the extent of incorporation of these protein sources in
Penaeus monodon diets. Moreover, also the EPA and DHA levels in the diets warrant
further research. Their growth promoting capacity is enhanced when an optimal balance of
both fatty acids are incorporated into the diet (Glencross and Smith, 2001; Tandler and
Koven, 2005). Besides, poorer performance of shrimp PL fed Artemia based diets
compared to animals fed commercial feed could be associated to different processing
techniques between experimental diets and commercial feed. Probably the commercial diet
is produced through the high technology of microencapsulation, which is high water
stability while the experimental diets are microparticulate diets with artisanal method, this
feed could be low water stability resulting in fast leaching of nutrients. According to Um
and Cuzon (1994), pellet water stability is an important parameter in the manufacture of
aquaculture diets especially for shrimp. Furthermore, the quality of shrimp diets is
determined not only by their nutritional make-up but also by their physical properties,
especially water stability. Pellet that break up into small particles and quickly leach nutrient
could reduce water quality of the culture medium and lead to poor animal growth,
inefficient feed conversion. Hence, shrimp pellets should be physically stable to minimize
disintegration and loss of water-soluble nutrients upon exposure to water, and during the
ingestion process (Obaldo et al., 2002; Dominy et al., 2003).
In summary, survival, growth performance and stress resistance of P. monodon larvae fed a
supplemental diet containing Artemia biomass seemed to be inferior to a commercial feed.
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Chapter 5
However, co-supplement of commercial feed with Artemia based diets gave the best results
among treatments. The results indicate that it is possible to substitute a live diet with
combination of 50% of the commercial feed and 50% a practical Artemia based diet
without negative effects on survival, growth and stress resistance in P. monodon
larviculture.
Economic aspects, more interesting to mention that Vietnam has a great potential of
Artemia biomass in the dry season at the coastal area (Brands et al., 1995; Hoa et al., 2007).
If a simple and cheap drying technique could be developed, Artemia biomass could be a
good and cheap protein source for formulated aquafeeds. This may stimulate producers to
use it for larvae and nursery culture to reduce the production costs, and to become less
dependent on expensive live foods.
Conclusion
From our results, it can be concluded that Penaeus monodon fed a combination of
commercial and experimental Artemia biomass based supplemental diets showed the best
growth and survival and stress resistance during the hatchery phase. In contrast, larvae
supplemented with only diets containing Artemia biomass displayed poorer performance
than the ones only receiving the commercial feed or a combination of both.
The practical Artemia based diets tested in this experiment can however successfully
replace 50% of the commercial feed used as live feed supplement in shrimp larviculture.
These practical Artemia based feeds most probably can be further improved by inclusion of
some essential amino acids and fatty acid supplements in order to meet the nutritional
requirements of Penaeus monodon larvae.
155
Chapter 5
156
Chapter 5
Chapter 5
Section II
Effect of fishmeal replacement with Artemia biomass as
protein source in practical diets for the giant freshwater
prawn Macrobrachium rosenbergii
Nguyen Thi Ngoc Anh1,2, Tran Thi Thanh Hien2, Wille Mathieu1, Nguyen Van Hoa2
and Patrick Sorgeloos1
1
Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience
Engineering, Ghent University, Belgium
2
College of Aquaculture and Fisheries, Cantho University, Vietnam
Paper published in Journal of Aquaculture Research 40, 669-680
Chapter 5
Chapter 5
Abstract
A 30-day feeding experiment was conducted in 160-L plastic tanks to evaluate the potential
use of Artemia biomass as protein source in practical diets for postlarval Macrobrachium
rosenbergii (initial-mean weight of 12.12-12.29 mg). Nine isoenergetic and isonitrogenous
experimental diets (approximately 40% crude protein) were formulated by replacing levels
of the fishmeal protein difference either with dried or frozen Artemia (0, 25, 50, 75 and
100%). The 0% Artemia treatment, in which Peruvian fish meal was the only main protein
source, was considered as the control diet. The results showed that prawn postlarvae fed the
fishmeal control diet had a lower survival (46%) compared to all Artemia diets. Significant
differences (p<0.05) were however only found at 75% and 100% Artemia protein inclusion
level (survival of 68-77%). A gradual increase in growth performance (live weight gain,
specific growth rate and total length) of the prawns was achieved with increasing dietary
inclusion of Artemia protein. Additionally, the size distribution exhibited the same response
as growth performance. However, prawns fed the frozen Artemia diets showed a better
performance than the ones fed the dried Artemia diets. It can be suggested that Artemia
biomass may totally replace fishmeal in practical diets for postlarvae of the freshwater
prawn Macrobrachium rosenbergii.
Keywords: Artemia biomass, fishmeal, growth, survival, size distribution, Macrobrachium
rosenbergii
1. Introduction
Presently, in Vietnam the aquaculture sector has been developing in terms of the culture
area, production, number of species and degree of management intensity (Edwards et al.,
2004). For example, the total aquatic production increased almost 7% in 2006, while
aquaculture production increased 14.6% (Huong and Quan, 2007). According to the
Ministry of Agriculture and Rural Development, Vietnam, the availability of fishmeal (FM)
is low and the price of imported FM from 2007 to the first six month of 2008 increased
significantly (1.1-1.4 USD/kg). Most feed manufactures are using expensive imported FM
as a protein source for aquafeeds (www.fistenet.gov.vn/details.asp?News). Therefore,
assessment of cheaper or more readily available alternative protein sources such as byproducts from fisheries, processing or other sources that may reduce the use of FM in feeds
is necessary.
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Chapter 5
Among the alternative animal protein sources, Artemia biomass (adult Artemia) may be
considered as a suitable ingredient for replacing fishmeal in fish and crustacean diets
because of its high nutritional value (Sorgeloos, 1980; Léger, 1986; Bengtson et al., 1991;
Lim et al., 2001; Maldonado-Montiel and Rodríguez-Canché, 2005). Frozen adult Artemia
included in a mixed diet improved reproductive performance of Penaeus vannamei
(Naessens et al., 1997; Wouters et al., 2001). In postlarval stages of penaeid shrimp and
clawed lobster, Artemia has been shown to provide excellent nutrition (Wickins and Lee,
2002; Tlusty et al., 2005b). Live adult Artemia is also an ideal food for ornamental fish
(Lim et al., 2001). According to Naegel and Rodriguez-Astudillo (2004), dried Artemia is a
well-suited feed for postlarval Litopenaeus vannamei.
Vietnam offers a potential source of Artemia biomass as a by-product derived from the
commercial cyst-oriented Artemia pond production systems which yield an average
Artemia biomass production of 0.2-0.3 ton ww ha-1 (Brands et al., 1995; Anh et al., 1997).
However, so far, only small amounts of this by-product have been utilized for production
purposes. Therefore, the use of Artemia biomass as protein source in feeds may contribute
to profitability of Artemia farmers and have a positive impact on the socio-economic
aspects in the coastal area. Thus, the potential of this product should be evaluated further.
There has been a very rapid global expansion of freshwater prawn farming since 1995. The
total global production of Macrobrachium is estimated at 750000-1000000 tons/year by the
end of this decade; most is produced in Asia, in which China is the leader, followed by
Vietnam and India (New, 2005). Currently, the giant freshwater prawn (Macrobrachium
rosenbergii), which is indigenous to the Mekong Delta, Vietnam, is becoming an
increasingly important target species for aquaculture. According to Sinh (2008), in 2006,
the total culture area of M. rosenbergii in the Mekong Delta was 9077 ha and production
arose to 9514 tons, with 111 hatcheries growing postlarvae (production: 107 million PL)
and about 300 millions of PL imported. Macrobrachium responds very well to extensive
and semi-intensive culture systems, and its culture, especially in rice fields, is considered to
have potential to raise income among impoverished farmers and contribute to enhance rural
development in Vietnam (Hai et al., 2003; Phuong et al., 2006). Because of the recent
serious disease outbreaks in tiger shrimp (Penaeus monodon), prawn culture has been
considered as an alternative to shrimp in low saline water areas and coastal saline soils as
they can grow comfortably in waters having a salinity up to 10 g L-1 (New, 2002; Cheng et
al., 2003). Increase in prawn culture has led to a growing demand for PLs from hatcheries.
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Chapter 5
Prawn farmers prefer to stock PL older than 10 days to ensure high survival rate in their
grow-out ponds. However, feed is the single largest cost item for Macrobrachium
rosenbergii culture, as it constitutes 40-60% of the operational costs (Phuong et al., 2003b;
Mitra et al., 2005). Moreover, the formulation of well-balanced diets and their adequate
feeding are of utmost importance for successful aquaculture (Watanabe, 2002). The
nutritive value of formulated feeds depends on the ingredient composition and considerably
affects the prawn performance especially in the indoor-nursery phase, where the prawns
rely solely on supplemented feed. Hence, development of formulated feeds to attain higher
survival, better growth and more efficient feed conversion ratios is needed. The main goal
of the study reported here was to evaluate the use of Artemia biomass as a high-quality
protein source to improve postlarval diets for M. rosenbergii.
2. Materials and Methods
2.1. Experimental diets
Peruvian FM was supplied by CATACO Company, Can Tho city; dried and frozen Artemia
biomass were obtained from the experimental Artemia biomass ponds in the experimental
station of Can Tho University in Bac Lieu province, Vietnam. Dried Artemia meal was
obtained by sun-drying thin layers of Artemia biomass. Other ingredients such as soybean
meal, soybean oil, squid oil, gelatine, wheat flour... were purchased from commercial
suppliers. The dietary ingredients were analyzed for chemical composition (Table 1) prior
to the formulation of the diets.
Nine experimental diets were formulated by replacing 0%, 25%, 50%, 75% and 100% of
the FM protein in a standard diet with either dried or frozen Artemia biomass (Table 2). In
the 0% Artemia treatment, Peruvian FM was the main protein source of mixed ingredients.
All diets were formulated to be approximately isolipidic, isoenergetic and isonitrogenous
(40% dietary protein). The ‘SOLVER’ program in Microsoft Excel was used to establish
the formulated feeds. In this program the proximate composition of the ingredients and
those of the diets are preset, in which the proportion of FM protein substituted by Artemia
protein must be precise. Based on the composition of fish meal and Artemia meal it is
therefore possible that other ingredients (e.g. amount of wheat flour, soybean oil and squid
oil) vary as well in order to keep the gross composition of the resulting diets as similar as
possible. The diets were made into sinking pellets (700µm) using a pellet machine, ovendried at 60°C and stored at 4°C.
159
Chapter 5
Table 1. Proximate composition (% of dry matter) of the ingredients used in the
experimental diets
Ingredients
Peruvian
fishmeal
Dried
Artemia meal
Frozen
Artemia
Soybean
meal
Wheat
flour
Dry matter
91.38
86.57
12.44
89.19
88.78
Crude protein
59.20
50.76
50.89
47.60
11.62
5.62
9.89
9.93
3.17
1.92
33.29
24.97
23.98
7.38
0.71
0.62
2.50
2.48
6.99
1.25
Crude lipid
Ash
Crude fiber
Table 2. Composition (g 100 g-1 dry matter) of the nine experimental diets
Ingredients
0% A
25%
DA
25%
FA
50%
DA
50%
FA
75%
DA
75%
FA
100%
DA
100%
FA
Fish meal
49.35
37.00
37.03
24.66
24.69
12.33
12.35
0.00
0.00
Dried Artemia
0.00
14.39
0.00
28.77
0.00
43.14
0.00
57.55
0.00
Frozen Artemia
0.00
0.00
14.36
0.00
28.73
0.00
43.11
0.00
57.40
Soybean meal
12.34
12.58
12.85
13.36
13.35
13.87
13.86
14.34
14.35
Wheat flour
25.08
23.03
22.89
20.98
20.71
18.94
18.52
16.90
16.86
Soybean oil
1.68
1.33
1.33
0.97
0.97
0.62
0.62
0.27
0.26
Squid oil
1.67
1.32
1.32
0.97
0.97
0.62
0.61
0.26
0.26
Lecithin
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
Vitamin Premix
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
Gelatin
2.50
2.50
2.50
2.50
2.50
2.50
2.50
2.50
2.50
Cellulose
2.38
2.58
2.73
2.78
3.08
2.98
3.43
3.18
3.37
A: Artemia, DA: dried Artemia, FA: frozen Artemia
(3) Vitamin produced by Rhone-Poulenc, namely VEMEVIT. In 1 kg of mixture, there are Vit. A:
2.000.000 UI; Vit. D3: 400.000 UI; Vit. E: 12.000 mg, Vit. K: 480 mg; Vit. B1: 800 mg; Vit. B2:
800 mg; Vit. B6: 500 mg, Nicotinic acid: 5.000 mg, Calcium: 2.000 mg, Vit. B12: 2.000 mg, Foli
acid: 160 mg; Microvit. H: 2000:1.000 mg; Vit.C: 100.000 mg; Fe++: 1.000 ppm, Zn++: 3.000 ppm;
Mn++: 2.000 ppm, Cu++: 100 ppm, Iodine: 20 ppm, Co++: 10 ppm and some others.
(4)
Gross energy was calculated based on protein = 5.65; lipid = 9.45 and NFE = 4.20 (kgcal g-1)
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Chapter 5
2.2. Chemical analysis
Proximate analysis (moisture, crude protein, total lipid, fibre and ash) of the ingredients and
experimental diets were carried out according to the standard methods of AOAC (1995).
Nitrogen-free extract (NFE) was estimated on a dry weight basis by subtracting the
percentages of crude protein, lipids, crude fiber and ash from 100%.
Table 3. Chemical compositions (% of dry matter) of the nine experimental diets
Ingredients
0% A
25%
DA
25%
FA
50%
DA
50%
FA
75%
DA
75%
FA
100%
DA
100%
FA
Dry matter
91.91
92.76
90.01
92.07
91.25
90.72
92.61
89.62
92.75
Crude Protein
39.95
39.87
39.64
39.69
39.79
39.78
39.81
39.91
39.94
Crude Lipid
7.47
7.93
7.95
8.03
7.96
7.99
8.10
8.78
8.15
NFE
29.19
28.40
27.63
26.43
26.96
27.12
27.52
26.74
27.96
Ash
20.57
20.69
21.49
21.77
22.04
21.09
21.74
20.73
21.42
Crude Fiber
2.82
3.11
3.29
4.08
3.25
4.02
2.83
3.84
2.53
Energy (Kcal/g)
4.19
4.20
4.15
4.11
4.13
4.14
4.17
4.21
4.20
The amino acid content of the test diets were analyzed by liquid chromatographic analysis
(HP, Hewlett-Packard AOS - 1090M, Germany) using conductivity of pre-column OPA
(O-phtal aldehyde) and FMOC (9-fluorenylmethyl chloroformate).
The fatty acid composition of the experimental diets was determined by gas
chromatography. Fatty acid Methyl esters (FAMEs) were prepared via a procedure
modified from Lepage and Roy (1984).
2.3. Experimental design
A feeding trial was conducted for 30 days in the shrimp hatchery of the College of
Aquaculture and Fisheries, Can Tho University, Vietnam. The test was set up as a
completely randomized design with 3 replicates per treatment.
The plastic 160-L tanks were filled with 120 L of de-chlorinated tapwater. Each tank was
provided with continuous aeration and black plastic nets were distributed throughout the
water column as substrate for PL prawns in order to reduce the opportunity for cannibalism.
Postlarvae from one single batch were purchased from a commercial hatchery in Can Tho
city and reared in 1m3 tank for 5 days before the start of the trial.
At the start of the experiment, all PL were deprived of food for 1 day. 120 PL (mean initial
weight of 12.16-12.29 mg) were transferred into each tank. 50% of the tank volume was
161
Chapter 5
exchanged every 3 days at 14:00 hour. Daily water temperature and pH were measured at
7:00 and 14:00 hrs using a thermo-pH meter (YSI 60 Model pH meter). Total ammonia
nitrogen (NH3/NH4+), NO2-N and NO3-N were monitored weekly using a spectrophotometer
according to APHA (1998).
Table 4. Amino acid composition of the experimental diets (% of dry matter)
Amino acid
0%
A
25%
DA
25%
FA
50%
DA
50%
FA
75%
DA
75%
FA
100%
DA
100%
FA
Essential amino acid
Arginine
1.83
1.67
1.71
1.89
1.96
2.07
2.10
2.11
2.18
Histidine
0.75
0.70
0.72
0.74
0.74
0.82
0.85
0.85
0.84
Isoleucine
0.95
1.03
1.08
1.02
1.08
1.20
1.14
1.21
1.26
Leucine
1.69
1.64
1.85
1.74
1.93
1.93
1.96
2.01
2.08
Lysine
1.43
1.39
1.53
1.42
1.58
1.37
1.68
1.46
1.65
Methionine
0.66
0.59
0.61
0.62
0.65
0.65
0.69
0.67
0.70
Phenylalanine
0.96
1.06
1.12
1.05
1.12
1.17
1.16
1.22
1.16
Threonine
0.98
1.04
1.11
1.11
1.24
1.26
1.26
1.28
1.27
Valine
1.15
1.22
1.21
1.12
1.13
1.34
1.25
1.32
1.33
Non- essential amino acid
Alanine
1.64
1.74
1.83
1.78
1.80
1.83
1.93
1.88
1.95
Aspartic
2.07
1.99
2.09
2.21
2.20
2.08
2.51
2.46
2.53
Glutamic
4.37
3.81
4.23
4.01
4.30
4.18
4.53
4.34
4.46
Glycine
1.28
1.69
1.73
1.45
1.69
1.79
1.67
1.73
1.72
Proline
1.35
1.45
1.40
1.59
1.60
1.71
1.87
1.74
1.95
Serine
1.26
1.35
1.38
1.38
1.42
1.46
1.52
1.67
1.67
Tyrosine
0.92
0.94
1.06
1.02
0.91
0.98
1.02
1.18
1.19
Prawns were fed four times a day at about 8:00, 12:00, 16:00 and 20:00 hrs at 15% of their
body weight. Uneaten feed and faecal matter were siphoned out before the first feeding in
the morning. Thirty prawns in each tank were randomly sampled at the start of the
experiment and at 10 day intervals during the experimental period. The amount of diet
given was adjusted according to these weight measurements. Prawns were weighed in
groups of 30 on a digital Mettler scale (Denver Instrument Co.) and mean weights were
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Chapter 5
determined. Prawns were then returned to their original tanks. At the end of the experiment,
the survival, mean individual weight and total length of the prawn were determined.
Table 5. Fatty acid profile (mg g-1 dry weight) of the experimental diets containing
different levels of dried or frozen Artemia protein
Experimental diets
Fatty
acids
0%
A
25%
DA
25%
FA
18:2n-6
13.17a
14.55b
18:3n-3
1.30a
20:4n-6
50%
DA
50%
FA
75%
DA
75%
FA
100%
DA
100%
FA
14.88bc 15.56cd 15.51cd 16.03de 16.32ef
16.69ef
16.93f
1.49b
1.56bc
1.59c
1.62c
1.73d
1.76d
1.87e
1.94e
0.56a
1.29b
1.26b
1.92c
1.94c
2.45d
2.67d
3.22e
4.43f
20:5n-3
5.99a
7.67b
8.24c
9.27d
9.46d
10.56e
10.91f
11.61g
12.14h
22:6n-3
2.74a
2.23b
2.14b
1.53c
1.58c
0.93d
1.00d
0.34e
0.43e
Total
SFA
26.54a
25.73ab 25.46ab 26.01ab 24.36bc 23.48bc 23.24cd
22.79cd 22.24d
Total
MUFA
19.20a
21.10b
21.68b
22.54c
24.15d
23.86d
25.34e
25.84e
27.47f
Total
MPUA
12.01a
14.43b
15.37b
16.91c
17.76c
19.08d
20.28e
20.99e
23.74f
Total n-3
11.19a
12.78b
13.67bc 14.23cd 14.96de 15.58ef
16.45f
16.52f
19.93g
Total n-6
13.99a
16.21b
16.58b
18.24c
18.31c
19.54d
20.15d
21.16e
22.74f
n-6/n-3
ration
1.25a
1.27a
1.21a
1.28a
1.22a
1.25a
1.22a
1.28a
1.27a
Data are mean values (±SD) of two determinations. Means in the same row with different
superscripts are significantly different (P<0.05). ARA: Arachidonic acid; EPA:
Eicosapentaenoic acid; DHA: Docosahexaenoic acid; SFA: saturated fatty acids; MUFA:
monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids.
2.4. Performance parameters
Performance was evaluated by live weight gain, specific growth rate (SGR), total length
and survival, which were calculated as follows:
Live weight gain (mg) = final weight - initial weight
SGR (% day-1) = 100 x [(ln final weight - ln initial weight)/days of experiment]
Survival (%) = 100 x (final prawn number/initial prawn number)
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Chapter 5
The total length (mm) of prawns was measured from the tip of the rostrum to the telson
with a calibrated ruler.
2.5. Statistical analysis
Data for all measured parameters were analysed using SPSS for Windows, Version 11.0.
Variations with dietary treatment were compared by one-way ANOVA. The Tukey HSD
post hoc analysis was used to detect differences between means. Significant differences
were considered at P<0.05. All percentage values were normalized through a square root
arcsine transformation prior to statistical treatment.
3. Results
3.1. Water quality
Daily water temperatures ranged from 27.5 to 30.5°C and pH fluctuated from 7.2 to 7.8;
variation in levels of total ammonia nitrogen, NO2-N and NO3-N were 0.02 - 0.61, 0.02 0.32 and 0.08 - 0.19 mg L-1, respectively, throughout the experimental period. Generally,
the water quality was not much different between treatments and remained within the
suitable range for the normal growth of M. rosenbergii PL (Niu et al., 2003; D’Abramo et
al., 2003).
3.2. Experimental diets
Proximate analysis showed that all diets used in this study were nearly isonitrogenous,
isolipidic and isocaloric (Table 3). However, a slightly lower lipid content in the FM basedcontrol diet compared to the 100% Artemia-based diets was noted.
The analysed amino acid concentrations in the experimental diets (Table 4) indicated that
the diets containing Artemia protein had for all amino acids equal or slightly higher levels
than the control diet. In general, these differences among the diets were negligible.
The fatty acid profiles of the experimental diets are shown in Table 5. Concentrations of
Linoleic (18:2n-6), Linolenic (18:3n-3), EPA (20:5n-3) and ARA (20:4n-6), total
monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-3 and n-6
fatty acids in the Artemia diets were significantly (p<0.05) higher than in the FM control
diet. The ratio of n-6/n-3 was similar among the treatments. In contrast, total saturated fatty
acid in the FM control diet was higher as compared to the Artemia diets. Especially,
docosahexaenoic acid (DHA) (22:6n-3) was also six to eight times higher in the control diet
than in the frozen and dried Artemia diets. Overall, the levels of unsaturated fatty acids
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Chapter 5
increased with inclusion of Artemia protein in the diets. In addition, amino acid and fatty
acid levels in the dried Artemia diets were slightly lower in comparison with the frozen
Artemia diets.
3.3. Prawn performance
The effect of the dietary FM replacement level on the survival and growth parameters of the
freshwater prawn M. rosenbergii is presented in Table 6. Prawn fed the FM control diet had
the lowest survival (46%). However, a significant difference (p<0.05) was only found at the
75% and 100% replacement level (68-77%). The polynomial regression (Figure 1) and Rsquare value (R2=0.9507) indicated that there was a positive correlation between the
survival of the prawn and the dietary Artemia protein inclusion level. After 30 days of
feeding, growth performance of the prawns was noticeably affected by the treatments
(Table 6). Overall prawn PL in all experimental groups grew at rates that were consistent
with values routinely obtained in our research facility.
Table 6. Survival, live weight gain, specific growth rate and total length of PL
Macrobrachium rosenbergii after a 30 day feeding trial with different Artemia protein
inclusion level
Treatment
Survival
(%)
Initial weight
(mg)
Final weight
(mg)
Live weight gain
(mg )
SGR
(% day-1)
Total length
(mm)
45.83±6.29a
12.16±0.15
84.58±23.93
72.41±5.70a
6.46± 0.25a
21.17±3.34a
25% DA
59.17±5.20ab
12.34±0.20
102.32±31.05
89.98±8.50ab
7.04±0.33ab
23.83±3.42b
25% FA
59.72±3.37ab
12.22±0.16
111.43±29.44
99.21±4.63ab
7.37±0.71b
24.51±3.41b
50% DA
61.67±3.33ab
12.25±0.15
117.04±26.91
104.79±4.20b
7.52±0.09b
25.38±3.64bc
50% FA
62.50±3.82abc
12.29±0.38
157.26±31.13
144.97±9.96
c
8.49±0.29c
26.47±3.58cd
75% DA
67.78±5.42bc
12.21±0.23
147.39±35.49
135.18±6.98
c
8.30±0.20c
26.67±3.60cd
75% FA
68.33±5.20bc
12.30±0.52
163.37±34.40
151.07±8.92c
8.62±0.20c
28.06±3.70d
100% DA
75.00±3.82bc
12.24±0.30
174.29±31.58
162.06±17.62 c
8.84±0.41cd
28.08±3.51d
100% FA
77.22±7.47c
12.23±0.47
211.33±36.08
199.10±12.97 d
9.50±0.27d
30.67±4.09e
0% A
Means with different superscripts in the same column are significantly different (P<0.05);
SGR: specific growth rate.
165
Chapter 5
A gradual increase in the growth performance (live weight gain, specific growth rate and
total length) of the prawn occurred with increasing dietary inclusion of Artemia protein.
Differences with the control however only became significant from the 50% replacement
level upwards. As for survival, there was a similar significant positive correlation between
the specific growth rate of the prawns and the dietary Artemia level. R2 values were 0.9580
and 0.9952 for frozen and dried Artemia, respectively (Figure 2). At the same level of
Artemia inclusion in the diet, no significant difference was observed between the dried and
frozen Artemia form. However, prawn fed frozen Artemia diets overall showed a better
performance than the ones fed dried Artemia diets.
90
y = -0.0008x2 + 0.3649x + 47.452
R2 = 0.9572
80
Survival (%)
70
60
Frozen Artemia
Dried Artemia
y = -0.001x2 + 0.3662x + 47.27
R2 = 0.9611
50
40
30
0
25
50
75
100
Artemia protein inclusion level (%)
Figure 1. The relationship between survival and dietary Artemia protein inclusion level in
Macrobrachium rosenbergii at the end of the 30 day feeding trial.
10
9
SGR (%/day)
Frozen Artemia
y = -0.0001x2 + 0.0414x + 6.4701
R2 = 0.9723
Dried Artemia
8
7
y = 2E-05x2 + 0.0216x + 6.4597
R2 = 0.9961
6
5
0
25
50
75
100
Artemia protein inclusion level (%)
Figure 2. The relationship between specific growth rate and dietary Artemia protein
inclusion level in Macrobrachium rosenbergii at the end of the 30 day feeding trial.
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Chapter 5
100%
80%
>0.25 g
0,21- 0,25 g
60%
0.16-0,20 g
0.10-0.15 g
40%
<0.10 g
20%
0%
A
FA
FA
FA
DA %FA
DA
DA
DA
%
%
%
0% 5%
%
%
0
0%
75
25
50
0
75
2
50
10
1
Treatment
Figure 3. Size distribution of Macrobrachium rosenbergii after 30 days fed diets with
different Artemia inclusion level based on weight categories
The size distribution based on weight categories of prawn PL revealed the same trend as for
growth performance (Figure 3). The proportion of prawns in the 0.21-0.25 g category was
higher in the treatments fed the diets with 50, 75 and 100% Artemia protein inclusion (15.631.1%) than animals fed FM control and 25% Artemia diet groups (2.2-7.8%). In addition,
the proportion of the large size class (>0.25 g) in the groups fed diets with 50% Artemia
inclusion and upwards ranged from 11.1-27.8% while this size was not found in the FM
control and 25% Artemia replacement diet groups. On the contrary, the proportion of the
small size class (<0.1 g) was highest (66.7%) in the FM control group and this size class
was not present in the treatment with a complete FM substitution.
4. Discussion
The result of the present study indicated that growth performance of a prawn M.
rosenbergii PL fed an FM control diet was inferior to prawn fed Artemia diets. Hari and
Kurup (2003) obtained the highest SGR (3.72 % day-1) of M. rosenbergii (0.264 g initial
weight) fed with diets containing 30% protein and varying amounts of trash fish meal and
groundnut oilcake. Du and Niu (2003), when evaluating dietary substitution of soya bean
meal for FM on growth of prawn (initial weight of 0.32 g), achieved the best SGR of 2.5 %
day-1 in the FM control diet. A study by Gitte and Indulkar (2005) evaluated the use of five
different marine fish meal diets (40% protein) for M. rosenbergii PL (initial weight of 10.8
mg) and found the SGR of prawn ranged from 4.82-5.86 % day-1.
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Chapter 5
Furthermore, Marques et al. (2000) studied stocking densities for a nursery phase culture of
M. rosenbergii PL in cages at densities between 2 and 10 PL L-1. The initial weight of the
prawns was 0.011 g. Prawns were fed with a 35% protein commercial pellet. After 20 days
of culture, survival and final mean weight ranged from 63-95% and 0.042-0.061 g (SGR:
6.70-8.65 % day-1), respectively. Hossain and Islam (2007) found that PLs of M.
rosenbergii (initial weight of 26 mg) raised in a recirculatory system for 60 days and fed a
commercial shrimp nursery diet (30% protein) achieved survival of 76%, a final weight of
502.33 mg and a SGR of 3.28 % day-1. A study by Phuong et al. (2003b) investigated the
nursing of M. rosenbergii PL in 400-1200 m² earthen ponds with stocking densities of 50
and 100 PL m-2. The initial weight was 6.8 mg. A commercial feed (Grobest company)
containing 33% crude protein was used. After 45 days, the survival of the prawns varied
around 76-77% and final weight was 0.33-0.68 g (SGR: 8.63-10.23% day-1).
From these results, it can be suggested that the SGR of M. rosenbergii PL fed the FM
control diet in the current study (6.46 % day-1) was comparable or better than the prawn fed
a fishmeal-based diet in other studies. Moreover, prawn fed the Artemia diets demonstrated
a similar range of survival (59-77%) and a SGR (7.04-9.50 day-1) that was equal or superior
than prawn fed commercial feeds in the studies reported above.
Polynomial regression analysis indicated that when the dietary Artemia protein inclusion
level increased from 0 to 100%, the survival and growth rate of prawn PL increased
significantly (P<0.05) and the best survival and growth performances occurred at 100%
replacement of FM protein by Artemia protein. The high survival and good growth
performance of prawn fed Artemia diets as compared with an FM control diet may be due
to an increased feed intake caused by a better palatability of the Artemia diets. Cook et al.
(2003) compared the transfer of erythromycin from bioencapsulated live and freeze-dried
adult Artemia and pellets to fry of sockeye salmon, Oncorhynchus nerka (Walbaum), and
found that both Artemia feeds are so palatable that they were more immediately and
completely consumed by the fish relative to the medicated pellets.
From observation of the feeding behaviour in the present study, it was observed that prawn
receiving diets containing 50% Artemia protein and more showed a faster response to the
feed pellets than animals in the FM control diet. Therefore, although feed intake was not
determined in this study, the Artemia diets are apparently more attractive to the prawns than
the FM control diet. This may result in better feed satiation of the prawns and hence high
survival (lower cannibalism) and good growth performance.
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Chapter 5
Previous studies demonstrated that the amino acid requirements of freshwater prawn are
relatively constant during larval life and can be satisfied by suitable protein sources that
resemble the larval amino acid profile (Roustaian et al., 2000). Moreover, M. rosenbergii
require the same ten essential amino acids as other crustacean and fish species, but
quantitative requirements have not been well established (D’Abramo, 1998; Mitra et al.,
2005). In the present study, all experimental diets had nearly the same protein content and
similar amino acid levels. It is therefore unlikely that this explains the different response of
the prawn PL to the diet.
Fatty acids play important roles in the nutrition of crustaceans. Their main functions are as
a source of energy and for the maintenance of functional integrity of biomembranes (Reigh
and Stickney, 1989). The highly unsaturated fatty acid (HUFA) content of artificial diets
has been found to have a strong impact on survival, growth, feed conversion, fecundity, egg
hatchability, moulting and stress tolerance of Penaeus and Macrobrachium species.
Particularly 18:2n-6 (linoleic acid), 18:3n-3 (linolenic acid), 20:5n-3 (eicosapentaenoic
acid, EPA) and 22:6n-3 (docoxahexaenoic acid, DHA) fatty acids are essential to increase
growth and survival of larvae and juveniles of these genera (Querijero et al., 1997;
D’Abramo, 1998; Roustaian et al., 1999; Mitra et al., 2005). For M. rosenbergii, the dietary
requirements for HUFA are relatively small: both n-3 and n-6 HUFA at dietary levels of
0.075% were found to increase remarkably the weight gain and feed efficiency of prawn
(Mitra et al., 2005). A similar observation by Teshima et al. (1992) found the highest
weight gain in M. rosenbergii fed diets containing a mixture of 18:3n-3 and 18: 2n-6. On
the contrary, M. rosenbergii did not show any significant increase in weight gain with
supplements of either 18:3n-3 or 18:2n-6 dietary fatty acids; however, their absence was
associated with significant reductions in the midgut somatic index (D’Abramo and Sheen
1993). Moreover, González-Baró and Pollero (1998) reported that prawns are unable to
synthesize either 20:4n-6 (arachidonic acid) or 20:5n-3 from shorter chain fatty acids and
these fatty acids must be supplied by the diet.
From a nutritional viewpoint in terms of fatty acids, most levels of important fatty acids
such as n-3, n-6 PUFA in the diets containing 50% or more Artemia protein were
significantly higher than those in the FM control diets which may have contributed to the
better survival and growth of PLs in this experiment.
The beneficial effect of Artemia protein inclusion level in practical diets on growth
performance of the prawn was consistent with the results of size distribution. Higher
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Chapter 5
proportions of prawns in the 0.21-0.25 g and >0.25 g categories were found mainly in the
diets with 50, 75 and 100% Artemia protein inclusion. The proportion of small (<0.1 g) size
classes was highest (66.7%) in the FM-control group and this category was not present in
the treatment with a complete FM substitution. Size variation in prawns is an important
factor affecting economic efficiency of nursing practice. Ra’anan and Cohen (1985) pointed
out that Macrobrachium rosenbergii express perceptible individual variation in growth
rates and the wide variation in growth rate is believed to be induced primarily by intrinsic
factors associated with social hierarchy rather than by environmental factors. Starting with
a cohort of PLs with a normal size distribution, the population at the time of harvest is
highly heterogeneous. However, a combination of batch-graded and size-graded postlarvae
can reduce heterogeneous individual growth in prawns (Ranjeet and Kurup, 2002). Size
grading of juveniles from a nursery-grown population before stocking into production
ponds is an effective method to increase mean individual weight and total yield at harvest
(D’Abramo et al., 2003; Tidwell et al., 2004).
Generally, the levels of amino acids and fatty acids in the dried Artemia diet were slightly
lower than in the frozen Artemia diet. This is probably due to the loss of nutrients during
the direct sun drying process. Similar results have been reported in the literature for sun
drying of various fruits and vegetables. According to Jayaraman and Gupta (1995) sun
drying cause increased loss of ascorbic acid. This may be due to the long drying time (1824 h) and the large surface area or open pore structure of the dried products that facilitates
oxidation of ascorbic acid. Considerable loss of ascorbic acid content in sun-dried aonla
fruit, and sun-dried tomatoes when compared with fresh samples were detected by Pragati,
et al., (2003); Latapi and Barrett (2006), respectively. In addition, Negi and Roy (2000)
found that sun drying resulted in high losses of β-carotene and ascorbic acid of leafy
vegetables, and the β-carotene content of sun-dried apricots was significantly lower than in
the original materials (Karabulut et al., 2007). In this study, besides the lower content of
amino acids and fatty acids in dried Artemia diet, other nutrients may also be less than that
of the frozen Artemia diet. This could explain why at the same replacement level, overall a
poorer performance was observed for prawns fed the dried Artemia diets.
The results of this study are in agreement with those of Abelin et al. (1989), who reported
that PL30-45 of Penaeus monodon and PL15 of P. vannamei fed a diet containing freezedried Artemia meal had a significantly better growth as compared to the FM based diet.
Naegel and Rodriguez-Astudillo (2004) demonstrated that feeding Litopenaeus vannamei
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Chapter 5
shrimp PL with dried Artemia biomass resulted in a significant higher survival and larger
size compared to four commercial feeds and three crustacean meals. Moreover, juvenile
American lobsters (Homarus americanus) fed enriched frozen adult Artemia showed the
greatest weight gain (>6% day-1) compared to formulated diets (Tlusty et al., 2005a).
Naessens et al. (1997) found an enhanced reproductive performance when using frozen
Artemia as a dietary supplement for P. vannamei. Similar to the above studies, our
experiment again demonstrates the potential of Artemia biomass as a diet ingredient in
aquaculture.
Overall, the formulated diets containing Artemia biomass ensured that the essential nutrient
requirements for M. rosenbergii PL were met and resulted in excellent growth performance
and a high survival. Among the experimental diets, total replacement of fishmeal protein
with frozen Artemia protein seems to be the best for prawn PL. On the other hand, more
work is needed to test other target species and to optimise the use of Artemia biomass as a
promising alternative feed that could bring about important socio-economic aspects. The
interesting issue is that in coastal areas in Vietnam, about 0.2-0.3 ton ha-1 of live Artemia
biomass, a by-product from commercial cyst-oriented Artemia production in solar saltpans,
can be harvested when the culture season ends (Brands et al., 1995; Anh et al., 1997a).
According to Hoa et al. (2007), the production area in the 2006 culture season was
approximately 350 ha, and thus more than 100 tonnes of Artemia biomass could be
obtained from these regions. Until now, this product has been not efficiently utilized.
Therefore, use of Artemia biomass as protein source for feeding aquatic species or other
animals may not only help the Artemia producers to improve cost-effectiveness of their
operations but may also contribute to reduce the use of fishmeal in aquafeeds. Although a
full economic comparison between the costs of FM and Artemia biomass in practical diets
for the M. rosenbergii PL was beyond the scope of this study, some considerations can be
made. In Vietnam labour cost is cheap and sun-drying or solar drying of Artemia biomass
are very inexpensive methods. In the present study, Artemia biomass was collected from
experimental ponds hence it is difficult to estimate its cost. It can be assumed Artemia
biomass; a by-product was used, even when labour and processing costs are accounted for
would still be cheaper or at most similar in price as imported FM (1.1-1.4 USD kg-1). The
results of this study moreover demonstrated that survival of prawn fed the Artemia diet was
about 1.5 times higher than prawn fed the FM diet; growth response was also better.
Therefore, the nursery phase can be shorter and more profitable.
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Chapter 5
172
Chapter 5
Chapter 5
Section III
Effect of different forms of Artemia biomass as a food
source on survival, molting and growth rate of mud crab
(Scylla paramamosain)
Chapter 5
Chapter 5
Abstract
An experiment was conducted to evaluate the effect of different forms of Artemia biomass
as a food source on survival, molting and growth rate of mud crab Scylla paramamosain.
Instar 1 crablets with a mean weight of 8.2±0.7 mg, were reared both individually and
communally, and fed different diets consisting of fresh shrimp meat (control feed), live
Artemia biomass, frozen Artemia biomass and a dried Artemia-based formulated feed for
40 days. The highest survival was obtained for crablets receiving live Artemia (92.5 and
75.8%) followed by the groups fed frozen biomass (90.0 and 47.5%), the control feed (72.5
and 24.2%) and the dried Artemia-based diet (60.0 and 21.7%) for individual and
communal cultures, respectively. The intermolt period, the total number of moltings and the
growth rate, which were determined on individually reared crabs, showed the same pattern
as for survival. The results suggest that crab performance decreased in the order live
Artemia>frozen Artemia>fresh shrimp meat>dried Artemia-based formulated feed. Live
Artemia biomass proved an ideal feed for nursery of Scylla paramamosain crabs. Frozen
Artemia biomass may be an alternative in times of shortage. Our findings illustrate the high
potential for local utilization of Artemia biomass in Vietnam for reliable production of mud
crab juveniles.
Keywords: Scylla paramamosain, Artemia biomass, shrimp meat, survival, growth,
individual and communal culture
1. Introduction
Artemia adults are believed to have a better nutritional value than freshly hatched nauplii.
They have higher protein content and are richer in essential amino acids (Léger et al., 1986;
Merchie, 1996; Lim et al., 2001). Live adult Artemia are an excellent feed for culturing fish
fry (Dhert et al., 1993; Kim et al., 1996), for ornamental fish (Lim et al., 2001; 2003) and
postlarval crustaceans (Conklin, 1995; Nair et al., 1995; Wickins and Lee 2002; Ut et al.,
2007a). Lobster farming relies on adult Artemia, and the best results are obtained using live
Artemia, compared with frozen or freeze-dried (Léger et al., 1986; Conklin, 1995; Tlusty et
al., 2005a,b). In addition, frozen adult Artemia have been used as a supplement or
replacement in maturation diets for various penaeid shrimp (Browdy et al., 1989; Nair et
al., 1995; Naessens et al., 1997). It is also possible to boost the Artemia with nutritional
supplements, which has been shown to improve the growth and reproductive performance
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Chapter 5
of respectively postlarvae and broodstock of penaeid shrimp (Sorgeloos et al., 1998; Lavens
and Sorgeloos, 2000a; Wouters et al., 2002). Also, dried Artemia is a well-suited feed for
postlarval Litopenaeus vannamei shrimp (Naegel and Rodriguez-Astudillo, 2004).
Several authors reported that indoor rearing of adult Artemia from cysts is time consuming
and labour intensive. The least expensive source of adult Artemia is commercial harvest
from natural and man-controlled salt-pond systems (Baert et al., 1996; Dhont, and
Sorgeloos, 2002). In the coastal area of the Mekong delta of Vietnam, seasonal culture of
Artemia cysts in solar saltworks has been successfully practiced for a long time (Brands et
al., 1995; Hoa et al., 2007). Apart from cysts, large quantities of Artemia biomass (on
average 0.2-0.3 ha-1) can be collected when the production season ends. Moreover, these
saltworks are located near farming areas of brackish and marine species (shrimp, mud
crabs, goby, etc.), and therefore potentially provide an excellent opportunity for the
utilization of Artemia biomass in these aquaculture operations. Demonstration of its
successful use could support the valorisation of Artemia biomass in the region.
Mud crabs of the genus Scylla are commercially important in several Indo-Pacific countries
and they provide an important source of income as well as food for many coastal fishing
communities (Keenan, 1999). Mud crab farming is well established throughout Southeast
Asia (Allan and Fielder, 2003). Vietnam has a great natural potential for developing mud
crab culture: about 858,000 ha of marine and brackish water areas can be used for shrimp
and mud crab culture, of which more than 300,000 ha lay in the Mekong Delta (Lindner,
2005; Ut et al., 2007b). During the past decade, aquaculture has rapidly increased with an
ever increasing number of species being cultured. Mud crabs (Scylla spp.) are one of the
most promising, and are often seen as an alternative to shrimp, which are plagued with
disease outbreaks, since crab are more tolerant to environmental stress than shrimp, and
little processing is required for crab before export (Johnston and Keenan, 1999; Macintosh
et al., 2002; Christensen et al., 2004). Several studies investigated the potential of crab
farming. Crab production was found highest in monoculture and lowest in mangroveshrimp integrated culture with average yields of 1,008 kg ha-1 and 75 kg ha-1, respectively
(Ut et al., 2007b). Thach (2007) showed the productivity of intensive crab culture can
reach 1.5-2.0 tons ha-1. Recently, large scale hatchery technology for Scylla paramamosain
has been achieved in Vietnam (Allan and Fielder, 2003; Lindner, 2005) and nowadays,
most crab aquaculture production relies on commercial hatchery-reared stocks (Lindner,
2005; Thach, 2007). Nonetheless, hatchery-produced seed supplied to farmers is typically
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Chapter 5
in the form of small postlarval crabs with a carapace width between 4 and 8 mm, resulting
in low survival and productivity (Ut et al., 2007a). Hence, a nursery period as an
intermediate step in between hatchery and grow-out, to grow postlarvae to a size
appropriate for transport and release into large extensive to intensive production systems, is
considered necessary (Ut et al., 2007a; Rodriguez et al., 2007).
In practice, the low survival during the nursery phase of crab may be influenced by several
factors in which underfeeding or unsuitable feed resulting in high cannibalism is one of the
major problems (Sheen and Wu, 1999; Hartnoll, 2001; Holme et al., 2009). Since feed costs
account for 34-40% of the total production costs, it is extremely important that the diet
given sustains good growth and survival (Wickins and Lee 2002; Holme et al., 2009). As
mentioned earlier, in regions of Vietnam where crab farming is practised, live Artemia
biomass is seasonally available in large quantities, which could potentially be used for
intensive nursery culture. Therefore, the aim of the present study was to evaluate the effect
of different forms of Artemia biomass as a food source on survival, intermolt period and
growth of mud crab Scylla paramamosain crablets, with the overall objective to support the
utilization of Artemia biomass in local aquaculture operations and in this way contribute to
the socio-economic development of the region.
2. Materials and Methods
2.1. Experimental design
A 40-day feeding experiment was performed in the shrimp hatchery of the College of
Aquaculture and Fisheries, Can Tho University, Vietnam.
A completely randomized design was set up with the following four feeding treatments:
-
Fresh shrimp (Metapenaeus ensis) meat (control treatment)
-
Live Artemia biomass
-
Frozen Artemia biomass
-
Formulated feed with dried Artemia as main protein source (approximately
50% protein and 10% lipid)
Treatments were simultaneously run on individually and communally cultured animals. For
both systems, an open culture system with daily water exchange was used.
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Chapter 5
•
Individual culture: individual instar 1 crablets were kept in 0.6L transparent
perforated plastic jars, and the 40 replicates in each treatment were suspended in a
500-L round composite tank with a floor area of 0.7m². This set-up served to
monitor intermolt period and growth rate of individual crablets.
•
Communal culture: 40 instar 1 crablets were stocked in 500-L tanks
(approximately 57 crabs m-2), the same tank volume as for individual culture. Every
treatment had three replicate tanks.
2.2. Source of postlarvae
Hatchery-reared postlarval mud crabs from a single batch were obtained from the
experimental hatchery of the Department of Marine Aquaculture, Can Tho University.
Uniformly sized instar 1 crabs with mean initial body weight and carapace width (CW) of
7.9-8.2 mg and 2.7-2.8 mm respectively, were selected.
2.3. Rearing systems and culture conditions
The composite 500-L tanks were filled with 300 L water with a salinity of 20 g L-1. Each
tank was provided with continuous aeration. For communal culture, 10 standard clay
construction bricks (17 cm x7 cm x 10 cm) with four circular holes of 2.5 cm diameter
along the length of the brick were placed on the tank bottom as shelters, providing at least
one hole per crab. The tanks were kept in natural light and at ambient temperature. The
culture systems are shown in Figure 1.
Overview culture system
Individual culture
Communal culture
Figure 1. Culture systems for mud crab postlarvae
Crabs were fed to satiation twice a day at 07:00 and 18:00 h. Dead crabs and uneaten feed
were removed before every feeding. 50-60% of the water volume was exchanged every 2
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Chapter 5
days to maintain good water quality in all treatments. Salinity was maintained at 20±2 g L-1.
For individual culture, molting of each crab was checked prior to feeding. Post-molt crabs
were measured (carapace width (CW) and body weight) after their carapace hardened (1248 h after moult). For communal culture, a random selection of 40 crablets was sampled at
the start of the experiment. Each crab was measured and weighed to calculate the mean
initial CW and body weight. At 10 day intervals, 10 crabs were randomly sampled in each
tank to measure CW and body weight. Crabs were then returned to their original tanks. At
the termination of the experiment, survival, CW and body weight were determined. Before
weighing the crabs, excess water was removed with a tissue-paper and the wet weight
recorded with a 0.0001 g precision analytical balance.
Daily water temperature and pH were measured at 07:00 and 14:00 hours using a thermopH meter (YSI 60 Model pH meter, Hanna Instruments, Inc. Mauritius).
2.4. Experimental feeds
Fresh shrimp (Metapenaeus ensis) were purchased every day from the local market.
Artemia biomass was obtained from the experimental Artemia biomass ponds in the
Experimental Station of Can Tho University in Bac Lieu province, Vietnam. Live Artemia
biomass was transported from the culture site to the laboratory every two days. One batch
of frozen Artemia biomass was stored at -15°C for use during the entire feeding experiment
and dried Artemia meal was obtained by solar-drying thin layers of Artemia biomass, and
then incorporated with other ingredients (soybean meal, wheat flour, squid oil...) to make a
formulated feed. The diet ingredients were analyzed for chemical composition (Table 1)
prior to the formulation of the feed. The feed was made into sinking pellets (700-1000µm)
using a pellet machine, oven-dried at 60°C and stored at 4°C.
Table 1a. Proximate composition (% of dry matter) of the ingredients used in the Artemia
based formulated diet.
Ingredients
Dried Artemia meal
Soybean meal
Wheat flour
Dry matter
56.88
46.62
11.62
Crude protein
19.54
3.02
1.92
Crude lipid
12.17
32.18
84.50
Ash
19.32
10.42
0.71
1.09
7.76
1.25
Crude fiber
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Chapter 5
Table 1b. Composition (g 100 g-1 dry matter) of the Artemia based formulated diet.
Ingredients
Quantity
Dried Artemia meal
79.23
Soybean meal
5.90
Wheat flour
5.75
Squid oil
1.00
Lecithin
1.00
Vitamin premix
3.00
Gelatine
3.00
Cellulose
1.12
2.5. Chemical analysis
Proximate composition (dry matter, crude protein, total lipid, fibre and ash) of the
ingredients and experimental feed were analyzed according to the standard methods of
AOAC (1995). Nitrogen-free extract (NFE) was estimated on a dry weight basis by
subtracting the percentages of crude protein, lipids, crude fiber and ash from 100%.
Table 2. Proximate composition (% of dry matter) of the experimental diets
Proximate
compositions
Shrimp meat
Live Artemia
Frozen
Artemia
Dry matter
17.78±0.69
10.55±0.10
10.63±0.07
89.83±0.45
Crude protein
62.28±5.33
56.45±5.17
55.28±0.57
49.48±0.50
1.64±0.77
11.24±3.42
10.73±0.31
10.26±0.24
Ash
19.72±4.28
18.63±2.32
17.89±0.91
18.86±0.31
Fibre
2.23±0.39
1.51±0.61
1.64±0.35
4.11±0.27
NFE
14.15±5.68
12.17±5.86
14.46±1.59
17.28±0.16
Ca
1.86±1.35
2.59±2.43
2.54±0.20
2.85±0.20
P
1.19±1.27
1.58±1.13
1.41±0.10
1.39±0.07
Crude lipid
Dried Artemia
based feed
2.6. Crab performance
For individual culture, intermolt period, percentage molting, total number of moltings,
percentage weight and size increment (wet weight) and survival were calculated for each
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Chapter 5
molt. At the end of the experiment, survival, CW and body weight were also recorded to
calculate the specific growth rate.
For communal culture, survival and growth were determined every 10 days and at the
termination of the experiment.
Growth performance of the mud crabs was evaluated using the following parameters:
Specific growth rate (% day-1) in terms of weight (SGRW) and carapace width (SGRCW)
was calculated as follow:
SGRW = (ln final weight - ln initial weight)/days of culture x 100
SGRCW = (ln final carapace width – ln initial carapace width)/days of culture x 100
Percentage weight increment = (post-molt W– pre-molt W)/pre-molt W x100
Percentage size increment = (post-molt CW – pre-molt CW)/pre-molt CW x100
2.7. Statistical analysis
Data for all measured parameters were analysed using SPSS for Windows, Version 13.0.
Variations with dietary treatment were compared by one-way ANOVA. The Tukey HSD
post-hoc analysis was used to detect differences between means. Significant differences
were considered at P<0.05. All percentage values were normalized through a square root
arcsine transformation prior to statistical treatment.
3. Results
3.1. Water quality
Daily water temperature did slightly vary during the day, with morning and afternoon
temperatures of 29.1± 0.7 and 31.8 ± 0.9°C, respectively. Water pH remained within the
range of 8.1-8.5 and salinity in all tanks was relatively stable, ranging from 20 to 22 g L-1
during the course of rearing. These parameters are within the suitable range for normal
development of Scylla paramamosain postlarvae (Trinö and Rodriguez, 2002; Ruscoe et
al., 2004).
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Chapter 5
3.2. Experimental diets
Proximate analysis showed that the crude protein content was highest in shrimp meat
(62.3%), followed by live and frozen Artemia biomass (55.3-56.5%) and lowest in the dried
Artemia-based feed (49.5%). On the contrary, crude lipid in the three Artemia diets (10.311.2%) was about 6-7 times higher than in shrimp meat (1.6%). Calcium level of shrimp
meat (1.9%) was slightly lower compared to the Artemia diets (2.5-2.9%) while Phosphorus
concentration of all feeds was similar (Table 2).
3.3. Crab performance
The effect of the feeding treatments on survival of individually and communally reared
crablets is presented in Table 3. For the individual culture, at the end of the experiment, the
groups fed live and frozen Artemia biomass showed considerably higher survival (9092.5%) than those receiving the control shrimp meat (72.5%) and the dried Artemia-based
feed (60%) (Table 3a). Similar results were obtained for the communal culture: survival of
crablets was significantly higher in the live Artemia treatment compared to the three other
groups for all sampling days (P<0.05). Animals fed the fresh shrimp meat and driedArtemia formulated feed showed a similar survival (P>0.05) and significantly lower than
the group receiving frozen biomass (Table 3b). With the same feeding treatment, survival
of crabs in the individual culture was always higher than in the communal culture.
Table 3a. Survival (%) of crablets fed different diets in the individual culture
Molt
number
Shrimp meat
Live Artemia
Frozen Artemia
Dried Artemia
based feed
First
100
100
100
100
Second
80.0
97.5
95.0
97.5
Third
75.0
97.5
92.5
87.5
Fourth
72.5
95.0
92.5
67.5
Fifth
72.5
95.0
92.5
60.0
Sixth
72.5
95.0
90.0
-
Seventh
-
92.5
90.0
-
Eighth
-
92.5
-
-
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Chapter 5
Table 3b. Survival (%) of crablets fed the different diets in the communal culture
Culture
period
Shrimp meat
Live Artemia
Frozen Artemia
Dried Artemia
based feed
100.0
100.0
100.0
100.0
Day 10
51.7 ± 9.3a
89.2 ± 5.2b
65.8 ± 9.5a
50.8 ± 8.8a
Day 20
40.8 ± 10.5a
81.7 ± 7.6c
60.2 ± 9.0b
38.3 ± 8.4a
Day 30
33.3 ± 6.4a
79.2 ± 5.2c
55.3 ± 6.6b
30.0 ± 6.6a
Day 40
24.2 ± 5.1a
75.8 ± 6.5c
47.5 ± 6.5b
21.7 ± 3.8a
Day 0
Values are means ± SD. Means with different letters within the same row are significantly
different (P<0.05).
Table 4. Intermolt period (day), percentage molting (%) and total number of molts of
crablets fed different diets for 40 days
Shrimp meat
Live Artemia
Frozen
Artemia
Dried Artemia
based feed
First
2.20±1.14a
(100%)
1.85±0.80a
(100%)
2.00±0.82a
(100%)
2.43±1.24a
(100%)
Second
5.34±1.26b
(100%)
3.39±0.68c
(100%)
4.00±0.74c
(100%)
6.67±1.72a
(100%)
Third
7.10±1.71b
(100%)
4.38±1.23c
(100%)
5.14±1.32c
(100%)
9.23±1.70a
(100%)
Fourth
8.41±2.13b
(100%)
5.03±1.00d
(100%)
6.05±1.25c
(100%)
10.89±1.34a
(100%)
Fifth
10.31±1.91b
(89.7%)
6.32±1.30d
(100%)
7.38±1.23c
(100%)
12.21±1.48a
(58.3%)
Sixth
10.75±1.39a
(27.6%)
7.39±1.53b
(100%)
8.34±1.21b
(97.2%)
-
Seventh
-
8.31±1.45a
(86.5%)
9.47±0.94b
(47.2%)
-
Eighth
-
8.45±0.69
(29.7%)
-
-
Total number
of molts
6
8
7
5
Molt number
Values are means ± SD. The numbers in parenthesis indicate the percentage of crabs going
through this molt. Means with different letters within the same row are significantly
different (P<0.05).
181
Weight increment (%)
Chapter 5
270
240
210
180
150
120
90
60
30
0
First
Shrimp meat
Live Artemia
Frozen Artemia
Dried Artemia based diet
(a)
Second
Third
Fourth
Fifth
Sixth
Seventh
Eighth
Moulting stage
Shrimp meat
Live Artemia
Frozen Artemia
Dried Artemia based diet
90
80
(b)
Size increment (%)
70
60
50
40
30
20
10
0
First
Second
Third
Fourth
Fifth
Sixth
Seventh
Eighth
Molting stage
Figure 2. Growth of crab postlarvae in the individual culture. (a) percentage weight
increment, (b) percentage size increment
Table 4 summarizes the intermolt period, total number of moltings and percentage molting
of the crablets fed the different diets. Results showed that the first intermolt period was
similar in all treatments (1.85-2.43 days). From the second molt onwards, the molting
interval was significantly different among treatments (P<0.05), with the shortest and
longest intermolt for the crabs reared on live biomass and the dried Artemia formulated
feed, respectively. Intermediate values were found for crabs fed frozen biomass and shrimp
meat. As a consequence, the total number of moltings over 40 days of rearing was also
highly affected by the dietary treatments: these values were 8, 7, 6 and 5 for the crab PL fed
live biomass, frozen biomass, fresh shrimp meat and dried Artemia-based feed,
respectively.
182
Mean body weight (g)
Chapter 5
9
8
7
6
5
4
3
2
1
0
Shrimp meat
Live Artemia
Frozen Artemia
Dried Artemia based diet
0
10
20
Culture period (days)
30
Shrimp meat
40
Carapace width (mm)
(a)
(b)
Live Artemia
35
40
Frozen Artemia
30
Dried Artemia based diet
25
20
15
10
5
0
0
10
20
30
40
Culture period (days)
Figure 3. Growth curve of mud crab postlarvae fed the different test diets in the communal
culture. The error bars stand for standard deviation. (a) body weight, (b) carapace width.
The average percentages weight and size increment were largest in the first molt and then
tended to decline with each molting stage (Figure 2). For most molts, the crablets receiving
the control (fresh shrimp meat) and the dried Artemia diet showed lower values compared
to the other groups.
The specific growth rates in terms of weight (SGRW) and carapace width (SGRCW) of the
crab PL fed the different diets for 40 days are presented in Table 5. The average final
weight and carapace width, and SGRW and SGRCW of crab PL reared separately were
significantly different among treatments (P<0.05). Growth decreased in the order live
biomass>frozen biomass>shrimp meat>dried Artemia-based feed. For the communal
culture, growth both in terms of weight and carapace width was rather steady over the
entire culture period in all treatments (Figure 3). There were no significant differences
among the test diets although these parameters were slightly higher in the group fed live
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Artemia biomass. Animals fed the formulated Artemia based feed had the poorest growth
performance (P>0.05).
Table 5. Growth parameters of crablets fed the different diets for 40 days.
Shrimp meat
Live Artemia
Frozen Artemia
Dried Artemia
based diet
Initial weight (mg)
8.10± 0.69
7.88 ± 0.52
7.88 ± 0.52
7.85 ± 0.55
Final weight (g)
0.66±0.17b
3.70±1.29d
2.03±0.84c
0.29±0.13a
SGRW (%day-1)
10.91 ±0.65b
15.21 ±1.01d
13.70 ±0.97c
8.85 ±1.02a
Initial CW (mm)
2.78±0.14
2.74±0.12
2.72±0.11
2.72±0.09
Final CW (mm)
15.5±1.4b
28.62±4.16d
23.33±3.63c
11.45±1.56a
4.29 ±0.25b
5.83±0.41d
5.35± 0.39c
3.57 ±0.35a
8.23±0.73
8.23±0.73
8.23±0.73
8.23±0.73
Final weight (g)
4.74±2.02a
5.85±2.17a
4.22±2.13a
3.63±1.93a
SGRW (%day-1)
15.90±1.62a
16.43±1.48a
15.60±1.37a
15.23±1.15a
2.77±0.09
2.77±0.14
2.75±0.10
2.74±0.12
Final CW (mm)
30.92±5.57a
32.47±4.46a
30.61±4.34a
28.77±5.05a
SGRCW (%day-1)
6.03±0.72a
6.15±0.65a
6.02±0.58a
5.88±0.76a
Diets
Individual culture
SGRCW (%day-1)
Communal culture
Initial weight (mg)
Initial CW (mm)
Values are means ± SD. Means with different letters within the same row are significantly
different (P<0.05).
4. Discussion
4.1. Effect of feeding different forms of Artemia biomass on survival of mud crab
In the present nursery experiment, survival of the crabs over 40 days was in the ranges of
60-93% and 22-76% for individual and communal culture, respectively. This result was
comparable to the study of Ruscoe et al. (2004), who individually reared instar 2 Scylla
serrata crablets with a mean weight of 18.43 mg at different combinations of temperature
(20-35°C) and salinity (5-40 g L-1) for 18 days. This author obtained survivals of 36-94%.
Similar findings were reported by Ut et al. (2007a) who conducted pilot-scale nursery
production in lined-ponds of instar 1 Scylla paramamosain, stocked at a density of 70m-2,
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fed live Artemia biomass and fresh chopped shrimp or tilapia for 30 days. In this study
survival rates of 52-66% were achieved. Additionally, Mann et al. (2007), when culturing
Scylla serrata from megalops to instar 8 stage at different densities and with different
artificial habitats in concrete tanks and lined ponds, using live sub-adult Artemia for the
first and second phase and commercial pelleted feed for the third phase, obtained survival
rates of 56% to 81%.
The results of the current study showed significant effects of the Artemia biomass form as
feed on survival of Scylla paramamosain crablets in both culture systems. For individual
culture, survival of the groups fed live and frozen biomass was similar, but higher than
those fed the control feed (fresh shrimp meat) and the dried Artemia-based formulated feed.
In particular, high mortality from the first molt to the second molt was in the control
treatment. The reason for this was unclear. Moreover, from daily observations, it was noted
that survival of crablets fed the Artemia formulated feed was reduced mainly by moltrelated mortality predominantly occurring between the third and the fifth molting stage.
Mann et al. (2001) investigated an Artemia-based diet for larvae of the mud crab Scylla
serrata. They proved that survival is useful for identifying the effect of inadequate diets on
the crab larvae. These authors found high incidence of molt death syndrome which refers to
larvae that have begun molting but further progress is halted before the exuvium is
completely shed. These larvae survive for a short period but can not swim or feed. Similar
observations were reported by Genodepa et al. (2004), which observed high mortality when
S. serrata megalopa molt to the first crab stage and also referred to it as ‘molting-deathsyndrome’. Although the cause of the problem is not fully understood, it is believed to be
associated with inappropriate nutrition (Williams et al., 1999; Hamasaki et al., 2002; Holme
et al., 2009). In view of these findings, it may be fair to speculate that the formulated feed
used in the current study is nutritionally imbalanced or fails to fulfil all requirements of the
crablets, leading to increased mortality.
Also for communal culture, noticeable differences in survival were observed among the
four feeding treatments. The sharp decline in survival in the three treatments with nonliving
diets at day 10 could be related to the transition from the hatchery using live feed combined
with cooked fish to the nursery phase, in which the animals received non-living and/or
formulated feeds. Apart from this weaning effect, it may also well be that feeding live
Artemia effecting reduced cannibalism, resulting in increased survival as compared to other
groups. Zmora et al., (2005), who investigated mass production of blue crab Callinectes
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sapidus, who found that from the megalopa stage onwards, heavy losses of crab juveniles
occurred due to cannibalism. Several studies reported that living prey can greatly reduce
cannibalism in Lobster (Conklin, 1995), penaeid shrimp (Wickins and Lee, 2002), fresh
water prawn (Phuong et al., 2003a); pike fish, Esox lucius (Wolska-Neja and Neja, 2006).
At the end of the experiment, the highest survival was achieved in crablets fed live Artemia.
This was 1.6, 3.1 and 3.5 times higher than in the groups receiving the frozen biomass, the
control feed and the dried Artemia diet, respectively. The phenomenon of conspecific
cannibalism was clearly observed at the later part of the experiment, when pieces of dead
crabs were observed in different tanks and most likely constitute crabs that were
killed/eaten by the bigger ones during molting. The results in the current experiment are in
accordance with the study of Mann et al. (2007), who observed in clear-water mud crab
nursery systems. Moreover, numerous investigations stated that loss of mud crabs during
communal rearing under semi-intensive to intensive conditions is generally attributed to
aggression-associated mortality and cannibalism, accounting for a large percentage of
mortality (30-50 %) which is considered to be the main constraints from these culture
systems (Quinitio et al., 2001; Wickins and Lee, 2002; Allan and Fielder, 2003; Mann and
Paterson, 2003).
4.2. Effect of feeding different forms of Artemia biomass on molting and growth of mud
crabs
The result of this study clearly demonstrated that the shortest intermolt period and highest
number of molts and percentage weight increment were obtained in the group fed the live
Artemia biomass, followed by frozen Artemia, control feed (shrimp meat) and dried
Artemia-based feed. The live Artemia group went through 8 molts within 40 days, while
this was only 5 to 7 in the other groups. Overall, the intermolt period increased while
percentage growth increment decreased with each molting stage. The present observations
are consistent with the study of Fumis et al. (2006) who found a significant decrease in
molt increment and an increase in intermolt intervals of the crab Dissodactylus crinitichelis
from juvenile to adult stages. Similar observations were found in other crab species such as
spider crabs Hyas coarctatus and Inachus dorsettensis (Hartnoll and Bryant, 2001),
estuarine crab Chasmagnathus granulatus (Luppi et al., 2004), stone crab Menippe sp.
(Susan and Bert, 2008).
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Moreover, growth parameters in terms of the final weight and CW, SGRW and SGRCW
reared individually followed the same pattern as growth increment. Again the highest
values were observed for the group fed live biomass followed by the group consuming
frozen Artemia, the control group and the group receiving the dried Artemia-based
formulated diet. Similarly, Jee et al. (2007) found the poorer growth rate in juvenile tiger
crab fed microencapsulated feed compared to live Artemia nauplii and to a range of other
diets (fresh clam meat, and four different combinations of mixed diets). Ut et al. (2007a)
confirmed that live Artemia biomass can be used as a feed for crabs reaching a size of 35
mm CW, and 10.5 g body weight from instar 1 up to 60 days. Moreover, analogous
findings were observed for other crustacean groups. Tlusty et al. (2005a), feeding frozen
adult Artemia to the American lobsters (Homarus americanus), found that live Artemia
results in good growth, but is expensive. Although frozen Artemia is lower in cost, it
generally resulted in decreased survivorship and growth relative to live Artemia. Another
study by the same authors showed that after 3 months of culture, juvenile American
lobsters (H. americanus) fed frozen adult Artemia diets had a significantly greater weight
gain (>6 % day-1) compared to formulated diets (Tlusty et al., 2005b). Van et al. (2008)
demonstrated those both live and frozen biomasses are a suitable feed for nursery culture of
postlarval Penaeus monodon.
It was noticed that in the present experiment, the effect of the feeding treatments on growth
performance of the crablets cultured communally was different as in those reared
individually. Significant differences among treatments in the terms of final weight and CW
as well as the SGRW and SGRCW were not detected in communal culture (P>0.05). As
mentioned earlier, the lower survival of the crabs in the communal culture was probably
due to a high incidence of conspecific cannibalism during molting. Thus, survivors not only
feed on the experimental feed but also consumed newly molted individuals in the culture
tank. Similar result was reported by Ut et al. (2007a): in pilot-scale nursery production in
lined-ponds with shelters, live Artemia biomass and fresh chopped shrimp or tilapia were
found to be equally effective feeds for juvenile crabs.
Differences in nutritional composition of various diets used in this study could be another
reason for the observed differences in crab performance. All Artemia biomass diets
contained between 49.5 and 56.5% protein and 10.3-11.2% lipid, which are within
recommended levels for the growth of mud crab reported by previous studies (Sheen and
Wu, 1999; Catacutan, 2002; Holme et al., 2009). In contrast, although having a higher
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protein level (62%), the lipid content of fresh shrimp meat was only 1.6%. This might be
another cause of the lower weight gain and reduced molting frequency of crabs consuming
this feed. Sheen and Wu (1999) investigated the effects of dietary lipid levels on the growth
response of juvenile mud crab Scylla serrata. They observed that crabs fed diets without oil
addition and diets with 2% added oil had significantly lower weight gains than those fed a
higher dietary lipid level and suggested a dietary lipid level ranging from 5.3 to 13.8%
appears to meet the lipid requirement for this crab species. Moreover, Catacutan (2002)
reported that the crab S. serrata grew well when fed diets with 32-40% dietary protein and
6-12% lipid at dietary energy levels ranging from 14.7 to 17.6 MJ kg-1. Although the dried
Artemia-based feed contained appropriate protein (49.5%) and lipid (10.3%) levels, it still
resulted in the poorest performance as demonstrated by the low survival and growth. This
may also be in part due to poor stability of the feed pellets in the water (visual observation).
The diet was formulated to contain approximately the same protein concentration as the
other test diets. As a result, the proportion of dried Artemia meal was high (79.2%) and the
fraction of wheat flour, a natural binder, low (5.8%). Several studies reported that most
diets formulated to suit the larvae and postlarvae of carnivorous species have high animal
protein content. These ingredients contain few natural binding agents, and therefore the
incorporation of an ‘external’ binder is important in the formulation of these feeds
(Melcion, 2001; Holme et al., 2009). In addition, Genodepa et al. (2007) stated that if feed
particles show relatively poor stability in water, there are potential problems relating to
water quality and bacterial proliferation, as well as nutrient deficiency resulting from
leaching. However, in the present study, due to the high rate of water exchange (about 30%
day-1), water quality deterioration in the culture tank could be minimized.
Conclusion
Data obtained from both individual and communal cultures indicated that crablets fed live
Artemia biomass had the best performance in terms of survival and growth rate, followed
by the group fed frozen Artemia, the control group and the group receiving the dried
Artemia-based formulated diet. Therefore, live Artemia biomass proved to be an excellent
feed for nursery culture of Scylla paramamosain crabs. Given that in the coastal regions of
Vietnam where crab farming is practiced, live Artemia biomass is seasonally available in
great quantities, this demonstrate the high potential for local use of Artemia biomass for
intensive nursery of crab. Moreover, improvements in the formulation and water stability of
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the Artemia based formulated feed are necessary to make it an effective nursery diet for
mud crab.
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Chapter 5
Section IV
Substituting fishmeal with Artemia meal in diets for goby
Pseudapocryptes elongatus: effects on survival, growth
and feed utilization
Chapter 5
Chapter 5
Abstract
The present study was performed to evaluate the effect of replacing fishmeal with Artemia
meal in the diet of goby Pseudapocryptes elongatus on survival, growth performance and
feed utilization. A control diet containing fishmeal as main protein source was compared
with four experimental diets in which fishmeal protein was replaced by increasing dietary
levels of Artemia protein, namely 25%, 50%, 75% and 100%. The five test diets were
compared with a commercial diet and dried Artemia. All diets were formulated to be
equivalent in gross energy (3.7-3.8 Kcal g-1), crude protein (36-37%) and lipid (5.8-6.5%),
except for the dried Artemia, which had a higher protein and lipid content (43.6 and 7.8%,
respectively). The study was conducted in 80 L plastic tanks filled with water with a
salinity of 15 g L-1. Goby fingerlings with 0.21g initial weight were fed the test diets for 30
days. Treatments were run in triplicate. The results showed that survival of the fish was not
influenced by the feeding treatments and varied from 79.2 to 85.8%. Weight gain and
specific growth rate of goby were positively correlated with total feed intake. Moreover, a
gradual increase in growth performance and better feed utilization were achieved with
increasing dietary inclusion of Artemia protein. Growth performances (mean final body
weight, weight gain, specific growth rate) and feed utilization (total feed intake, feeding
rate, feed conversion ratio, feed efficiency and protein efficiency ratio) in the goby fry
receiving the commercial feed and fishmeal control diet were similar, both were inferior to
the groups fed dried Artemia and the based formulated diets. Significant differences
(p<0.05) were however only found from 50% Artemia protein inclusion level onwards.
These results illustrate that both dried Artemia and Artemia based-feeds can be used for
feeding goby Pseudapocryptes elongatus fingerlings, indicating the high potential of using
locally produced Artemia biomass, which could contribute to reduce the reliance on
fishmeal and improve profits for Artemia producers.
Keywords: Pseudapocryptes elongatus, fishmeal, Artemia biomass, commercial feed,
survival, growth, feed utilization
1. Introduction
Seasonal culture of Artemia cyst in the coastal saltworks in Vietnam has proven successful
and has considerably improved the standard living of salt farmers since 1990s (Brands et
al., 1995). After about twenty years of development, Artemia culture is widespread over
1000 ha and the households’ livelihood strategy has become more diversified and reliable
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(Son, 2008; Nam et al., 2008). Besides the highly valuable Artemia cyst production, a sideharvest of Artemia biomass of 0.2-0.3 ton ha-1 can be obtained in these ponds after
termination of the production season (Brands et al., 1995; Anh et al., 1997a). This indicates
large quantities of Artemia biomass as by-product from the cyst-oriented pond production
are available seasonally in these regions. Nonetheless, so far, this product has not been
efficiently utilized for aquaculture purposes. When the culture season ends, most farmers
simply discharge the Artemia biomass into reservoirs for feeding wild shrimp and fish.
Numerous studies reported that Artemia biomass is an excellent feed for ornamental fish
(Lim et al., 2001; 2003); as a nursery food for marine fish (Sorgeloos et al., 2001; Le et al.,
2008), shrimp (Naegel and Rodriguez-Astudillo, 2004; Van et al., 2008), prawn (New,
2002) and crab (Ut et al., 2007a); as a high-protein ingredient for aquaculture feeds as well
feed attractant (Dhont, and Sorgeloos, 2002), and as a maturation trigger in shrimp
broodstock diets (Wouters et al., 2002; Gandy et al., 2007). These kinds of aquaculture
activities are also found in the same coastal areas where Artemia culture in Vietnam is
practiced. Therefore, utilization of Artemia biomass as protein source in feeds for local
commercial culture of fish or shrimp could enhance the income of Artemia producers and
contribute to reduce the reliance on the use of fishmeal (FM) in aquafeeds. The FM in high
quality feeds for fish fingerlings and crustaceans is currently imported and represents about
90% of the total FM used for aquaculture in Vietnam (Edwards et al., 2004). Ideally,
alternative ingredients should have good availability and satisfactory nutritional quality for
the species to be fed, and moreover should be cost effective (Watanabe, 2002; Glencross et
al., 2007).
Recently, disease outbreaks in shrimp farming areas in Vietnam have revived the call for
diversification of cultured species. Apart from the freshwater prawn Macrobrachium
rosenbergii and mud crab Scylla spp., the goby Pseudapocryptes elongatus has been
considered an attractive candidate for culture. This species is a benthic fish, distributed in
the Indo-Pacific region (Rainboth, 1996; Froese and Pauly, 2006) and is common in the
coastal areas of the Mekong delta, Vietnam (Dinh et al., 2007). It is a highly valuable fish
for domestic consumption as well as for export and has a good tolerance to a wide range of
salinity conditions. For these reasons goby is now becoming an important candidate for
integrated or alternative culture models in brackish water aquaculture (Khanh, 2006;
Chung, 2007). Furthermore, recent studies reported that semi-intensive and intensive
culture of goby can deliver high profits within 4-5 months, with limited risk of disease and
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low inputs compared to shrimp culture (Nga and Long, 2005; Nhon, 2008). Pond culture of
goby is currently completely dependent on wild-caught seed and, in some areas; further
expansion of goby farming is now limited by fisheries for fingerlings (Dinh et al., 2005;
Nhon, 2008). Additionally, mortality is size-specific in the goby P. elongatus; with large
individuals having higher survival than smaller ones in grow-out culture (Khanh, 2006;
Chung, 2007). In practice, although 1-2 cm fry could be stocked directly in predator-free
grow-out ponds, this size is not acceptable to some goby culturists due to the longer culture
period and lower survival (Nhon, 2008; Anh et al., 2009). Thus, nursery culture of goby to
attain fry of larger size and high quality prior to transport for stocking in grow-out ponds is
necessary. The objective of this study was to evaluate the possibility of replacing fishmeal
in the diet of goby Pseudapocryptes elongatus with locally available by-product and to
detect possible effects on survival, growth and feed utilization.
2. Materials and methods
2.1. Culture system
A 30 day feeding experiment was carried out in the experimental hatchery of the College of
Aquaculture and Fisheries, Can Tho University, Vietnam. The test was set up as a
completely randomized design with 3 replicates per treatment. The 80-L plastic tanks were
filled with 60 L water with at salinity of 15 g L-1. Each tank was provided with continuous
aeration and feeding trays were distributed in each tank for collecting uneaten feed. Every
other day about 50% of the tank volume was exchanged. Daily water temperature and pH
were recorded at 7:00 and 14:00 h using a thermo-pH meter (YSI 60 Model pH meter).
2.2. Experimental fish
Wild fingerlings of goby were purchased from a reliable provider in Bac Lieu province and
visually checked for signs of disease and parasites. Before starting the feeding trial, they
were first reared in a 1m3 tank for one week in order to acclimate the fish to the laboratory
conditions and to get them acquainted with the feeding method (feed on a feeding tray)
before starting the feeding trial.
After acclimation, 40 uniformly sized fish with initial individual weight of 0.20-0.22 g were
placed in each tank. Fish were fed three times a day at 7:00, 12:00 and 17:00. The initial
feed ration was 15% of the biomass, but this was adjusted daily based on the presence or
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absence of residual feed. About 1.5 h after feeding, unconsumed feed was removed,
transferred to aluminium cups and dried to a constant weight.
2.3. Feeds and experimental design
Artemia biomass was collected from commercial Artemia cyst-oriented ponds in Vinh Hau
village, Bac Lieu province, Vietnam at the end of the culture season. Dried Artemia was
obtained by solar-drying thin layers of biomass and then grinding it into particles of 500
µm. Kien Giang fishmeal (manufactured in Kien Giang province, Vietnam) was purchased
from CATACO Company, Can Tho city. Other ingredients such as soybean meal, rice bran,
squid oil, gelatine, wheat flour... were purchased from commercial suppliers. The dietary
ingredients were analyzed for their proximate composition (Table 1) prior to the
formulation of the diets. Commercial feed (GROBEST-GB640) is produced by Grobest &
I-MEI Company, Dong Nai province, Vietnam. According to the label, the feed had the
following composition: protein ≥40%, lipid ≥ 6%, Ash ≤14%, fiber ≤ 6% and moisture
≤11%.
Five experimental diets were formulated by replacing 0, 25, 50, 75 and 100% of the
fishmeal protein in a standard diet with dried Artemia meal (Table 2). In the 0% Artemia
treatment (control treatment), Kien Giang fish meal was the main protein source. All diets
were formulated to be approximately isolipidic, isoenergetic and isonitrogenous with the
commercial feed. The diets were made into sinking pellets (700-1000µm) using a pellet
machine, oven-dried at 60°C and stored at 4°C before use. The five experimental feeds
were compared with two other feeds, namely pure dried Artemia biomass (ground to the
same particle size as the formulated diets) and the commercial feed.
Table 1. Proximate composition (% of dry matter) of the ingredients used in the test diets
Ingredients
Fish meal
Artemia meal
Rice bran
Wheat flour
Dry matter
89.57
89.73
88.45
89.19
Crude protein
50.16
43.57
15.16
10.05
Crude lipid
5.68
7.78
10.21
1.64
NFE
7.11
11.82
55.34
85.35
Ash
35.43
35.98
9.07
1.71
Fibre
1.62
0.85
10.22
1.25
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2.4. Chemical analysis
Proximate analysis (moisture, crude protein, total lipid, fibre and ash) of the ingredients and
experimental diets was carried out according to the standard methods of AOAC (1995).
Nitrogen-free extract (NFE) was estimated on a dry weight basis by subtracting the
percentages of crude protein, lipids, crude fibre and ash from 100%.
Table 2. Composition (g 100 g-1 dry matter) of the dried Artemia-based formulated diets
Ingredients
0%A
25%A
50%A
75%A
100%A
Fish meal
62.92
48.02
31.54
15.81
0
0
19.10
36.32
55.59
70.58
Rice bran
15.21
14.91
14.07
11.64
12.75
Wheat flour
14.34
11.31
11.73
10.80
10.47
Squid oil
1.50
1.02
0.87
0.65
0.50
Vitamin premix
2.00
2.00
2.00
2.00
2.00
Gelatine
3.00
3.00
3.00
3.00
3.00
Cellulose
1.03
0.64
0.47
0.51
0.70
Artemia meal
Vitamin mix was VEMEVIT produced by Rhone-Poulenc. In 1 kg of mixture, there is Vit. A: 2.000.000 UI;
Vit. D3: 400.000 UI; Vit. E: 12.000 mg, Vit. K: 480 mg; Vit. B1: 800 mg; Vit. B2: 800 mg; Vit. B6: 500 mg,
Nicotinic acid: 5.000 mg, Calcium: 2.000 mg, Vit. B12: 2.000 mg,
++
Foli acid: 160 mg; Microvit. H:
++
2000:1.000 mg; Vit.C: 100.000 mg; Fe : 1.000 ppm, Zn : 3.000 ppm; Mn++: 2.000 ppm, Cu++: 100 ppm,
Iodine: 20 ppm, Co++: 10 ppm.
The fatty acid profile of the experimental diets was determined by gas chromatography.
Fatty acid methyl esters (FAMEs) were prepared via a procedure modified from Lepage
and Roy (1984).
2.5. Growth performance and feed utilization
To estimate the growth performance during the experimental period, initial and final as well
as intermediate samples were taken to measure average individual fish weight. Sampling
was conducted at a 10-day interval. Ten fish in each tank were randomly sampled and
weighed in groups of 10 using an electronic balance with an accuracy of 0.001g and mean
weights were determined. Feed intake was also estimated over a 10-day period.
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Weight gain (WG), specific growth rate (SGR), feed intake, feeding rate, feed conversion
ratio (FCR), protein efficiency ratio (PER) and survival were calculated using the following
equations:
Weight gain (g) =Final body weight - Initial body weight
(ln final weight - ln initial weight)
Specific growth rate (% day-1) = ------------------------------------------ x 100
Days of culture
Total feed supplied - Total feed remaining
Total feed intake (g fish-1) = ---------------------------------------------------------- x 100
(Initial number of fish + Final number of fish)/2
Feed intake
Feeding rate (%BW day-1) = ---------------------------------------------------- x 100
(Initial body weight + Final body weight)/2
Feed intake (dry weight)
Feed conversion ratio = --------------------------------Weight gain (wet weight)
Weight gain
Feed efficiency = -------------------Feed intake
Weight gain
Protein efficiency ratio = -------------------Protein intake
Final number of fish
Survival (%) = ----------------------------- x 100
Initial number of fish
2.6. Statistical analysis
Data for all measured parameters were analysed using SPSS for Windows, Version 13.0.
Variations with dietary treatment were compared by one-way ANOVA. The Tukey HSD
post-hoc analysis was used to detect differences between means. Significant differences
were considered at P<0.05. All percentage values were normalized through a square root
arcsine transformation prior to statistical treatment.
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3. Results
3.1. Water quality
Average water temperature over the experiment was 27.8 ± 0.9°C and 29.7 ±1.2 °C at 7:00
and 14:00, respectively. Salinity was 15 ± 2 g L-1 and pH 7.6 ± 0.5. These water parameters
were generally within the suitable range for nursing of goby Pseudapocryptes elongatus
(Khanh, 2006; Chung, 2007).
3.2. Experimental diets
Table 3 shows the proximate composition of the seven experimental diets. All formulated
diets used in the present experiment were nearly isonitrogenous, isolipidic and isocaloric.
The protein content of the dried Artemia (DA) (43.57%) was higher than that of the other
diets (36.15-36.98%). The lipid level was slightly lower in the commercial feed (CF), while
it was higher in the dried Artemia (DA).
Table 3. Proximate composition (% of dry matter) of the seven experimental diets
CF
DA
0%A
25%A
50%A
75%A
100%A
Dry matter
89.57
88.62
90.92
89.47
91.21
90.54
89.73
Crude protein
36.48
43.57
36.98
36.41
36.18
36.49
36.15
5.83
7.78
6.13
6.54
6.47
6.25
6.58
NFE
27.44
14.82
28.79
25.72
27.13
28.24
27.21
Ash
24.51
32.98
23.68
26.11
25.16
24.42
25.33
Crude fiber
5.74
0.85
4.42
5.22
5.06
4.60
4.73
Energy (Kcal g-1)
3.73
3.78
3.84
3.72
3.76
3.81
3.77
Crude lipid
CF: commercial feed, DA: dried Artemia, A: Artemia meal.
Gross energy was calculated based on the physiological values: protein = 5.56; lipid = 9.54
and NFE = 4.20 (Kcal g-1)
The fatty acid profile of the experimental diets is summarized in Table 4. The linoleic
(18:2n-6) and linolenic acid (18:3n-3) levels in the commercial feed (CF) were respectively
about 2-3 and 4 times higher than in the other diets (P<0.01). The archidonic acid (ARA,
20:4n-6) level of the CF and control diet (0%A) proved slightly higher than in the dried
Artemia (DA) and Artemia-based diets, in which the CF was significantly different from the
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50%A and 75A diets (P<0.05). The lowest and highest eicosapentaenoic acid (EPA, 20:5n3) concentrations were found in the CF and DA, respectively and moreover the level of this
fatty acid increased with the percentage Artemia in the diet. In addition the CF, DA and
100%A contained the lowest level of docosahexaenoic acid (DHA, 22:6n-3) among the
different feeds. DHA level was highest in FM control diet. In the formulated Artemia-based
diets, DHA level decreased with Artemia inclusion. In contrast, total saturated fatty acid
(∑SFA) and total monounsaturated fatty acids (∑MUFA) content in the CF was
significantly higher than in the other test diets (P<0.05). In most cases, the content of total
polyunsaturated fatty acids (∑PUFA) and ∑n-3 and the ratio of n-3/n-6 of all experimental
diets was not significantly different (P>0.05). However, the ∑n-6 PUFA in the CF was
significantly higher than in the control and the Artemia formulated feeds.
Table 4. Fatty acid profile (mg g-1 dry weight) of the seven experimental diets
Fatty acids
CF
DA
0%A
25%A
50%A
75%A
100%A
18:2n-6
18.01f
6.55a
11.08e
9.79d
8.79c
8.44bc
7.51b
18:3n-3
6.23b
1.58a
1.49a
1.64a
1.63a
1.52a
1.50a
20:4n-6
3.76b
3.49ab
3.65ab
3.41ab
3.18a
3.23a
3.44ab
20:5n-3
1.12a
5.84c
4.30b
4.66b
4.74b
4.86b
5.09bc
22:6n-3
0.23a
0.25a
1.02d
0.88d
0.79cd
0.53b
0.29a
Total SFA
26.32b
21.24a
22.00a
23.29a
21.68a
24.48ab
22.87a
Total MUFA
30.14c
25.85b
22.06a
23.51a
24.33a
23.42a
24.71ab
Total PUFA
13.81ab
14.28b
13.36a
13.18a
13.38a
12.42a
13.13a
Total n-3
8.33ab
8.97b
8.55ab
8.56ab
8.26ab
7.86a
8.40ab
Total n-6
5.48b
5.31ab
4.81a
4.62a
5.12a
4.56a
4.73a
n-6/n-3 ratio
1.52a
1.69a
1.78a
1.86a
1.62a
1.73a
1.78a
Mean values in each row with different letters are significantly different from each other
(P<0.05). Data are means ± SD of two replicates.
3.3. Survival
Survival of goby P. elongatus fed the different diets is shown in Figure 1. Results showed
that the survival of fish was not affected by the feeding treatments, ranging from 79.2% in
the group fed 100%A to 85.8% in the 50%A group (P>0.05).
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CF
100
DA
0%A
Survival (%)
80
25%A
60
50%A
75%A
40
100%A
20
0
10
20
30
Experimenatl period (days)
Figure 1. Survival of goby fed different test diets over 30 days
3.4. Growth and feed utilization
Growth performance and feed utilization of goby fingerlings P. elongatus fed the different
experimental diets over 30 days are given in Table 5. Mean final body weight (FBW),
weight gain (WG) and specific growth rate (SGR) of the goby fed the various test diets
were in the ranges of 1.84-3.15 g, 1.54-2.95 g and 7.01-9.16 % day-1, respectively. The
FBW and WG in the groups fed the commercial feed (CF) were similar to the group
receiving the control diet. Values for these diets were however significantly lower than for
fish fed the 50, 75 and 100% Artemia feed (P<0.05), in which the best growth rate was
found in the 100%A diet. Dried Artemia (DA) resulted in intermediate growth values. The
SGR in the different treatments showed the same pattern as observed for the FBW and WG.
y = 1.6473x + 3.9711, R² = 0.8063
WG (g), SGR (%/day)
10
Specific growth rate
8
6
y = 0.9833x - 0.2136, R² = 0.8413
4
Weight gain
2
0
1.5
2.0
2.5
3.0
3.5
-1
Total feed intake (g fish )
Figure 2. Linear regression between weight gain/SGR and total feed intake of goby fed
different test diets over 30 days.
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Chapter 5
Table 5. Growth performance and feed utilization of goby Pseudapocryptes elongatus fry fed different experimental diets over 30 days.
Parameters
CF
DA
0%A
25%A
50%A
75%A
100%A
Initial body weight (g)
0.21±0.01
0.22±0.01
0.22±0.01
0.20±0.01
0.21±0.02
0.21±0.01
0.21±0.01
Final body weight (g)
1.84±0.19a
2.51±0.27c
1.75±0.19a 1.93±0.16ab 2.38±0.19bc 2.85±0.18cd
3.15±0.17d
Weight gain (g)
1.54±0.20a
2.29±0.26c
1.63±0.21a
2.95±0.19d
Specific growth rate (%day-1)
7.01±0.37a 8.18±0.31bc 7.12±0.42ab 7.50±0.29ab 8.11±0.53bc
Total feed intake (g fish-1 )
2.10±0.16a 2.22±0.16ab
Feeding rate (%BW day-1)
8.94±0.58c
5.96±0.31a 8.58±0.65bc
9.28±0.64c 8.74±0.73bc 7.23±0.60ab 7.60±0.43bc
Feed conversion ratio (FCR)
1.37±0.08c
0.97±0.05a 1.30±0.12bc
1.34±0.08c 1.10±0.04ab
1.07±0.10a
Feed efficiency
0.73±0.04a
1.03±0.05c
0.75±0.04a 0.91±0.04bc
0.94±0.09c 0.93±0.05bc
Protein efficiency ratio (PER)
1.48±0.17a
2.45±0.24c 1.63±0.29ab 1.54±0.17ab 2.28±0.18bc
1.7±0.15ab 2.17±0.20bc 2.64±0.13cd
8.76±0.29c
9.16±0.24c
2.11±0.23a 2.31±0.17ab 2.40±0.32ab 2.83±0.36bc
3.17±0.15c
0.77±0.07a
2.44±0.50c
1.08±0.05a
2.39±0.24c
Mean values in each row with different letter are significantly different from each other (P<0.05). Data are means ± SD of three replicates.
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The average total feed intake was between 2.10 and 3.17 g fish-1 with the highest values
found in the 75%A and 100%A treatments, which was statistically different from the other
test diets (P<0.05). Moreover, Figure 2 indicated that the WG and SGR were positively
correlated with the total feed intake (R²=0.80 and 0.84 respectively). Mean feeding rate
varied from 5.96 to 9.28 %BW day-1, in which larger percentages were observed in the
goby fed the CF, 0%A, 25%A and 50% diets; the other two Artemia-based diets showed
intermediate values and the lowest rate was found in the DA group. Interestingly, linear
regression analysis for the Artemia-based diets showed that a gradual increase in total feed
intake (R²=0.85) and growth performance (WG and SGR, R²= 0.91 and R²= 0.83,
respectively) of fish occurred with increasing dietary inclusion of Artemia protein (Figure
3a). When comparing all feeding treatments, the feed conversion ratio (FCR) in the groups
fed the CF, 0%A and 25%A treatments (1.30-1.37) were significantly higher than in the
four remaining, in which the 50%A, 75%A and 100%A treatments were in the range of
1.07-1.10 and the lowest FCR was observed in the DA-fed group (0.97). Similarly, feed
efficiency (FE) and protein efficiency ratio (PER) were best in the DA, 50, 75 and 100%A
groups. The effect of Artemia protein inclusion on FCR and FE and PER can be clearly
seen in Figure 3b: FCR decreases (R²=0.79) while FE and PER increase (R²= 0.80 and
0.72) with increasing Artemia protein inclusion level. Overall, it can be concluded that
growth performance and feed utilization in the CF-fed groups was similar to the control-fed
group and both were inferior to the Artemia-fed groups.
2
y = 0.0321x + 6.203, R = 0.832
10
9
8
7
6
5
4
3
2
1
0
2
y = 0.0118x + 1.9669, R = 0.8525
Specific growth rate
Total feed intake
Weight gain
2
y = 0.0151x + 1.4565, R = 0.9122
0
25
50
75
100
Artemia protein replacement level (% )
Figure 3a. Linear regression between total feed intake/weight gain/SGR and dietary
Artemia protein inclusion level in goby
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Chapter 5
2
3.0
y = 0.0106x + 1.5341, R = 0.8251
Protein efficency ratio
2.5
2.0
2
1.5
y = -0.0027x + 1.3111, R = 0.7153
1.0
Feed conversion ratio
Feed efficency
2
y = 0.0024x + 0.7469, R = 0.8059
0.5
0.0
0
25
50
75
100
Artemia protein replacement level (%)
Figure 3b. Linear regression between feed conversion ratio/feed efficiency/protein
efficiency ratio and dietary Artemia protein replacement level in goby
4. Discussion
4.1. Goby performance
In the present experiment, the mean survival was 82.5 and 85.0%, the specific growth rate
(SGR) 7.12 and 7.01 % day-1, the feed conversion ratio (FCR) 1.30 and 1.37 and the protein
efficiency ratio (PER) 1.63 and 1.48 for goby P. elongatus fingerling fed the FM control
diet and commercial feed (CF), respectively. These results are comparable to those obtained
in recent laboratory studies. Nhon (2008) and Anh et al. (2009) stocked fry of P. elongatus
(0.56 and 0.74 g initial weight) at different densities between 50 and 250 fish m-2 in
fibreglass and concrete tanks and fed them the same commercial feed as used in this study.
After 30 days of culture, the survival was of 83-95% and 76-85%, the SGR of 6.7-7.5 and
5.5-6.3 % day-1 and the FCR of 1.3-1.5 and 1.4-1.6, respectively.
Considering the weight gain and SGR of P. elongatus fingerlings fed the CF, DA and
various formulated diets during 30 days of indoor nursery rearing, the DA and the Artemiabased formulated diets resulted in better growth of goby compared to the CF and the
control diet. Linear regression analysis revealed that when the dietary Artemia protein
inclusion level increased from 0 to 100%, the total feed intake and growth rate of goby
increased significantly (P<0.05); the greatest feed intake and the best growth performance
occurred at 100% replacement of FM protein by Artemia protein. This result was similar to
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the result obtained for postlarvae of the freshwater prawn Macrobrachium rosenbergii
(Section II of chapter 5).
The superior growth performance of the fish fed Artemia diets as compared to the FM
control and the CF could be due to an increased total feed intake caused by a better
palatability of the Artemia diets. In fact, visual observations in the present experiment
revealed that goby fingerlings displayed a better feeding response to the dried Artemia and
the Artemia based formulated diets than to the other two diets, the CF and the FM control.
Fish fed the Artemia feeds attained satiation within 20-30 min, and by then most of the fish
displayed a bulging belly. This was however not observed in fish fed the CF or FM control
feed. Our results are in agreement with earlier reports that feed intake considerably impacts
the growth rate of fish and crustaceans (Teshima et al., 2000). According to Glencross et al.
(2007), issues relating to feed intake are the key performance criteria in palatability
assessments. These authors pointed out that significant differences in feed intake between
the reference and test diets reflect the apparent palatability of the test ingredient. Wouters,
(2001) reported that incorporation of freeze-dried Artemia biomass into an artificial
broodstock diet improved the performance of the artificial diet by increasing diet ingestion
and improving gonad maturation in female and male Litopenaeus vannamei shrimp. It
furthermore increased reproductive performance. Cook et al. (2003) compared the transfer
of erythromycin from bioencapsulated live and freeze-dried adult Artemia and pellets to fry
of sockeye salmon, Oncorhynchus nerka (Walbaum), and found that both Artemia feeds are
so palatable that they were more immediately and completely consumed by the fish relative
to the medicated pellets. Similar findings were found by Abelin et al. (1989) who reported
that PL 30-45 Penaeus monodon and PL15 P. vannamei receiving a diet containing freezedried Artemia meal had a significantly better growth as compared to the FM based control
diet. Naegel and Rodriguez-Astudillo (2004) illustrated that feeding Litopenaeus vannamei
shrimp PL with dried Artemia biomass resulted in a significant higher survival and larger
size compared to four commercial feeds and three crustacean meals.
Numerous studies have been conducted concerning the nutritional value of formulated
diets, especially on the level of essential fatty acids (EFA) as a principal factor affecting the
dietary value of artificial feeds for normal growth of cultured species (Bureau et al., 2000;
Watanabe, 2002). The EFA requirements differ principally between freshwater and marine
species, but also from species to species. In general, freshwater fish require either dietary
linoleic acid (18:2n-6), or linolenic acid (18:3n-3), or both, whereas marine fish require
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long-chain n-3 highly unsaturated fatty acids (HUFA), such as eicosapentaenoic acid (EPA,
20:5n-3), and/or docosahexaenoic acid (DHA, 22:6n-3) in their diets. EFA deficiency has
been shown to reduce growth rate, reduce feed efficiency, and increase mortality in larvae
and juveniles of fish and crustaceans (Sargent et al., 2002; Craig and Helfrich, 2002;
Tocher, 2003).
The slower growth of the CF-fed group compared to the Artemia-fed groups is probably
caused by a combination of factors, such as poor feed intake, palatability and feed quality.
Chemical analysis indicated that the fatty acid composition of the commercial feed used in
this study seems more appropriate for freshwater fish as it contains high levels of linoleic
and linolenic acids (18.01 and 6.23 mg g-1 DW), but low levels of EPA and DHA (1.12 and
0.23 mg g-1 DW), respectively (Table 4). The goby P. elongatus is a marine fish so this
feed may not meet the nutritional requirements of this species. Goby culture in Vietnam is
relatively new, and the nutritional requirements of this species have not been determined so
far, thus a pellet feed specific for goby is not yet available on the market. The CF feed used
in the current study is to date however the most popular for grow-out culture of goby in
Vietnam (Nhon, 2008; Anh et al., 2009).
Comparing the FM control and Artemia-based diets, it was noted that levels of most fatty
acids were similar. Only the levels of EPA and DHA were different in which a lower EPA
and higher DHA levels was found in the control diet compared to the Artemia-based feeds.
It is however unlikely that this explains the different response of the fish to these diets.
Therefore, as discussed above the difference in feed intake is here the most plausible
explanation. In contrast, the feeding rate (%body weight day-1) was lowest in the dried
Artemia group. This may be attributed to the higher protein and lipid in this diet compared
to the other diets.
Feeding juvenile stages of marine fish is generally less challenging as compared to larvae.
Nevertheless there is a need to maximize the utilization of the feeds in order to improve
cost effectiveness. An important issue during the on-growing stages, when food demand is
high and a major component of production costs is the need to identify and develop
alternative protein sources for fishmeal. Since different fish species have different
nutritional requirements, farmers should ensure that they use a feed specifically formulated
for the species that they are culturing (Day and Howell, 1997). Furthermore, Williams and
Rimmer, (2005) confirmed that different fish species respond differently to different feed
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formulations. For example, barramundi (Lates calcarifer) respond to increasing protein or
lipid levels by growing faster, whereas groupers (Epinephelinae) respond by depositing fat
in the intestinal cavity. Consequently, it is necessary for feed manufacturers to develop and
market specific diets for specific species, or groups of species. However, this tends to
increase the production cost of the diets, because production runs of each formulation are
smaller and savings through economies of scale are reduced.
4.2. Feed utilization
With respect to feed utilization, higher growth rates of fish lead to decreased feed
conversion ratios (FCR) and improved feed efficiency (FE) and protein efficiency ratios
(PER). According to Craig and Helfrich (2002), feed conversion ratios (FCR) will vary
among species, sizes and activity levels of fish, environmental parameters and the culture
system used. FCRs of 1.5-2.0 are considered “good” growth for most species. Additionally,
FCR ranged between 2.3 and 2.6 in the integrated and alternative culture of freshwater
prawn (Lan et al., 2008) and varied from 1.6 to 3.0 in the intensive shrimp farming (Phuong
et al., 2008). The results of the present study indicate that growth rate and feed conversion
improved markedly when Artemia protein was used at inclusion rates above 25% as a
replacement for fish meal protein. The good FCR values (0.97-1.10) obtained in these
treatments are thought to reflect the quality and palatability of the diets formulated. The
high FE and PER achieved in fish fed the Artemia diets indicate they meet the nutrient
requirements for this species.
Conclusions
The results revealed that replacement of fishmeal protein with Artemia protein results in
superior growth performance and better feed utilization compared to a FM control and a
commercial feed. The final body weight of goby fed dried Artemia or a formulated feed
with complete replacement of fish meal by Artemia meal was respectively 1.4 and 1.7 times
higher as compared to the group fed the control feed. Using these feeds one could thus
considerably shorten the nursery phase of goby. The use of dried Artemia or Artemia-based
diets for feeding goby P. elongatus provides good opportunities for the use of locally
available Artemia biomass in the region, reduce the requirement of the aquaculture feed
industry for fishmeal and enhance the sustainability of the industry.Valorization of Artemia
biomass moreover can help Artemia producers to enhance their profits.
205
206
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CHAPTER
General discussion and conclusions
Chapter 6
Chapter 6
General discussion, conclusions and future research
1. Introduction
Aquaculture in Vietnam has developed considerably, which can be attributed to both an
expansion in the production area and improvements in production techniques. Particular
focus is given to diversification of high-value species, improvements in the efficiency of
culture methods, and development of areas for intensive aquaculture. Development and
success in these activities is largely dependent on the availability of high quality food for
larval and postlarval stages of the culture species.
Aside from the successful production of Artemia cysts in the coastal saltworks in Vietnam
to meet the live food demand for the development of shrimp farming, the culture of Artemia
biomass for local use has been practiced since the late 1980s. Most early studies on Artemia
were production-oriented i.e. focused on the culture system, on production parameters and
on population parameters while data collection mainly concerned environmental parameters
(e.g. salinity, temperature, depth, turbidity), which influence the cyst and biomass
production capacity. In biomass-oriented production, the adult abundance is a determinant
parameter, which is, however, still difficult to control. Additionally, the recruitment and
renewal of the Artemia adult population may be low. In this case, Artemia growth could be
limited by the dominant environmental factors and by food availability and thus standard
methods for pond management strategies should be applied. So far these approaches have
not been intensively studied. Moreover, knowledge on processing techniques for Artemia
biomass as feed used for aquaculture species is still limited. Therefore, this thesis covered
several aspects related to Artemia biomass with a focus on optimization of culture
techniques in salt ponds, development of simple drying methods for Artemia biomass and
its application for larviculture and nursery of a number of important aquaculture species in
the Mekong delta, Vietnam.
2. Culture of Artemia biomass in saltworks (Chapter 3)
In order to optimize the culture technique of Artemia biomass, many factors need to interact
in a concerted way, such as technical aspects, population management, weather conditions,
etc. Among these, harvesting strategies and pond management in terms of feed
supplementation in the culture ponds and nutrient management in the fertilization ponds to
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stimulate blooming of appropriate microalgae as natural food for Artemia seem to be the
most important factors affecting biomass productivity.
The experiment on harvesting strategies (Section I) showed that total biomass yield was
linked to adult abundance and its standing stock in culture ponds. The highest values of
these two parameters were generally observed when using a 3-days harvesting interval
compared to three other harvesting frequencies (1-day, 6-days and 9-days interval) resulting
in higher biomass productivity. However, it should be noted that methods for harvesting
Artemia biomass are specific to each location. Not all harvesting strategies are appropriate
for each situation. Factors such as pond management, pond size and morphology, the water
level in the pond, estimated harvestable surplus and climatic conditions often dictate which
harvesting strategies can be used.
In practice, food availability in Artemia ponds almost completely depends on external input
and several methods have been used to increase the food level e.g. adding organic
fertilizers, pumping enriched water, stirring up bottom sediments and supplementing extra
food such as rice bran or other agricultural by-products. Section II evaluated the effect of
different feed supplements on Artemia biomass production in the culture ponds. The results
proved that supplementation with pig manure and rice bran or soybean meal is more
beneficial than the use of natural food alone, as the biomass yield obtained was twice as
high in the former case. It is logical to assume that a higher diversity of supplied feeds leads
to better growth, survival and productivity; firstly these products are a direct food source,
but they also act through the hetero-trophic food chain by supplying organic matter and
detritus to the pond ecosystem; the manure serves mainly as a substrate for the growth of
bacteria and protozoa, which in turn serve as a protein-rich food for Artemia (Intriago and
Jones, 1993). Although the C/N ratios of the supplemented products used in the present
works were in the recommended ranges for bacterial growth, the effects of different C/N
ratios of supplemental feeds on the food available for Artemia production has not been
investigated in this study.
It is well known that microalgae are the best natural food for Artemia. However, it is
unlikely that a stable growth of algae of suitable size in the Artemia culture ponds can be
maintained as food for Artemia due to the high salinity (70-100 g L-1) and low water level
(20-40 cm above the platform) in the ponds. Moreover, when inorganic fertilizers are added
to the culture ponds, filamentous algae (lab-lab: mainly consists of filamentous algae; they
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can float after developing on the pond bottom) most certainly develop. These filamentous
algae can not be taken up by Artemia and they interfere with brine shrimp swimming and
filtering activity because animals are trapped in the algae filaments, furthermore, this lablab interferes with biomass collection. On the other hand, primary productivity is strongly
affected by the total nitrogen:total phosphorus (TN:TP) ratio present in the water column.
Therefore, as a practical solution to avoid this limitation, the management of the algal
bloom by utilizing different N:P ratios in the fertilization ponds to provide “green water”,
in combination with supplemental feeding to the Artemia ponds, can be the best approach
to optimize culture techniques, as has been described in Section III. Our results indicated
that using inorganic fertilizer with a ratio of N:P =5 combined with pig manure for
microalgae production as feed for Artemia appears to be more cost-effective than a ratio
N:P=10, as illustrated by the higher biomass yield and economic return in the former case,
although significant difference was not detected.
When comparing our three experiments conducted on Artemia culture in saltworks, our
results revealed that total Artemia biomass yield improved year by year. For instance, the
highest biomass yield (1.6 ton ha-1) achieved in Section I, was lower than the average
biomass production in Section II (1.9 ton ha-1) and Section III (2.2 ton ha-1). This could be
due to some specific aspects of pond management related to the experimental design. As
the experiment in Section I focused on partial harvesting strategies, water exchange was not
done throughout the experiment whereas in the two other experiments about 10-20% of
pond water was exchanged once a week from week 6 onwards. In practice, after 6-7 weeks
of inoculation the Artemia density became higher, with a dominance of larval and juvenile
stages and culture pond conditions are becoming worse (low food, high salinity and
temperature, oxygen stress, etc.). Hence, water exchange is necessary so that part of the
Artemia population (mainly nauplii and metanauplii passing through the mesh of the net) is
also discharged to reduce density, new water is added to the pond and more natural food is
introduced to favour growth and renewal of the Artemia population.
It was observed in Section III that when green water was produced from the N:P=5
treatment combined with a sole supplement of pig manure or rice bran, biomass yield (2.3
ton ha-1) was better than or equal to the treatments in Section II with a single supplement of
pig manure (1.8 ton ha-1) and a co-supplement of pig manure and rice bran or soybean
meal (2.3 ton ha-1), respectively. Moreover, the amount of feed supplement utilized in
Section III was 30% lower than in Section II suggesting that a proper management of the
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fertilization pond not only improves biomass productivity but also decreases the utilization
of inert foods in the culture ponds and thus reduces the production costs.
With regard to abiotic parameters, especially temperature appears to be one of the main
uncontrollable factors affecting biomass productivity. Temperature increases from January
to May in the dry season, and high production of Artemia cysts and biomass usually occurs
in the period February-March when water temperature is less than 35°C. Towards the end
of the dry season between April and May, lethally high temperatures often occur in the
afternoon (>37°C at 14:00). In that period, Artemia lives in an environment with high
salinity and temperature meaning that at the same time they have to cope with low oxygen
levels and limited food as a result of which they spend more energy to adapt to these
extreme conditions, which cause high mortality, slow growth and poor recruitment rate.
Consequently, productivity is low towards the end of the culture season. In particular, the
reproductive mode also seems to be influenced by temperature i.e. ovoviviparous
reproduction increases with increasing temperature. This is illustrated by the linear
regression analysis for the three experiments, which showed that the percentage of
ovoviviparous Artemia females was positively correlated with water temperature (R² =
0.74-0.86, P<0.01) leading to a booming population density, mainly dominated by larvae
and juveniles as observed in Section I. Because in that period low trophic and more extreme
pond conditions can happen, it is recommended that animal densities should be reduced by
either partial harvesting in the early morning, when they concentrate in the pond corner on
windless days, or at the time of water exchange. These animals can be used for feeding
larvae and postlarvae of shrimp or mud crab in the local hatcheries, which are located
nearby the Artemia farming units. By this procedure, less food competition occurs and
over-carrying capacity of the ponds may be minimized and both productivity and economic
efficiency of the culture system can be improved.
In the Artemia population always two modes of reproduction exist: oviparity (production of
dormant eggs or cysts) and ovoviviparity (production of free-swimming nauplii). Data
obtained in our three experiments indicated that the highest percentage of ovoviviparous
Artemia females seldom exceeded 50%. Therefore, biomass harvesting is done partially
during the culture period and the remaining egg-bearing females release cysts, which can
then be harvested. Earlier studies found that for biomass production in a culture area
≥700m², side-harvest of Artemia cysts can also amount to 17 to 60 kg cysts wet weight ha-1
crop-1 corresponding with 30-75% as compared to the pure cyst-oriented production
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(Brands et al., 1995; Anh et al, 1997a; Anh and Hoa, 2004). This product can contribute
significantly to enhance the income from the biomass production system, because top
quality Vietnamese Artemia cysts are sold at approximately 230 US$ kg-1 dry product in
2009. However, in the present experiments negligible amounts of cysts were collected due
to the small size of the experimental ponds (300m²).
In this study the ponds were constructed 2 m wide, 30-40 cm deep, with a peripheral ditch
and with maximum water levels attaining 50-60 cm from the platform by the end of the
culture period. Although the yield of Artemia biomass in the present work increased year
by year, the highest productivity is still lower than the results obtained in previous studies
(Brands et al., 1995; Anh et al., 1997a). These authors reported that the higher yield
obtained in their studies are because of the deeper ponds and the more intensive
management conditions. Anh et al. (1997a) conducted experiments at two different water
depths of 30 and 60 cm in 200m² flat bottom ponds, without central platform to evaluate the
possibility to increase biomass productivity by culturing Artemia at higher water levels, and
achieved 4.9 and 8.1 ton ha-1 after 5 months of culture. A similar finding was observed for
cyst production, where the average Artemia cyst yield in deep ponds (60 cm) was 46.3%
higher than in shallow ponds (30 cm) (Brands et al., 1995). As the pond volume is larger in
deep ponds than in shallow ponds, it is possible to improve the yields by increasing the
water depth and thus with the same population density the Artemia standing stock per
surface unit is increased which leads to enhanced production capacity. Increasing the water
level may also help to diminish the negative impact of high water temperatures during the
mid and late dry season.
3. Drying Artemia biomass (Chapter 4)
Because Vinh Chau and Bac Lieu saltworks where Artemia farming is practiced, are
located in remote areas of South Vietnam, transportation of live Artemia biomass over long
distances from the culture sites to the other places is costly and may only be economically
feasible for application in the culture of highly valuable species. Moreover, newly
harvested Artemia biomass has a very high moisture content (approximately 90% water),
and is highly perishable. Consequently, developing long shelf-life products is extremely
important for the further application of Artemia biomass (Léger et al., 1986; Baert et al.,
1996). Among the different preservation methods for plant and animal products, drying can
211
Chapter 6
be a good approach to maintain a stable product quality (FAO, 1992; Brennand, 1994;
George et al., 2004).
Data obtained in our study (Section I) indicated that the quality of Artemia biomass, in
terms of total lipid and fatty acid profile, dried by intermittent microwave combined with
conductive hot air drying was comparable to frozen Artemia (control). In addition, this
technique resulted in faster drying and better quality as compared to the convective hot air
and oven drying, but required high capital investment and power supply for operation
which may be economically prohibitive for large-scale application or impractical for remote
regions where electricity is not always available. Results in Section II revealed that when
Artemia biomass was dried by a natural convective solar dryer on sunny days or on days
with sunny intervals, the drying time was considerably reduced and the product quality
(total lipid and fatty acid contents and lipid classes) was superior to open sun drying and
less different to fresh Artemia (control). Therefore, this preliminary study suggests that
under Vietnamese climatic conditions an experimental solar dryer can be considered a
cheap and simple method for drying biomass on the production site. However, a limitation
in outdoor drying is the need to add a preservative (an antioxidant such as butylated
hydroxytoluene) to prevent product spoilage during the drying process while Artemia
biomass dried by the indoor drying techniques can be kept free from preservative.
Application of Artemia biomass for some target aquaculture species (Chapter 5)
Our results revealed that for larval rearing of tiger shrimp Penaeus monodon (Section I),
growth performance (length and dry weight) and stress tolerance were significantly lower
in larvae supplemented with practical feeds containing fresh or dried Artemia as compared
to larvae receiving only commercial feeds or the ones fed a combination of both. This could
be associated to a lack of essential fatty acids in the Artemia-based formulated diets
because the nutritional value of Artemia used in this study indicated a low level of DHA
(see Section I of chapter 4). Moreover, different processing techniques between the
commercial INVE feed and the experimental diets also could have affected feeding
treatments i.e. the commercial feed is a microencapsulated feed which has a high water
stability while the Artemia-based practical diets were microparticulate diets which may
have a high leaching rate resulting in loss of water-soluble nutrients.
212
Chapter 6
For nursery culture of Macrobrachium rosenbergii both survival and growth performances
of prawn PL were positively correlated with the dietary Artemia protein inclusion level.
The results also showed that the performance of prawn PL fed diet with complete
replacement of fishmeal protein with Artemia protein was superior to those fed the fishmeal
control diet (Section II).
Goby Pseudapocryptes elongatus fry fed Artemia-based formulated feeds or dried Artemia
biomass were significantly larger than fish consuming fish meal control diet or a
commercial pellet feed produced in Vietnam; moreover feed utilization was also more
efficient (Section IV).
Furthermore, performance of mud crab Scylla paramamosain crablets fed live or frozen
Artemia biomass was superior to the group consuming fresh shrimp meat. However, crabs
fed the dried Artemia-based formulated feed showed the poorest performance in survival
and growth (Section III), which could be because this diet had poor water stability resulting
in leaching of water-soluble nutrients. It was observed that when dried Artemia meal
obtained by different drying methods were used for the feeding trials, the results indicated
that animals fed the dried Artemia-based diets had a poorer performance than the groups
fed the fresh or frozen Artemia-based diets, although differences were generally not
statistically significant. These findings demonstrated that Artemia biomass can be applied
either as direct food (live, frozen and dried forms) or as an ingredient in formulated diets, to
make a suitable food for various species. Moreover, it is apparent that Artemia biomass is a
high-quality protein source, which can potentially totally replace fishmeal in practical diets
for highly valuable cultured species.
In Vietnam, in Artemia cyst-oriented production ponds, apart from cysts, large quantities of
Artemia biomass (on average 0.2-0.3 ha-1) can be collected when the culture season ends
(Brands et al., 2005; Hoa et al., 2007). As a large amount of Artemia biomass is thus locally
available, the use of this product in aquaculture can help the Artemia farmers to diversify
their production and enhance their income. This could also contribute to a reduced use of
fishmeal in aquafeeds.
213
Chapter 6
5. Future research
The present study was conducted to address the critical technical issues of Artemia biomass
production, to contribute to the development of a simple cheap drying method for Artemia
biomass, as well as to optimize its application in target aquaculture species in the Mekong
Delta, Vietnam. While this study has met the research objectives, the results generated
suggestions for further research:
-
Evaluating the effect of different C/N ratios of the supplementary feeds on the growth
of heterotrophic bacteria in water and benthos can provide useful information on the
importance of natural biota, particularly the flocculated particles as food source for
Artemia production.
-
Investigating the intensive production of Artemia biomass in deep ponds to enhance
productivity.
-
For safe use of outdoor-dried product in aquaculture, further research should be
conducted on the co-drying of Artemia biomass with other ingredients, e.g. Artemia
biomass mixed with rice bran, soy bean meal or other ingredients, to absorb water in
order to shorten its drying time and to maintain its quality without using chemical
substances, thus producing a more suitable ingredient for aquafeeds.
-
Aside from drying to sustain the shelf-life of Artemia biomass, future investigations
need to evaluate other processing methods such as hydrolyzing and liquefying or
fermenting the fresh Artemia biomass in order to produce other added-value Artemia
products such as high-quality feeds for aquaculture species, Artemia sauce for human
consumption, etc.
-
Formulation of Artemia-based formulated feeds for larval rearing must take into
account both the supplement of essential fatty acids and improvement of water stability.
-
Further studies are needed for the application of Artemia biomass for other aquaculture
species in the larviculture and nursery phases, as well as for broodstock, which could
contribute to further commercial production of Artemia biomass as high quality feed in
aquaculture in Vietnam.
214
Chapter 6
215
CHAPTER
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238
Summary/Samenvatting
Summary
This thesis provides new information on various aspects of Artemia biomass production in
solar salt ponds in the Mekong Delta, Vietnam, more specifically on culture techniques,
drying methods and its applications for target aquaculture species.
With the aim to optimize Artemia biomass production in the salt ponds (chapter 3), firstly
we evaluated different harvesting strategies on Artemia biomass production; the results
showed that the highest biomass yield was achieved when using a 3-days harvesting
interval, i.e. 17%, 31% and 39% higher compared to the 1-day, 6-days and 9-days
harvesting frequencies, respectively. Hence, partial harvest of Artemia biomass done every
3 days seems to be an appropriate strategy to enhance biomass productivity in the Mekong
delta salt ponds (Section I). Secondly, the effect of different supplemental feeds on the
production and quality of Artemia biomass was assessed. The results illustrated that
different supplemental feeds had a significant effect on growth and total yield, but did not
affect the proximate composition of the Artemia biomass. Artemia ponds supplied with pig
manure alone or in combination with rice bran or soybean meal as supplemental feeds gave
significantly higher biomass yields compared to the control, only receiving green water as
natural food. Our results show that the co-supplementation of pig manure and rice bran or
soybean meal can be applied for culture of Artemia in salt ponds (Section II). Furthermore,
we investigated the effect of different ratios of N:P on primary productivity, combined with
feeding strategies for Artemia biomass production in salt ponds. The results revealed that
when applying ratios of N:P=5 and 10 in the fertilization pond, the chlorophyll a
concentration and algal composition was similar. Bacillariophyta (diatoms) were the
dominant group over the sampling period. When using this green water combined with rice
bran or pig manure for rearing Artemia, the result illustrated that total biomass yields in the
N:P=5 treatment were better than in the N:P=10 treatment but not statistically different at
P=0.05. An economic analysis showed that higher profits could be obtained by using the
N:P=5 ratio to produce green water as natural food and pig manure as feed supplement for
production of Artemia biomass in salt ponds (Section III).
Investigation of drying methods for Artemia biomass was performed to find out an
appropriate drying method in terms of economic aspects and product quality (chapter 4). A
thin layer of Artemia biomass dried by outdoor sun drying was compared with three indoor
drying techniques namely convective hot air drying (HA), intermittent microwave
239
Summary/Samenvatting
combined with convective hot air drying (MWHA) and oven drying at temperatures of 50,
60 and 70°C. The results showed that among the three indoor drying techniques, the
shortest drying times were observed in MWHA followed by HA and oven drying,
respectively, while sun drying showed the longest dehydration times compared to other
drying methods. Moreover, the drying time decreased with increasing temperature. Overall,
for the three indoor drying methods total lipid content and fatty acid profile of the dried
Artemia in most cases was not significantly different from the frozen Artemia (control).
Although sun drying resulted in significant reductions of these nutrients, it is much less
energy consuming. The intermittent MWHA drying is a promising technique, which could
produce high quality dried products in short drying times. However, it may not be
appropriate for large-scale application in the coastal area of the Mekong delta because of
high capital investment and operating costs (Section I). Therefore, another experiment was
conducted to compare the performance of convective solar drying with open sun drying for
Artemia biomass under various weather conditions in the South of Vietnam. The results
indicated that when drying Artemia biomass on sunny days or days with sunny intervals,
the drying time was substantially reduced with 45% and 25%, respectively, compared to
open sun drying. However, on cloudy/rainy days the reduction was only 7%. Generally,
total lipid content, lipid class composition and fatty acid profile was better in solar-dried
Artemia than in sun-dried Artemia samples. This preliminary work has proven that the use
of a natural convective solar drier for drying Artemia biomass can be a cheap and easily
adoptable method for farmers at household level, which could produce an acceptable
quality of dried Artemia (Section II).
The use of different Artemia biomass preparations as feeds in the larviculture and nursery
phases was also investigated for a few important cultured species in the Mekong delta
(Chapter 5). A first study evaluated the effect of formulated feeds containing fresh or dried
Artemia biomass as live food supplement in the larval rearing of black tiger shrimp,
Penaeus monodon (Section I). We found that the time of metamorphosis of the shrimp
larvae in the different stages was the same and shrimp survival in all developmental stages
showed no statistical differences among feeding treatments (P>0.05). Nonetheless,
postlarval performance in the combination treatments (commercial INVE feed and Artemiabased formulated diets) were better or equal compared to those fed commercial feed alone
as seen by the better growth rate and higher resistance to formalin stress. The results
240
Summary/Samenvatting
indicate that feed containing fresh or dried Artemia can partially supplement live feeds for
larval rearing of P. monodon.
A second study dealt with the effect of fishmeal replacement with Artemia biomass as
protein source in practical diets for the giant freshwater prawn Macrobrachium rosenbergii.
Nine experimental diets were formulated by replacing 0, 25, 50, 75 and 100% of the
fishmeal protein in a standard diet with either dried or frozen Artemia biomass. In the 0%
Artemia treatment, Peruvian fishmeal was the main protein source. Results demonstrated
that survival and growth performance of prawn PL were enhanced with increasing dietary
Artemia protein inclusions. It can be suggested that Artemia biomass may totally replace
fishmeal in practical diets for PL of the prawn M. rosenbergii (Section II).
Furthermore, the use of different forms of Artemia was tested as feed for mud crab, Scylla
paramamosain. Instar 1 crablets were reared both individually and communally. Data on
survival and growth suggested that crab performance decreased in the order: live
Artemia>frozen Artemia>fresh shrimp meat>dried Artemia-based formulated diet. Our
findings illustrate the high potential for local utilization of Artemia biomass in Vietnam for
reliable production of mud crab juveniles (Section III).
In the following, feeding trial Artemia biomass was evaluated for fingerlings of the goby
Pseudapocryptes elongatus. The results proved that survival of the goby was not affected
by the feeding treatments. The goby fed the fishmeal control diet and the commercial feed
had a similar growth performance (mean final body weight, weight gain, specific growth
rate) and feed utilization (total feed intake, feeding rate, feed conversion ratio, feed
efficiency and protein efficiency ratio). Both were however significantly poorer compared
to the groups fed dried Artemia and the groups fed the Artemia based-diets from the 50%
replacement level onwards. This suggests that both dried Artemia and Artemia based-diets
can be used for feeding goby P. elongatus fingerlings (Section IV).
All the feeding trials in the current study demonstrated that Artemia biomass can be used
either as direct feed or as ingredient in formulated feeds. Moreover, valorisation of Artemia
biomass for local commercial culture of fish or shrimp could enhance the income of
Artemia producers and contribute to reduce the reliance on fishmeal in aquafeeds, which is
of high socio-economic relevance in the Mekong delta, Vietnam.
241
Summary/Samenvatting
The knowledge obtained through this thesis work could contribute to the optimization of
Artemia pond production and a more sustainable development of Artemia farming, leading
to a better understanding of the drying methods for Artemia biomass, and to the
development of applications of Artemia biomass in the culture of aquaculture species in the
region.
242
Summary/Samenvatting
Samenvatting
Deze thesis verschaft nieuwe informatie over verschillende aspecten van Artemia biomassa
productie in zoutpannes in de Mekong delta in Vietnam, meer specifiek over
kweektechnieken, droogtechnieken en de toepassing ervan voor aquakultuursoorten.
Met het doel de productie van Artemia biomassa in zoutpannes te optimaliseren (hoofdstuk
3), werden eerst verschillende strategieën voor de oogst uitgetest op de Artemia biomassa
productie; de resultaten toonden aan dat de hoogste biomassa opbrengst verkregen werd
wanneer een 3-dagen oogstinterval gehanteerd werd. Hierbij was de biomassa productie
17%, 31% en 39% hoger in vergelijking met respectievelijk oogstintervallen van 1 dag, 6
dagen en 9 dagen. Dus gedeeltelijk oogsten om de 3 dagen lijkt een geschikte strategie om
de biomassa productiviteit in Vietnamese zoutpannes te verbeteren (Deel I). Ten tweede
werd het effect nagegaan van verschillende voedersupplementen op de productie en
kwaliteit van de Artemia biomassa. De resultaten illustreerden dat verschillende
voedersupplementen een significant effect hadden op groei en opbrengst, maar dat deze de
samenstelling van de Artemia biomassa niet beïnvloedden. Artemia vijvers voorzien van
enkel varkensmest of een combinatie van varkensmest en rijstzemelen of sojameel als
voedersupplement resulteerden in significant hogere opbrengsten in vergelijking met een
controle, die enkel groen water als natuurlijk voedsel kreeg. Onze resultaten tonen aan dat
supplementatie met varkensmest samen met rijstzemelen of sojameel kan toegepast worden
voor de kweek van Artemia in zoutpannes (Deel II). Verder onderzochten we het effect van
verschillende N:P verhoudingen op de primaire productiviteit, gecombineerd met
voederstrategieën voor Artemia biomassa productie in zoutpannes. De resultaten toonden
aan dat wanneer een N:P verhouding van 5 of 10 gebruikt werd in de bemestingsvijver, de
chlorophyl a concentratie en algensamenstelling gelijk bleef. Bacillariophyta (diatomeeën)
waren de dominante groep gedurende de periode van staalname. Wanneer dit groen water
gecombineerd werd met rijstzemelen of varkensmest voor de kweek van Artemia, toonden
de resultaten aan dat een N:P verhouding van 5 beter was dan een verhouding van 10; dit
verschil was echter niet statistisch significant (P>0.05). Een economische analyse toonde
aan dat een hogere winst kan bekomen worden door gebruik te maken van de N:P = 5
verhouding om groen water te produceren als natuurlijk voedsel en varkensmest te
gebruiken als voedersupplement voor de productie van Artemia in de zoutvijvers (Deel III).
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Summary/Samenvatting
Onderzoek naar droogmethodes voor Artemia biomassa werd uitgevoerd om een geschikte
droogtechniek in termen van ecomische aspecten en productkwaliteit te vinden (hoofdstuk
4). Het in dunne lagen buiten drogen van Artemia biomassa werd vergeleken met drie
technieken voor binnenshuis drogen, namelijk “convective hot air drying” (HA),
“intermittent microwave gecombineerd met “hot air drying” (MWHA) en ovendrogen bij
50, 60 en 70 °C. De resultaten toonden aan dat van de drie technieken van binnenshuis
drogen, de kortste droogtijden bekomen werden met MWHA, gevolgd door HA en
ovendrogen, terwijl zondrogen de langste droogtijden had vergeleken met de andere
droogtechnieken. Verder verminderde de droogtijd met toenemende temperatuur. Over het
algemeen was het totale vetgehalte en de vetzuursamenstelling van de gedroogde Artemia
voor de drie verschillende technieken voor binnenshuis drogen niet verschillend van
ingevroren biomassa (controle). Hoewel zondrogen resulteerde in een significante afname
van deze nutriënten, verbruikt het veel minder energie. MWHA drogen is een
veelbelovende techniek, die gedroogde producten van hoge kwaliteit kan produceren in
korte tijd. Het lijkt echter minder geschikt voor toepassing op grote schaal wegens de grote
kapitaalsvereisten en operationele kosten (Deel I). Voor die reden werd een ander
experiment uitgevoerd om de werking van “convective solar drying” te vergelijken met
open zondrogen voor Artemia biomassa onder verschillende weerscondities in ZuidVietnam. De resultaten toonden aan dat wanneer Artemia gedroogd wordt op zonnige dagen
of dagen met zonnige perioden, de droogtijd significant gereduceerd werd met 45% en 25%
respectievelijk, in vergelijking met open zondrogen. Op bewolkte/regenachtige dagen
echter was deze reductie slechts 7%. Over het algemeen was het totale vetgehalte,
lipideklassen-samenstelling en vetzuurprofiel beter in de solar-gedroogde Artemia dan in de
zongedroogde Artemia stalen. Dit preliminair werk heeft bewezen dat het gebruik van een
natuurlijke “convective solar droger” voor het drogen van Artemia biomassa een goedkopen
en gemakkelijk toepasbare methode kan zijn voor individuele boeren, die een aanvaardbare
kwaliteit van gedroogde Artemia kan opleveren (Deel II).
Het gebruik van verschillende Artemia bereidingen als voedsel in de larvale en nursery
kweek van belangrijke gekweekte soorten in de Mekong delta werd ook nagegaan
(hoofdstuk 5). Een eerste studie onderzocht het effect van geformuleerde voeders op basis
van verse of gedroogde Artemia biomassa als supplement voor levend voedsel voor de
larvale kweek van tijgergarnalen Penaeus monodon (Deel I). We vonden dat het tijdstip van
metamorfose in de verschillende stadia niet statistisch significant verschillend was tussen
244
Summary/Samenvatting
de behandelingen (P>0.05). Echter werd er een betere of gelijke groei en stressresistentie
tegen formaline vastgesteld in de postlarven in de combinatie-behandelingen (commercieel
Inve voeder en Artemia voeders) in vergelijking met de behandeling met enkel
commercieel voeder. De resultaten tonen aan dat voeder op basis van verse of gedroogde
Artemia gedeeltelijk het levend voedsel kunnen supplementeren voor de larvale kweek van
P. monodon (Deel I).
Bijkomend werd het effect bestudeerd van de vervanging van vismeel door Artemia
biomassa als eiwitbron in praktische voeders voor Macrobrachium rosenbergii. Negen
experimentele voeders werden geformuleerd door 0, 25, 50, 75 en 100% van het vismeeleiwit in een standaard voeder te vervangen door ofwel gedroogde of ingevroren Artemia
biomassa. In de 0% Artemia behandeling was Peruviaans vismeel de enige eiwitbron. De
resultaten toonden aan dat overleving en groei van de postlarven verbeterd werd met
toenemend Artemia-eiwitgehalte in het voer. Er kan gesteld worden dat Artemia biomassa
volledig het vismeel kan vervangen in praktische voeders voor M. rosenbergii (Deel II).
Verder werd het gebruik van verschillende vormen van Artemia als voeder voor mangrove
krab Scylla paramamosain getest. Instar I krabben werd zowel individueel als in groep
gekweekt. Data van overleving en groei suggereren dat de resultaten daalden in de volgorde
levende Artemia > ingevroren Artemia > vers garnaalvlees > geformuleerd voeder op basis
van gedroogde Artemia. Onze resultaten tonen het grote potentieel aan van lokaal gebruik
van Artemia biomassa voor de betrouwbare productie van mangrove krab juvenielen (Deel
III). Verder werd er ook een voedertest uitgevoerd met goby Pseudapocryptes elongatus
juvenielen. De resultaten toonden aan dat overleving van de goby niet beïnvloed werd door
de behandelingen. De goby die gevoederd werden met het vismeel-controle voeder en het
commerciële voeder hadden een gelijke groei (gemiddeld finaal lichaamsgewicht,
gewichtstoename en specifieke groeisnelheid) en voedergebruik (totale voederinname,
voedingsniveau, voederconversie, voederefficiëntie en eiwitefficiëntie). Beide waren echter
significant slechter dan de groepen die gevoederd werden met gedroogde Artemia en de
groepen gevoederd met voeders op basis van Artemia vanaf een vervangingsniveau van
50% en hoger. Dit toont aan dat zowel gedroogde Artemia als geformuleerde voeders op
basis van Artemia kunnen gebruikt worden voor het voederen van goby Pseudapocryptes
elongatus juvenielen (Deel IV).
Al de voedertesten in de huidige studie toonden aan dat Artemia biomassa ofwel kan
gebruikt worden als direct voeder of als ingrediënt in geformuleerde voeders. De valorisatie
245
Summary/Samenvatting
van Artemia biomassa voor lokale commerciële kweek van vis of garnaal zou het inkomen
van Artemia producenten kunnen verbeteren en bijdragen tot het verminderen van de
afhankelijkheid van vismeel in visvoeders, welke een hoge socio-economische relevantie
hebben voor de Mekong delta in Vietnam.
De kennis behaald in deze thesis kunnen bijdragen tot de optimalisatie van Artemia kweek
in vijvers en de duurzame ontwikkeling van Artemia kweek, leiden tot een betere kennis
van de droogtechnieken voor Artemia biomassa en de ontwikkeling van toepassingen van
Artemia biomassa in de kweek van aquakultuur soorten in de regio.
246
Curriculum vitae
PERSONAL DATA
Name: NGUYEN THI NGOC ANH
Born: in Can Tho city (Vietnam), February 16th, 1966.
Status: married
Address: 114/4A- Tam Vu Street, Ninh Kieu ward, Can Tho city, Vietnam
Telephone: + 84.7103.738 511, Mobile: + 84. 947 445 886
Language: Vietnamese and English
Office: College of Aquaculture and Fishery- Can Tho University
Campus 2, 3/2 Street, Can Tho city, Vietnam
Telephone : +84.7103.834 307
Fax : + 84. 7103.830 323
E-mail: [email protected]
EDUCATION
-
1990: MBA degree, College of Aquaculture & Fishery - Can Tho University,
Vietnam.
-
2000: Certificate of MSc in Aquaculture - Ghent University, Belgium
PROFESSIONAL ACTIVITIES
1990-1998:
-
Research on production of Artemia cyst/biomass in the Vinh Chau salt fields
-
Research on the development of primary production (algae) in the brackish
ponds, and integrated system of aquaculture.
-
Attend the extension and transfer culture techniques of Artemia for farmer in
Soc Trang and Bac Lieu provinces.
1998-2000: Study MSc program at Ghent University, Belgium.
247
2001-2005:
-
Study on the effect of abiotic and biotic factors on Artemia biomass
production in the salt works.
-
Attend the extension and transfer culture techniques of Artemia for farmers in
Bac Lieu salt woks.
-
Study on the effect of food and hormonal substances on the reproductive
performance of goby, Pseudapocryptes elongatus.
-
Study on intensive grow-out culture of goby, Pseudapocryptes elongatus.
2005-2009: Study PhD program at Ghent University, Belgium.
TEACHING
-
Training course of integrated system of rice-fish, pig-fish for farmers.
-
The practical course of the brackish aquaculture for students in University and
College.
-
The culture techniques of seaweed for students in University and College.
CONFERENCE ATTENDED
-
The First Symposium on Artemia Culture in Vietnam, Can Tho University,
Vietnam. April 16-18, 1993.
-
The First National Symposium on Marine Biology, Nha Trang Institute of
Oceanography, Vietnam. October 26-28, 1995.
-
The International Training Course on Artemia. Ghent University, Belgium.
July-August, 1996.
-
The final Workshop of JIRCAS Mekong Delta Project. Can Tho University,
Vietnam. November 25-26, 2003.
-
Scientific Conference on Aquaculture. College of Aquaculture & Fishery, Can
Tho University, Vietnam, May 28, 2004.
-
Larvi’05 Fish and Shellfish Larviculture Symposium. Ghent University,
Belgium. September 5-8, 2005.
248
-
Scientific Conference on Aquaculture. College of Aquaculture & Fishery, Can
Tho University, Vietnam. April 28, 2006.
-
Asian-Pacific Aquaculture Conference. Ha Noi, Vietnam, August 5-8, 2007.
-
The ViFINET International Aquaculture Workshop, Can Tho University,
Vietnam. December 5-8, 2008.
-
The International Catfish Symposium, Can Tho University, Vietnam.
December 5-7, 2008.
PAPER PRESENTED
-
ANH, N.T.N, Hien, T.T.T., Hoa, N.V., Wille, M. and Sorgeloos, P. 2007.
Effect of fishmeal replacement with Artemia biomass as protein source in
practical diets for the giant freshwater prawn Macrobrachium rosenbergii.
Paper presented at Asian-Pacific Aquaculture Conference. Ha Noi, Vietnam,
August 5-8, 2007.
-
ANH, N.T.N, Nhi, N.T., Thanh, V.Q., Hoa, N.V., Van Stappen, G.,
Baerdemaeker, J.D. and Sorgeloos, P. 2008. Effect of different indoor and
outdoor drying techniques on lipid & fatty acid composition of dried Artemia
biomass. Paper presented at the ViFINET International Aquaculture
Workshop, December 5-8, 2008, Can Tho University, Vietnam.
NATIONAL PUBLICATIONS
-
Anh, N.T.N., Quynh V.D., Hoa, N.V., Baert, P., 1997. Potential for Artemia
biomass production in Vinh Chau salterns. Proceedings of the First National
Scientific Symposium on Marine Biology, Scientific and Technology
Publishing House, Vietnam (Abstract in English), 410-417.
-
Anh, N.T.N., Quynh V.D., Hoa N.V and Baert, P., 1997. Present situation of
Artemia and salt production in the coastal salinas from Soc Trang and Bac
Lieu provinces. Scientific Journal of Can Tho University, Vietnam (Abstract
in English).
-
Anh, N.T.N., Hoa N.V. 2004. Effect of harvest strategies on Artemia biomass
production in the saltfields. Scientific Journal of Can Tho University, Vietnam
(Abstract in English).
249
-
Hoa, N.V., Van, N.T.H., ANH, N.T.N., Ngan, P.T.T., Toi, H.T. and Le, T.H.
2007. ARTEMIA - Research and application in aquaculture. Technical book.
Can Tho University, Agricultural publishing house, Vietnam (in Vietnamese),
134 pp.
INTERNATIONAL PUBLICATIONS
-
Baert, P., ANH, N.T.N., Quynh, V. D and Hoa, N. V., 1997. Increasing cyst
yields in Artemia culture ponds in Vietnam: the multi-cycle system.
Aquaculture Research 28, 809-814.
-
Baert, P., ANH, N.T.N., Burch, A. and Sorgeloos, P. 2002. The use of
Artemia biomass sampling to predict cyst yields in culture ponds.
Hydrobiology 477, 149-153.
-
ANH, N.T.N. 2004. Culture techniques for Artemia biomass production in
saltpans of the Mekong delta, Vietnam (Aquaculture compendium–cab
international).
-
ANH, N.T.N., Hoa, N.V., Van Stappen, G. and Sorgeloos, P. 2009. Effect
of different supplemental feeds on proximate composition and Artemia
biomass production in salt ponds. Aquaculture 286, 217-225.
-
ANH, N.T.N., Hien, T.T.T. Mathieu, W., Hoa, N.V. and Sorgeloos, P. 2009.
Effect of fishmeal replacement with Artemia biomass as protein source in
practical diets for the giant freshwater prawn Macrobrachium rosenbergii.
Aquaculture Research 40, 669-680.
250