1. business and industrial
2. agriculture and forestry
3. aquaculture

Algae Meals as a substitute forfish meal and fish oil in practical diets for the Pacific white
shrimp (Litopenaeus vannamei)
by
Lorena Garcia
B.Sc., Jorge Tadeo Lozano University, 2009
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES
(Applied Animal Biology)
THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)
December 2013
© Lorena Garcia, 2013
Abstract
Traditionally, marine fish meal (FM) and fish oil (FO) have been the major feed
ingredients used as protein and lipid sources in the formulation of aquaculture feeds. However,
these commodities are limited by their availability, high cost as well as negatively impacting the
sustainability of wild fish stocks. Thus, finding alternatives sources to replace or reduce the use
of FM and FO in aquafeeds is critical. The primary goal of this research was to determine the
effects incorporating algal meals into diets fed to the Pacific white shrimp (Litopenaeus
vannamei) on production for and organoleptic qualities. Twenty circular tanks composed an
outdoor closed system with a volume capacity of 530-L. Specific Pathogen-Resistant (SPR)
Pacific white shrimp (mean weight ± SD, 1.31± 0.029) were stocked at a density of 28
shrimp/m2 in a green water system.Shrimp were assigned five dietary treatments.The
experimental diets,fed twice daily, were isolipidic (8%), and contained three protein levels: 34%,
37% and 48%. Spirulina and Schizochytrium were used to substitute FM and FO in the
experimental diets. Two FM based feeds were used as controls. The production parameters: final
weight (9.4 to 14.9 g), percent weight gain (479-680%), growth rate (0.7-1.3 g/week), feed
conversion ratio (1.6-2.5) and survival (85-95%) were estimated. Treatment effects for
production parameters were evaluated using one-way analyses of variance (ANOVA).
Differences (P ≤ 0.05) among the treatments existed for all production parameters except
survival. Organoleptic qualities of shrimp wereanalyzed by analysis of variance (ANOVA).
Differences existed for the desirable attributes of aroma, color and sweetness, but not for the
attributes of firmness, moistness, and fattiness. Differences were not detected for the undesirable
attributes off-odor,fish flavor, earthiness, off-flavor, and overall unpleasantness. The diet
containing Spirulinaat a 37% protein level with the inclusion of DHA from Schizochitrium, was
ii
perceived as not containing undesirable attributes and thus shows promise. Results from this
study show that Pacific white shrimp can accept algal meals in their feeds and that the inclusion
of certain algae ingredients in shrimp feeds could be commercially viable when considering
consumer acceptance.
iii
Preface
Chapter 2 will be submitted for publication [Chapter 2: Garcia-Hoyos, L.M., Quintero, H.,
Forster, I., Cliff, M., and McKinley, S. 2013. Algae meals as a substitute to fish meal and fish oil
in practical diets for the Pacific white shrimp (Litopenaus vannamei)].
Dr. Herbert Quintero and Ian Forster formulated the diets of this experimental study. Diets were
manufactured at the Center for Aquaculture and Environmental Research (CAER).
Dr. Herbert Quintero and I created thehusbandry protocol of the feeding trial and the
experimental design. I conducted the experimental trial for 3 months in Colombia where I was
responsible for experiment set up, general husbandry, sampling sessions,collection and analysis
of data. Co-authors Dr Herbert Quintero, Ian Forster and Margaret Cliff provided guidance and
commentary in the statistical analysis of data and preparation of the final manuscript. Dr.
Margaret Cliff contributed with assistance in the analysis and interpretation of sensory data.
iv
Table of Contents
Abstract ..................................................................................................................................... ii
Preface .......................................................................................................................................iv
Table of Contents ........................................................................................................................ v
List of Tables ............................................................................................................................vii
List of Figures ............................................................................................................................. x
List of Abbreviations.................................................................................................................xii
Acknowledgements .................................................................................................................. xiv
Dedication................................................................................................................................. xv
1
Introduction ........................................................................................................................... 1
1.1
Shrimp aquaculture .........................................................................................................2
1.2
The use of fish meal and fish oil in aquaculture feeds .....................................................3
1.3
Plant protein and oil sources for aquaculture feeds as alternatives ...................................7
1.4
Microalgae as an alternative ingredient for aquaculture feeds .......................................11
1.5
Thesis objectives ..........................................................................................................15
1.5.1.1 Research questions and hypothesis ......................................................................16
2 Algae meals as a substitute for fish meal and fish oil in practical diets for the Pacific white
shrimp (Litopenaeus vannamei)................................................................................................. 18
2.1 Introduction .....................................................................................................................18
2.2
Materials and methods ..................................................................................................21
2.2.1 Source of shrimp and experimental design .............................................................21
2.2.2 Feed formulation and management.........................................................................22
v
2.2.3 Chemical analyses..................................................................................................23
2.2.4 Sensory evaluation .................................................................................................24
2.2.5 Statistical analyses .................................................................................................25
2.3
Results and discussion ..................................................................................................26
2.3.1 Water quality .........................................................................................................26
2.3.2 Growth performance and production parameters ....................................................26
2.3.3 Chemical composition of shrimp body ...................................................................31
2.3.4 Sensory evaluation .................................................................................................32
2.4
Tables ...........................................................................................................................35
2.5
Figures .........................................................................................................................51
3 General discussion, limitations and future directions .............................................................. 54
3.1
Research contributions..................................................................................................54
3.2 Approaches for the evaluation of sensory characteristics in aquaculture products using a
three factor Analysis of Variance ...........................................................................................55
3.3
Research limitations and future directions .....................................................................56
3.4
Analysis of thesis work in future research directions .....................................................58
Bibliography ............................................................................................................................. 60
vi
List of Tables
Table 1.1.General composition of different algae (% of dry matter)………………………….36
Table 2.1. Nutritional composition of practical diets for Litopenaus vannameiKJ:
Kilojoules/100g. FM-37 (Fish meal based feed, main protein source: fish meal at 37%); AS-37
(Algae based feed, main protein sources: Spirulina at 37%); ASS-48 (Algae based feed, main
protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein
source:
fish
meal
at
36%)………………………………………………………………………………………..……37
Table 2.2. Average of water quality parameters during the 10-week culture period for
Litopenaeus vannamei. Values are means ± SD. FM-37 (Fish meal based feed, main protein
source: fish meal at 37%); AS-37 (Algae based feed, main protein sources: Spirulina at 37%);
ASS-48 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 48%); ASS-34
(Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%) and COM -36
(Fish
meal
based
feed,
main
protein
source:
fish
meal
at
36%)……………………………………………38
Table 2.3. Average production parameters at the end of a 10-week culture period for
LitopenausVannamei fed practical diets with varying levels of algae meal and fish meal diets 1.
Values are means ± SD.FM-37 (Fish meal based feed, main protein source: fish meal at 37%);
AS-37 (Algae based feed, main protein sources: Spirulina at 37%); ASS-48 (Algae based feed,
main protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main
vii
protein sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed,
main
protein
source:
fish
meal
at
36%)……………………………………………………………….39
Table 2.4. Amino acid composition of practical diets for Litopenaus vannamei (%)1. FM-37 (Fish
meal based feed, main protein source: fish meal at 37%); AS-37 (Algae based feed, main protein
sources: Spirulina at 37%); ASS-48 (Algae based feed, main protein sources: Spirulina and
Schizochitrium at 48%); ASS-34 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 34%)…………………………………………………………………………..40
Table 2.5. Whole body chemical composition of Litopenaeus Vannamei fed various diets1. KJ:
Kilojoules/100g. FM-37 (Fish meal based feed, main protein source: fish meal at 37%); AS-37
(Algae based feed, main protein sources: Spirulina at 37%); ASS-48 (Algae based feed, main
protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein source: fish meal at 36%)………………………………………………………………..42
Table 2.6. Fatty acid composition (% of FAME) of diets1. FM-37 (Fish meal based feed, main
protein source: fish meal at 37%); AS-37 (Algae based feed, main protein sources: Spirulina at
37%); ASS-48 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 48%);
ASS-34 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%) and
COM -36 (Fish meal based feed, main protein source: fish meal at 36%)………………………43
viii
Table 2.7. Fatty acid composition (% FAME) in shrimp muscle tissue 1. FM-37 (Fish meal based
feed, main protein source: fish meal at 37%); AS-37 (Algae based feed, main protein sources:
Spirulina at 37%); ASS-48 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 48%); ASS-34 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 34%) and COM -36 (Fish meal based feed, main protein source: fish meal at
36%)……………………………………………………………………………………………...45
Table 2.8. Mean sensory characteristics (n = 20; 5 judge ×4 replications) of shrimp at the end of
the feeding trial. Values are means ± SE mean. R represents the standard expected value for each
attribute. FM-37 (Fish meal based feed, main protein source: fish meal at 37%); AS-37 (Algae
based feed, main protein sources: Spirulina at 37%); ASS-48 (Algae based feed, main protein
sources:
Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein source: fish meal at 36%)………………………………………………………………..47
Table 2.9. Analysis of variance of sensory characteristics ratings (5 judges): degrees of freedom
(df), F-ratios, and error mean squares (MSE). J*S: Judge and sample interaction. J*R: Judge and
replicate interaction. S*R: Sample and replicate interaction…………………………………….49
Table 2.10. Correlation matrix among the sensory attributes (df = 3)…………………………..50
ix
List of Figures
Figure 2.1. Final body weight of Pacific white shrimp fed two control (FM-37; COM-36) and,
three experimental (AS-37, ASS-48, ASS-34) diets reared under a green water system. FM-37
(Fish meal based feed, main protein source: fish meal at 37%); AS-37 (Algae based feed, main
protein sources: Spirulina and Schizochitrium at 34%); ASS-48 (Algae based feed, main protein
sources:
Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein
source:
fish
meal
at
36%)
Error
bars
represent
SD.……………………………………………………………………………………………….51
Figure 2.2. Mean scores for the sensory attributes aroma, color and sweetness. Bars represent
aroma, color and sweetness of the various treatments. FM-37 (Fish meal based feed, main
protein source: fish meal at 37%); AS-37 (Algae based feed, main protein sources: Spirulina and
Schizochitrium at 34%); ASS-48 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 48%); ASS-34 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 34%) and COM -36 (Fish meal based feed, main protein source: fish meal at
36%). Treatments that do not share the same letter are significantly different at P ≤ 0.05. Error
bars
represent
SE
(standard
error).
……..……………………………………………………………………………………………..52
Figure 2.3. Cobweb with descriptive profiles of undesirable attributes for Litopenaeus vannamei
indicating the results of sensory tests where judges evaluated shrimp that had been reared on
experimental and control diets. FM-37 (Fish meal based feed, main protein source: fish meal at
x
37%); AS-37 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%);
ASS-48 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 48%); ASS-34
(Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%) and COM -36
(Fish meal based feed, main protein source: fish meal at 36%). The center of the figure
designates low intensity. Intensity of each attribute increases with distance……………………53
xi
List of Abbreviations
Abbreviations in text
ANOVA
Analysis of variance
AOAC
Association of Official Analytical Chemists
ArA
Arachinodic acid
AS-37
Algae based diet (main protein source: Spirulina-37% with inclusion of algal oil
DHA)
ASS-34
Algae based diet (main protein source: Schizochitrium, spirulina-34%)
ASS-48
Algae based diet (main protein source: Schizochitrium, spirulina-48%)
BHT
Butylated hydroxytoluene
CAER
Center for Aquaculture and Environmental Research
COM-36
Commercial fish meal feed (36% protein)
DHA
Docosahexaenoic acid
DPA
Decosapentaenoic acid
EPA
Eicosapentaenoic acid
FA
Fatty acid
FAME
Fatty acid methyl ester
FAO
Food and Agriculture Organization of the United Nations
FCR
Feed conversion ratio
FM
Fish meal
FM-37
Fish meal based diet (main protein source: fish meal-37%)
FO
Fish oil
xii
HUFA
Highly unsaturated fatty acids
IAA
indispensable amino acids
LC
Long-chain fatty acids
LSD
Least-significance difference
LNA
Linolenic acid
PCB
Polychlorinated biphenyls
PLS
Post-larval shrimp
POP
Persistent organic pollutants
PUFA
Polyunsaturated fatty acids
SBO
Soybean oil
SDA
Specific dynamic action
SPR
Specific-pathogen resistant
USA
United States of America
Symbols
NH4+
Ammonium
xiii
Acknowledgements
I would like to express my deepest gratitude to Dr. Scott McKinley for the opportunity he has
offered, and for his intellectual contribution in the completion of this research. I am especially
grateful to Dr. Herbert Quintero for his support during this research, time, advice and some very
stimulating scientific discussions. I would like to extend my gratitude to Ian Forster for his
willingness to help whenever needed and for following the progress of this research.
Also, I would like to thank Dr. Margaret Cliff for providing advice on statistical analysis of the
sensory evaluation component of this research. I am also very grateful to my committee
members Drs. Zhaoming Xu, James Thompson and Marina von Keyserlingk for their promptness
and assistance throughout the preparation of this thesis.
I would like to acknowledge my gratitude to Andres Suarez, Jaime Faillache and all personnel
from CENIACUA, Colombia, for their technical assistance during fieldwork. I would like to
further thank Mahmoud Rowshandeli from CAER and Bianca Arney for their patience and help
during laboratory analysis.
Special thanks must be given to my parents Alba and Raul, who have supported me in the
direction of following my dreams. Thanks to my sister Ana Maria, who is always willing to help
and cheer me up. Finally, thanks to my husband Michael Rosaine,for hisincredible support,
patience, constant encouragements and sweet love.
xiv
Dedication
To my husband, Michael and my family: Ana, Raul and
Alba.
xv
1 Introduction
Fish meal (FM) and fish oil (FO) have traditionally been considered essential ingredients
in aquaculture feeds for carnivorous (e.g. salmon, trout, sea bass, eel) and omnivorous (e.g.
tilapia, catfish, common carp, shrimp) species. Numerous aquaculturefeed formulations continue
to include FM and FO at levels that surpass 50% and 30%, respectively (Tacon and Metian,
2008a). Generally, these commodities are the primary and most costly ingredients in aquaculture
feeds. Other challenges encountered when using FM and FO are associated with environmental
and safety issues linked to the use of potentially contaminated by-products and the negative
effect of decreasing wild fish stocks from FM production from natural fish stocks. Food safety
risks in commercial aquafeeds may include salmonellae, mycotoxins, veterinary drug residues,
persistent organic pollutants (POP), heavy metals, and excess mineral salts. These contaminants
can have a potential effect on the health of cultured species, and may be passed along the food
chain via contaminated aquaculture produce to humans (Tacon and Metian, 2008b).
Recently, an average of 25 million metric tons of raw material, approximately 25% of the
world’s capture fisheries, has been used to produce FM and FO (De Silva, 2010). The utilization
of this volume of raw material has been the subject of continued controversy as the wild capture
fisheries reaches its maximum sustainable levels. In an attempt to address these issues, the
aquaculture industry has endeavored to find alternative protein and oil sources that can partially
or entirely substitute FM and FO in feeds of farmed aquatic animals. Hundreds of studies have
tested alternative protein and lipid sources in aquaculture feeds without compromising the
production and economic performance of farmed fish and crustaceans; therefore, there is great
promise and need for incorporating alternative and sustainable protein and lipid sources in
aquafeeds. FM and FO replacements are of paramount importance to the aquaculture sector.
1
1.1 Shrimp aquaculture
The commercial production of farmed shrimp has been expanding rapidly over the past
two decades and this trend is expected to continue as global population increases and the demand
for quality seafood continues to rise (Tacon and Forster, 2001). In 2001, marine shrimp were the
second highest farmed world aquaculture species with an estimated value of US$8.4 billion
(FAO, 2003). Measured by production, Litopenaeus vannamei is the most successful
internationally introduced marine crustacean species for aquaculture. In 2010, it accounted for
71.8% of world production of all farmed marine shrimp species of which 77.9% was produced in
Asia, with the rest in North and South America(FAO, 2012).
Several factors have led to the growth of shrimp farming. Growing demand for shrimp
from countries in North America, Europe and Japan, together with a leveling off of production
from capture fisheries, gave rise to high market prices in the 1980s. At the same time, the
emergence of new production technologies (e.g. hatchery-reared post larval shrimp (PLS) and
improvedartificial feeds) allowed for large-scale production (Neiland et al., 2001).However,
although the production and financial performance of the shrimp aquaculture industry has been
outstanding, its rapid growth has been accompanied by a decrease in shrimp price, due in part to
depressed markets and overproduction. As shrimp aquaculture is expected to continue to increase
in coming years, shrimp prices are likely to continue to fall as production exceeds demand,
thereby challenging the profitability of this industry (Amaya et al., 2007).
Commercial shrimp feed formulations generally include between25% and 50%fishmeal,
which represents the primary and most expensive ingredient (Dersjanti-Li, 2002; Tacon and
Barg, 1998; Gonzalez-Rodriguez, 2004). FM and FO have been preferred as the number one
protein and lipidsource as they arerich in marine oils, essential aminoacids, vitamins,
2
minerals,which all lead to good growth performance in shrimp (Patnaik et al., 2006; Samocha et
al., 2004). Fish oils are typically characterized by an array of fatty acids in chain length from C12
to C24. Fish oils are best known and highly regarded for their high proportions of n-3 LC-PUFA
(long chain-polyunsaturated fatty acids). In comparison to oils of terrestrial origin, fish oils
contain an abundance of EPA (20:5n-3) and DHA (22:6n-3). The fatty acid composition of
individual fish oils varies considerable, influenced by factors including age, size, species,
reproductive status, geographic location and the time of the year when harvested. Fish oils are
naturally rich in fat-soluble vitamin A and D. They also contain small concentration of dietaryderived vitamin E, which plays an important role in the prevention of PUFA oxidation (De Silva
et al., 2010).Fish meal is a natural source of vitamins such as choline, biotine, B12, and includes
various trace elements like selenium and iodine. Fish meal of good quality normally contains
between 60-72% crude protein by weight with a balanced amount of all essential amino acids
(Cho and Kim, 2010).However, limited availability and high demand make FM a costly
ingredient (Amaya et al., 2007).
1.2 The use of fish meal and fish oil in aquaculture feeds
By and large, marine FM and FO have been the major feed ingredients used as dietary
protein and lipid sources, respectively, in the formulation of commercial aquafeeds. It is
estimated that aquaculture feeds currently use about 90% of the global supply of FO and it has
been predicted that the demand for FO from the aquaculture industry will imminently outstrip
supply (Turchini et al., 2010). In fact, according to Tacon and Metia (2008b) there is a global
decrease in reported dietary FM and FO inclusion levels in commercial aquafeeds due to the
increasing prices of these commodities since 2000. The cause of the escalating prices are often
attributed to several factors, including static global supplies of FM and FO, strong market
3
demand by the aquaculture and livestock sector in the major importing countries and in particular
China (GAIN, 2007). It is evident then, that there is great urgency within the aquafeed industry
in finding suitable protein and lipid sources to replace or reduce the use of marine FM and FO,
and that economics is one of the principal drivers of using alternative feed ingredients.
Fish meal is a finite global resource and as the human population expands the demand for
seafood and overall seafood consumption will increase proportionally. Annual per capita supply
from aquaculture has increased from 0.7 kg in 1970 to 7.8 kg in 2006, an average annual growth
rate of 6.9 %. From a production of less than 1 million tones per year in the early 1950s,
production in 2006 was reported to be 51.7 million tonnes with a value of US$ 78.8 billion,
representing an annual growth rate of nearly 7 percent (FAO, 2005). This exceptional growth
will require a larger input of feeds, which depend upon higher quantities of various feed
ingredients, including protein and lipid sources. Since FM and FO are limited global
commodities, the demand for FM and FO in aquafeeds will certainly exceed the global
production of these products. This realization has led to significant efforts at research centers
throughout the world for the past 15 years or more to develop and test protein and lipid sources
to replace FM and FO in the diets of farmed fish (Hardy and Tacon, 2002). These research
efforts have focused mainly on the replacement of FM and FO by other protein and lipid sources
rather than examining what the animal requirements are and how to thoroughly meet those
specific requirements. There is a knowledge gap on the chemical composition and nutritive
profiles of some alternative feed ingredients, which will have to be addressed to achieve
formulated diets that can be used optimally for farmed fish.
Although FM and FO have been the ingredients for excellence in aquaculture feeds,
contamination of farmed fish via that contain FM and FO, which are sometimes derived from the
4
use of contaminated raw materials, is one of the biggest global concerns for food safety. Some of
the contaminants that may be present in FO are the persistent organic pollutants (POP), which
consist of a wide range of lipophilic compounds including polychlorinated biphenyls,
organochlorine pesticides, polycyclic aromatic hydrocarbons, polybrominated diphenyl ether,
and dioxins. Persistent organic pollutants bioaccumulate in the lipid component of fish tissues
and are subsequently persevered in extracted FO. Polychlorinated biphenyls represent the highest
risk in that they potentially increase the incidence of cancer and affect reproductive viability in
humans (De Silva et al., 2010).
Although these drawbacks strongly suggest that it is vital to reduce dependence on FM
and FO in aquaculture feeds in order to reduce health risks to humans, Mozaffariam and Rimm
(2006) reported that the benefits of fish consumption outweigh the potential risks of the
contaminants in fish. However, a key responsibility of an aquaculture feed manufacturer and
aquaculture food supplier should always be to ensure food safety and product quality as well as
protect the health of consumers.
Fish oils are best known and highly regarded for their abundance of LC-PUFA of the n-3
series-EPA, DPA, and DHA. Fish oils contain high proportions of EPA (20:5n-3) and DHA
(22:3n-6). The main species used in the production of FO are the Peruvian anchovy (Engrauhs
ringens), mackerel (Trachurus/scomber spp.), sand eel (Ammodyte spp.), capeling (Mallotus
spp.), menhaden (Brevoorita spp.), herring (Clupa Harrengus) and pollock (Pollachius spp.).
These species are known to be pelagic and have a lipid content of 8% or more. Peruvian anchovy
has EPA contents that range from 7.6% to 22% and DHA from 9.0 to 12.7%. Capelin oil levels
of EPA range from 6.1% to 8.0% and 3.7 to 6.0% for DHA. Herring oil contains levels of EPA
and DHA ranging from 3.9 to 15.2% and from 2.0 to 7.8 %, respectively (Turchini et al., 2010).
5
When fish and shrimp are fed diets containing FO they become a unique rich source of n3LC-PUFA that are critical components of the human diet. The importance of the fatty acid
composition of FO arises from the extensive evidence showing potential health benefits
associated with its consumption. Therefore, there is a high demand and increased consumption of
FO by humans that could potentially reduce this resource availability for commercial feed
production. The consumption of LC omega-3s derived from FO has been shown to help prevent
cardiovascular disease, cancer, inflammatory autoimmune diseases, and support brain function
and mental health (Ruxton et al., 2007). As for shrimp and other crustacean, it has been shown
that lipid content and the associated C18 polyunsaturated fatty acids, linoleic (18:2n-6) and
linolenic (18:3n3) as well as n-3 and n-6 HUFA (EPA, DHA, ArA) are required in their feeds.
Another possibly detrimental aspect of continued reliance on FO is the predicated effects
of climate change on fisheries, which could affect the availability of this raw material for use as
an aquafeed. Of great significance are the predicted changes in ocean circulation patterns that, in
turn, will have an influence on the occurrence and frequency of El Nino events. Barret et al.
(1995) have documented the devastating effects of El Nino on the Peruvian sardine and anchovy
stocks, subsequently impacting global FM and FO supplies and prices. As a potential indication
of limited supply, the price of FM roughly tripled from 2002 to 2010, and supply remains limited
while the demand for fish feed ingredients is expected to continue to rise (Rust, 2011). In
addition, it has been suggested that El Nino events will continue to modify the biomass structure
of pelagic resources (Niquen and Bouchon, 2004). In essence, potential climate changes can put
key stocks at risk for the production of FM and FO. Needleless to say, the aquaculture sector
seems to be compelled to move forward to develop alternatives to FM and FO. If the aquaculture
feed industry seeks to replace these commodities with alternatives, pragmatic approaches on the
6
nutritional benefits of ingredients, including digestibility and palatability, technology, price,
market availability and sustainability, will need to be considered.
It has been suggested that one way to reduce shrimp production costs and increase
profitability, is todevelop plant-protein feeds. In recent years, there had been a global initiative to
evaluate a number of different plant alternative sources owing to the great urgency within the
aquafeed industry to find sustainable alternative to fish meal and fish oil (Turchiniet al., 2010).
Nonetheless, the challenge of finding suitable alternative ingredients remains with regards to
maximizing sustainability, shrimp performance, and economic benefits.
1.3 Plant protein and oil sources for aquaculture feeds as alternatives
Fish meal and FO derived from wild-harvested whole fish and shellfish including bycatch
currently constitute the major aquatic protein and lipid sources available for animal feed (FAO,
2012). However, during 15 years (1994-2009), analysis of data collected by the Food and
Agriculture Organization (FAO) of the United Nations has indicated that global FM and FO
production from marine capture fisheries has been decreasing at annual average rates of 1.7 and
2.6 %, respectively. It is evident that the increasing demand for these commodities places great
pressure on marine ecosystems leading to a depletion of fish stocks. Therefore, there is a drive to
adopt more ecologically sound decisions regarding the future of protein and oil sources for
aquaculture feeds. It is extremely important to keep a holistic research focus that involves all the
components (ingredient digestibility, ingredient palatability, nutrient utilization, final product
quality and price) that are necessary to establish the viability of new ingredients.
Hundreds of studies have been conducted to evaluate numerous alternative plant proteins
such as soybean meal, corn gluten meal, rice protein, rapeseed protein, and plant oils such as;
soybean, rapeseed, linseed, sunflower, peanut, olive and palm oil among others in aquaculture
7
feeds. For example, Sooking and Davis (2010) found that soy protein concentrate inclusion up to
12% in soybean-based diet can be used in commercial feed formulations for Litopenaeus
vannamei without causing negative effect on feed conversion ratio (FCR), survival and growth.
In another study (Amaya et al., 2007), FM replacement using soybean meal at 37% protein level
in commercially manufactured feed for the Litppenausvannamei was performed. These authors
demonstrated that FM could be completely replaced using alternative vegetable protein sources
without compromising production and economic performance.
Similarly, Lin et al. (2012) investigated the replacement of FM with fermented soybean
meal in diets for pompano (Trachinotus ovatus) at a 46% protein level, and reported that
fermented soybean meal did not have a negative effect on fish growth, FCR, and body
composition. In another study carried out by Walker et al. (2010), partial replacement of FM
with soy protein concentrate at a 47% protein level in diets of Atlantic cod was evaluated, with
survival and growth not affected.
In addition, the use of soybean as a protein source has been examined for many other
commercially important fish species, such as red drum Sciaenops ocelltus (McGoogan and
Gatlin 1997), Atlantic salmon, Salmo salar (Refstie et al., 1998), Asian seabass, Lates calcarifer
(Tantikitti et al., 2005), sharpsnout sea bream, Diplodus puntazzo (Rondan et al., 2004), red sea
bream, Pagrus major (Biswas et al., 2007), and gilthead sea bream, Sparus aurata (Venou et al.,
2006; Bonaldo et al., 2008). Compared to other plant proteins, soybean meal is one of the most
promising alternatives due to its abundant availability, reasonable price, and high digestibility
(Lemos et al., 2000). However, in terms of lipid composition, soybean meal contains 90% fewer
n-3 fatty acids than FM and contains anti-nutritional factors such as trypsin inhibitors and lectins,
which are known to inhibit digestive enzyme activity (Zhou et al., 2011).
8
Another limitation of soybean protein is its amino acid profile, which does not match the
nutrient requirements of farmed fish (Wilson, 1989; Floreto et al., 2000); thus limiting its
inclusion rate compared to FM (i.e. deficiency in the essential amino acids methionine, lysine
and tryptophan), presence of antinutritional factors, and poor palatability (Lim and Dominy,
1990 and Tacon and Akiyama, 1997). Although most of the antinutritional factors do not lead to
mortality, they could produce adverse effects and decrease productivity as well as influence the
nutritional and organoleptic quality of the final product (Francis et al., 2001). There is a gap in
the literature regarding the assessment of organoleptic characteristics of farmed species fed
plant-derived ingredients that needs to be assessed.
As for FO replacement, a number of studies have suggested that certain vegetable oils
could be considered as good alternative lipid sources in aquaculture feeds. The most widely used
vegetable oils for inclusion in animal feeds are soybean, soybean lecithin, corn, cottonseed, and
sunflower (Gunstone, 2010). Soybean oil (SBO) and soybean lecithin oil, enriched with linoleic
(18:2n-6,LOA) and linolenic (18:3n-3, LNA) acids, have been used extensively in aquafeeds.
Soybean mealhas been used as lipid source forLitopenaeus Vannamei (Hu et al., 2011),
common carp (Cyprinus carpio) (Fontagne et al., 2000), japanese flounder (Paralichthys
olicaceus) (Lee et al., 2000), giant fresh water prawn (Macrobrachium rosenbergii) (Labao et
al., 1995), Asian seabass (Lates calcarifer) (Catacutan and coloso, 1997), black carp
(Mylopharyngodon piceus) (Dai et al., 1988), and red belly tilapia (Tilapia Zillii) (El-sayed and
garling, 1998) among others. These studies showed a general acceptance of SBO as a source of
dietary lipid for growth. Nonetheless, according to Gunstone (2010), the fatty acid composition
of tissues can be altered. This same author has reported that species fed SBO incorporate 16:0,
18:1n-9, and 18:2n-6 into tissue lipids as well as longer chain fatty acids of the same families,
9
but the n-3 fatty acid concentration in tissues tends to be lower. This in particular brings to light
a challenge for the aquaculture industry since aquatic animals are regarded as healthy foods for
humans, mostly because of the n-3 fatty acids found in edible portions, but there is an increasing
number of farmed species identified with high concentrations of n-6 fatty acids, which are not
appropriate from a human nutrition standpoint and promote the pathogenesis of many diseases
(Gonzalez-Felix et al., 2010).
Seafood is recommended in human diets to increase the n3/n6 ratio and to promote
human health by preventing cardiovascular disease, cancer, inflammatory autoimmune diseases,
and supporting brain development and function (Ruxton et al., 2007). For that reason, it is
critical to ensure that farmed species retain high levels of n-3 HUFA and high n-3/n6 ratios as
found in wild caught species. As diets are continually modified to meet the nutritional
requirements of targeted species and least-cost formulations become more common, there is a
possibility that diets will provide lower concentrations of n-3 fatty acids (Gunstone, 2010),
thereby affecting the final quality (and arguably acceptability) of farmed aquaculture species.
Moreover, the evaluation of sensory characteristics of alternative ingredients for
commercial aquaculture species needs further research in order to guarantee final product quality
and consumer acceptability. Literature on the impact of FO replacement on sensory quality in
farmed fish is rather scarce and mainly related to studies in salmonids. In salmon, replacing FO
with SBO had no effect on fillet texture (Rora et al., 2003). In addition, no differences were
observed in Atlantic halibut fed either FO or SBO (Haugen et al., 2006), neither in Atlantic cod
(Morkore et al., 2007), nor in European bass (Izquierdo et al., 2003).
Flesh pigmentation is an important factor in perception of flesh quality in salmonids (Bell
et al., 1998). The flesh levels of lipid-soluble nutrients such as astaxanthin are dependent on the
10
dietary composition (Lie, 2001), and it has been shown that both total lipid and type of oil can
affect carotenoid absorption in Atlantic salmon (Bjerkeng et al., 1999), and color characteristics
in raw and smoked Atlantic salmon fillets (Regost et al., 2004). However, the majority of studies
show no effects on flesh astaxanthin levels by using up to 100% vegetable oil (Bell et al., 2001;
Torstensen et al., 2004),but none of these replaced FO with SBO. As reported by Regust et al.
(2004) dietary SBO as a replacement of FO in salmon resulted in reduced flesh astaxanthin and
texture (Rora et al., 2003).
Perhaps, special consideration should be directed towards the inclusion of algae products in
aquaculture feeds, which are known to have high concentrations of n-3 fatty acids, as well as the
development of culture technology with regards to commercial large scale production, in order to
assure market availability and sustainability. Alternative ingredients that can provide the n-3
HUFA requirement for human consumption need to be found. Likewise, a comprehensive study
of nutrient composition, digestibility, and bioavailability of new ingredients is yet to be
conducted.
1.4 Microalgae as an alternative ingredient for aquaculture feeds
Microalgae are valuable in aquaculture and have been used as live feed for larval and
juvenile crustaceans and finfish, all bivalve mollusks including oysters, scallops, clams and
mussels, and as feed for zooplankton (Ju et al., 2009). Microalgae have received particular
interest in the aquaculture sector due to their nutrient profile. Microalgae are rich sources of
vitamins, essential amino acids, minerals, essential fatty acids, and carotenoid pigments for
aquatic animals (Takeuchi et al., 2002).The chemical composition of different algae sources is
now readily available in the literature (Table 1.1; Becker, 2007). Heterotrophically algal groups,
such as chrysophytes, cryptophytes and dinoflagellates have been known to produce lipids with
11
high levels of EPA, DHA and ArA (Cohen et al., 1995; Behrens and Kyle 1996). As microalgae
biomass is a rich source of nutrients such as n-3 and n-6 fatty acids, (Becker 1994), it has great
potential to be an alternative ingredient for sustainable aquaculture feeds.
Micoralgae have diverse uses in aquaculture, their applications are mainly to provide
nutrition and to enhance the color of the flesh of salmonids. The most frequently used species are
Chlorella, Tetraselmis, Isochrysis, Pavlova, phaeodactylum, Chaetoceros, Nannochloropsis,
Skeletonema and Thalassiosira (Hemaiswarya et al., 2011). Also, the potential use of DHA-rich
oils derived from single-cell microalgae (i.e Schizochytrium spp., Crypthecodinium cohnii, and
Phaeodactylum trictornutum) has been successfully tested on gilthead sea bream (Atalah et al.,
2007; Ganuza et al., 2008). Similarly, Schizochytrium spp. oil has been successfully tested on
Atlantic salmon (Miller et al., 2007).
Special attention should be paid to understanding the effects of DHA inclusion in the
fatty acid composition of aquaculture species. If species fed diets rich in DHA from microalgae
allow the retention of this fatty acid in animal’s tissue, microalgae might become one of the most
viable alternative ingredients for aquaculture feeds.
Although it is argued that producing algae is expensive, successful commercial utilization
of microalgae has been established in the production of nutritional supplements, antioxidants,
cosmetics, natural dyes and polyunsaturated fatty acids (PUFA) (Spolaore et al., 2006). To date,
several sophisticated technologies are employed worldwide for mass production and processing
of microalgae. The annual production of all species is estimated at about 10,000 t/y (Richmond
2004). The blue-green algae Spirulina spp. is used in substantial amounts (over 100 t/y) as a fish
and shrimp feed, and even larger markets could be projected as production costs may be reduced
in the long term (Hemaiswarya et al., 2011).
12
Research findings have shown that feedingSpirulina spp. results in improvements in
animal growth, fertility, aesthetic and final nutritional quality.Intake of Spirulina spp. or its
extracts intake has also been linked to an improvement in animal health, including accelerated
development of the immune system of farmed animals such as chickens, pigs, ruminants, cattle,
sheep and rabbits especially during the early stages of their lives (Holman and Malau-Aduli,
2012). Successful inclusion of microalgae in aquaculture feeds has been reported (Zhou et al.,
1991; Langdon and Onal 1999). More recently, Palmegiano et al. (2005) investigated the use of
Spirulina spp. as a nutrient source in diets for growing sturgeon (Acipenser baeri), and Kiron et
al. (2012), examined two marine algal products (i.e., Nanofrustulum and Tetraselmis) for their
suitability as FM protein substitutes in feeds of three important aquaculture species (i.e Atlantic
salmon, common carp and Pacific white shrimp). Growth performance and feed utilization of
these species did not exhibit any differences between those species provided with the algaebased feed and those provided with FM-based feed, indicating that algal meal is an effective
replacement of FM.
Diatom (Thalassiora weissfloggi) and Nannochlorpsis cultures have been added to
shrimp feeds, increasing the fatty acid and astaxanthin contents of shrimp tail muscles as well as
improving growth rates (Ju et al., 2009). Rincon et al. (2012) looked at substitution levels of FM
by Spirulina maxima in experimental diets for red tilapia fingerlings (Oreochromis sp.). They
found that FM could be substituted with up to 30% Spirulina maxima meal without impacting
growth performance in red tilapia fry. These studies provide evidence that microalgae are
suitable sources of protein and lipid for some aquaculture species. However, the inclusion of
microalgae in feeds should be evaluated at a species level, including a deeper understanding of
their digestibility and bio-availability.
13
Another potential species for aquaculture feeds is Haematococcus pluvialis. This alga has
frequently been added in diets to test for pigmentation of shrimp, salmon and trout (Chien and
Shiau, 2005). Astaxanthin production by this microalga is important to many farmed species
since, for example, crustaceans are unable to synthesize carotenoids de novo, and astaxanthin or
appropriate precursors must be supplied in the diet (Meyers and Latscha, 1997). Dietary
supplementation of astaxanthin or its precursors improve the color of penaeids (Liao et al.,
1993), especially those intensively cultured for a better market price (Liao and Chien, 1994).
One of the biggest advantages of using microalgae in aquaculture feeds is that in practice,
different strategies can be used to improve the PUFA content in microalgae by manipulation of
processing conditions such as light intensity, nutrient status and temperature. This in turn allows
for the modulation of the lipid composition and consequent optimization of their overall yield
productivity (Hemaiswara et al., 2011). The lipid content of microalgae is affected by
environmental conditions such as light intensity, nutrient limitation, salinity, temperature, pH
and culture age(Solovchenko et al., 2008). Under unfavorable conditions, microalgae growth is
arrested; photosynthetic activity decreases, and the excess energy might be stored in form of
lipids and carbohydrates (Khozin-Goldber et al., 2011). For example, Nannochloropsis
cultivated under conditions of nitrogen starvation, is a potent source of saturated and
monounsaturated oils (Rodolfi et al., 2009) Apart from this benefit, the high production cost of
microalgae remains a constraint. Nonetheless, larger markets could be projected as production
costs may be reduced in the long term.
Finally, as suggested by Raja et al. (2004) and Patil et al. (2007), in order to be used in
aquaculture, a microalgal strain has to meet various criteria, such as ease of culturing, lack of
toxicity, high nutritional value with correct cell size and shape, and a digestible cell wall to make
14
nutrients available. There is great promise for microalgae to become an alternative ingredient
that can greatly contribute to meet the essential nutrient requirements of farmed species and to
provide a high quality final product.
This thesis is primarily focused on evaluating the suitability of two types of algae (i.e.
Spirulina and Schizochitrium) as the primary sources of FM and FO replacement in practical
diets for Pacific white shrimp Litopenaus vannamei, an important and widely produced
aquaculture species. The effectiveness of algae meals on the source of dietary protein and fat is
normally determined by the estimation of growth rate, feed conversion ratio (FCR) and survival.
In addition, the sensory characteristics of shrimp muscle were assessed in order to elucidate if the
inclusion of algae meals in shrimp feeds could be commercially viable when considering
consumer acceptance.
1.5 Thesis objectives
Given the great need to findsuitable alternative ingredients to replace and/or reduce the use of
FM and FO in aquaculture feeds, the main objective of this research was to investigate if two
species of algae (i.e. Spirulina and Schizochitrium) could be optimal replacements for FM and
FOin practical diets for the Pacific white shrimp (Litopenaeus vannamei). A further aim was to
obtain shrimp of comparable nutritional value to shrimp traditionally fed with FM and FO diets
and then to analyze the impact of the inclusion of algae ingredients on the sensory characteristics
of shrimp muscle, as one of the most critical factors to consider when evaluating the viability of
alternative feeds is that of consumer acceptance of the product.
15
1.5.1.1 Research questions and hypothesis
1) Is the body chemical composition of shrimp fed algae feeds comparable to shrimp fed
FM feeds?
H01: The three essential fatty acids (DHA, EPA and ArA) in the body composition of
shrimp will be different between shrimp fed algae and FM feeds.
HA1: The three essential fatty acids (DHA, EPA and ArA) in the body composition of
shrimp will be the same between shrimp fed algae and FM feeds.
2) Can FM and FO be completely replaced with alternative algae protein and oil sources
in shrimp feeds without negatively compromising the production performance of
Litopenaeusvannamei?
H02:Growth parameters (i.e. growth rate, FCR and survival) of Litopenaeus vannameiwill
be different between shrimp fed algae and FM feeds.
HA2: Growth parameters (i.e. growth rate, FCR ratio and survival) of Litopenaeus
vannameiwill be the same between shrimp fed algae and FM feeds.
3) Does the inclusion of algae ingredients have an impact on the sensory characteristics of
shrimp muscle?
H03: The sensory attributes of shrimp muscle will bedifferent between shrimp fed algae
and FM feeds.
16
HA3: The sensory attributes of shrimp muscle will be the same between shrimp fed algae
and FM feeds.
17
2 Algae meals as a substitute for fish meal and fish oil in practical diets for the
Pacific white shrimp (Litopenaeus vannamei)
2.1 Introduction
In recent years, the increase in cultured production of Pacific white shrimp (Litopenaeus
vannamei) has led to a number of critical environmental and economic challenges. Consequently,
the foremost concerns are with respect to emerging environmental and safety issues associated
with the use of potentially contaminated animal by-products in aquaculture feeds and the
negative effect of FM production from natural fish stocks (Tacon and Metian, 2008a). Food
safety risks in commercial aquafeeds may include salmonellae, mycotoxins, veterinary drug
residues, persistent organic pollutants (POP), heavy metals, and excess mineral salts. These
contaminants can have a potential effect on the health of cultured species, and may be passed
along the food chain via contaminated aquaculture produce to humans (Tacon and Metian,
2008b). Therefore, the use of FM in aquaculture feedshas been criticized due to its negative
environmental impact and itslimited availability.
Commercial shrimp feeds generally contain between 25% and 50% FM, which accounts
for the primary and most costly protein ingredient (Dersjanti-Li, 2002; Tacon and Barg, 1998;
Gonzalez-Rodriguez, 2004). These high inclusion rates and associated costs create challenges
forthe profitability of the shrimp aquaculture industry.Furthermore, the rapid expansion in
aquaculture production has led to an increase in demand for marine animal meals and oilswhich,
in turn, has caused the price of fish meal and oil to steadily increase (Pike and Barlow 2003).
Collectively this has resulted in anincreased need for identification of alternative ingredients to
replace marine derived ingredients (Davis and Arnold 2000; Amaya et al., 2007).
18
In an attempt to address these issues, the aquaculture industry has succeeded in reducing
the inclusion of FM and FO in the feeds of farmed aquatic animals (Kiron et al., 2012).
Microalgae have received particular interest,as they are a rich source of vitamins,
essential amino acids, minerals, essential fatty acids, and carotenoid pigments for aquatic
animals (Takeuchi et al., 2002). Moreover, as microalgae biomass is also a rich source of some
essential nutrients such as n-3 and n-6 fatty acids (Becker 1994), it has potential to be an
alternative ingredient for sustainable aquaculture feeds. In addition, alternative oil sources for
shrimp feed formulations are being pursued given that marine oil sources are limited in supply,
and could contain undesirable contaminants (Patnaik et al., 2006). Given that the use of algae as
a protein and oil source in aquaculture feeds is a recent concept, the associated research is in its
infancy.
Some of the early work wascarried out by Zhou et al. (1991), who investigated the
application of Spirulina mixed feed in the breeding of Bay scallop. These authors demonstrated
that Spirulina feed proved to promote the normal development of scallop gonads, thereby
achieving a higher fecundity and hatchery rate. Palmegiano et al. (2005) investigated the use of
Spirulina as a nutrient source in diets for growing sturgeon (Acipenser baeri), and found that
Spirulina inclusion improves growth and that an inclusion level of 50% exhibited the greatest
growth rate, a more favorable FCR and the highest protein efficiency. More recently, Kiron et al.
(2012), examined two marine algal products (i.e. Nanofrustulum andTetraselmis) for their
suitability as FM protein substitutes in feeds for farmed Atlantic salmon, common carp and
Pacific white shrimp. Growth performance and feed utilization of all threespecies were similar
between those species provided with the algae-based feed and those provided with fish mealbased feed, indicating that algal meal is an effective replacement of FM. Silva-Neto et al.(2012),
19
studied the attractiveness of Spirulina for Litopenaeus vannamei by supplementing experimental
diets with Spirulina meal and control diets with a FM commercial attractant and reported no
differences in growth, but interestinglySpirulina meal attractant was preferred more by shrimp
than the commercial one.
In another study, Ju et al. (2009) evaluated the effects of supplementing two species of
marine algae (Thalassiora weissflogii and Nannochloropsis sp) to a formulated diet on growth,
survival and composition of shrimp (Litopenaeus vananamei). The results indicated that adding
algae biomass to the diets improved shrimp growth and survival as well as enhanced
pigmentation in shrimp tail muscle. Furthermore, Rincon et al. (2012) evaluated three levels of
substitution of FM with Spirulina maxima meal as a protein source in experimental diets for red
tilapia fingerlings (Oreochromis sp.) and found that FM can be substituted with up to 30%
Spirulina maxima meal in diets for red tilapia fry.
Although product costs are high, successful commercial utilization of microalgae has
been establishedin the production of nutritional supplements, antioxidants, cosmetics, natural
dyes and PUFA is well established (Spolaore et al., 2006). Worldwide,several sophisticated
technologies are employed for mass production and processing of microalgae. The annual
production of all species is estimated to be about 10,000t/y (Richmond, 2004). The blue-green
algae Spirulinais used in substantial amounts (over 100 t/y) as a fish and shrimp feed, and even
larger markets could be projected as production costs may be reduced in the long term
(Hemaiswarya et al., 2011). Research findings have associated Spirulinato improvements in
animal growth, fertility, and nutritional product quality. Spirulina intake has also been linked to
an improvement in animal health in farmed animals such as chickens, pigs, ruminants, cattle,
sheep and rabbits (Holman and Malau-Aduli, 2012). To my knowledge, no study has been
20
carried out on the use of Spirulina and Schizochytrium as replacement sources of FM and FO in
feeds for Litopenaeus vannamei. Therefore, this research was undertaken to investigate the
effectiveness of FM and FO substitution using Spirulina and Schizochytrium as the principal
protein and lipid sources in the practical diets of Litopenaeusvannameion growth and production
2.2 Materials and methods
2.2.1 Source of shrimp and experimental design
Research was conducted at the Colombian Aquaculture Research Center CENIACUA, in
Punta Canoa, Bolivar, Colombia. Twenty circular fiberglass tanks composed an outdoor closed
system with a volume capacity of 530-L; tanks were filled with green water from a shrimp
production pond to replicate pond production conditions. Each tank was fitted with a ring air
diffuser connected to a common air supply from two 1-hp regenerative blowers (Sweetwater
Aquaculture, Apopka, USA). Specific Pathogen-Resistant (SPR) Pacific white shrimp (mean
weight ± SD, 1.31± 0.029) were stocked at a density of 28 shrimp/m2. Diets were assigned to the
tanks according to a completely randomized design, where each treatment was replicated four
times (5 diets × 4 replicates = 20 tanks).
Daily water changes and a cleaning routine (siphoning) were performed. Water quality
parameters including pH, salinity, temperature, and dissolved oxygen were monitored twice daily
at 0830 and 1600 using a HI 255 Hanna combined meter (Woonsocket, USA), Biomarine
ABMTC meter (Hawthorne, USA), and YSI Pro 2030 meter (Yellow springs, USA) respectively.
Additionally, ammonium (NH4+) was determined on a weekly basis with a spectrophotometer
(Genesys-Thermo Scientific, Massachusetts, USA) using the Indophenol blue method (Tovar and
Erazo, 2009).
21
2.2.2 Feed formulation and management
The main target for the formulation of the experimental diets was the entire replacement
of FM, while including algae meal from Spirulina and Schizochytriumas the main protein and oil
sources, respectively. Experimental diets were isolipidic (8%) and designed to contain three
protein levels: 1) 37% containing fish meal (FM-37) used as a control, 2) 34% containing
Spirulina and Schizochytrium (ASS-34), 3) 37% containing Spirulina and algal oil (AS-37), 4)
48% containing Spirulina and Schizochytrium (ASS-48). A commercialfish meal feed (Nicovita)
was used as control as well (COM-36). The experimental diets were prepared in the nutrition
laboratory at the Center for Aquaculture and Environmental Research (CAER), West Vancouver,
Canada. Feed was manufactured as follows: dry ingredients were mixed for 15 min in a food
mixer (Butcher Hobart, troy, USA), and hot water (60°C) was added and mixed to obtain a
consistency appropriate for pelleting. The diet mash was passed through a commercial grinding
machine (Butcher Boy, Montebello, USA) with 2.4 mm diameter holes. After pelleting, the feeds
were dried targeting moisture content at 10% using an air-drying oven. Finally, feeds were stored
in a cold room at 15°C until use.
Initial feed rations were calculated assuming 100% survival, FCR of 1:1.5 and expected
growth of 1.5 g/week. Subsequent feed inputs were back calculated, based on an expected weight
gain of 1.5 g per week, FCR of 1:1.5 and a mortality rate measured every 2 weeks during a 10week period. Shrimp growth was monitored on a bi-weekly basis by weighing all the animals
and estimating the average weight. Feed was provided twice daily (40% at 0830; 60% at 1530).
In addition, four aquariums containing five shrimp each were set up with clear water and fed the
experimental diets to confirm the acceptability of feed throughout the study period. At the end of
22
the growth trial, shrimp production parameters were evaluated by a mean final weight, weekly
weight gain, FCR and survival.
2.2.3 Chemical analyses
At the end of the 10-week trial, shrimp were harvested and freeze-dried. Shrimp and
feeds were analyzed according to the Association of Official Analytical Chemists (AOAC,
1995). Moisture was determined by drying 2 gsamples in an oven (Isotemp-Fisher Scientific,
Waltham, USA) at 100°C for 16 h. Ash was obtained by incinerating 0.5 gsamples (dried
sample) in a muffle furnace (Isotemp-muffle furnance, Fisher Scientific, Waltham, USA) at
600°C for 2 h. Crude protein was determined from total Kjeldhahl-N method by means of an
automated spectrophotometric flow injection analyzer (FIAstarth5000, Hillerond, Denmark),
using a multiplication factor of 6.25. Gross energy was measured by bomb calorimetry (IKA C
5000 control). Lipids were determined according to the Bligh and Dyer (1959) method. Total
carbohydrate content was determined following the phenol sulphuric acid method. Amino acids
as well as chemical composition of whole body were analyzed by the laboratory Maxxam
Analytics, Burnaby, Canada. Proximate composition of practical diets is shown in Table 2.1.
Fatty acids (FA) were determined based on the one-step method described by Abdulkadir
and Tsuchiya (2008). Fatty acid methyl esters (FAME) of diets and shrimp tissue were prepared
in two replicates. Butylated hydroxytoluene (BHT) was added to the hexane solvent to prevent
lipid oxidation. FAMEwere quantified using a Varian 3900 Gas Chromatograph (Toronto,
Canada) with a Chrompack capillary column (CP-Sil 88 for FAME). Fatty acids were identified
by comparison of retention times to those of known standards and they were quantified by
comparing the peak area of individual FA in the sample with the peak of the internal standard
(Nonadecanoic acid, C19:0).
23
2.2.4 Sensory evaluation
At harvest, Pacific white shrimp heads were removed and tails were refrigerated (4°C)
overnight for sensory analysis the next day. Sensory analysis of shrimp tails was carried out at a
commercial shrimp processing plant by a panel of seven expert judges, who conduct shrimp
quality analysis on a daily basis. Shrimp tails were cooked for 1.5 min in boiling water, then
refrigerated to stop the cooking process before serving (5°C).
Samples from each tank (5 treatments×4 replications) were presented to the judges in a
pairwise manner, where each treatment was evaluated with every other treatment repeated four
times. There was a total of 10 combinations among the five treatments (FM-37 with AS-37,
ASS-48, ASS-34 and COM-38; AS-37 with ASS-48, ASS-34 and COM-38, ASS-48 with ASS34 and COM-38 and ASS-34 with COM-38).
Shrimp tail samples were served for each treatmentonwhite chinaware plates labeled
according to their treatment code (1-5). A sensory ballot was given to each judge consisting of 9point rating scales, one for each of the desirable (aroma, color, firmness, moistness, fattiness,
sweetness)and undesirable (off-odor, fish flavor, earthiness, off-flavor, overall unpleasantness)
sensory attributes.Judges evaluated shrimp based on the magnitude of sensory characteristics,
relative to the anchor (i.e. reference value) provided (Table 2.8). Immediately following each
testing, judgeswere given water and a piece of cracker to cleanse the palate. Sensory forms were
collected at the end of the sensory evaluation.
Descriptive analysis was performed using a collection of six desirable (aroma, color,
sweetness, firmness, moisture, fattiness) and five undesirable (off-odor, earthiness, off-flavor and
overall unpleasantness) sensory attributes. All quality attributes were evaluated on 9-point
structured rating scales, (Botta, 1995), anchored with references to delineate between acceptable
24
and unacceptable portions of the scale.The desirable attributes:aroma, color, sweetness, firmness,
moisture, fattiness, were anchored at 3.0, 3.5, 5.0, 6.0, 6.0 and 4.0, respectively. All the
undesirable attributes: off-odor, fish flavor, earthiness, off-flavor and overall pleasantness, were
anchored at 1.0, with the exception of fish flavor, which had an anchor at 4.0. These scores were
placed as anchors on the linescales, allowing each judge to score the samples relative to
previously established reference shrimp values recognized as a point of reference.
2.2.5 Statistical analyses
Treatment effects for water quality and production parameters were evaluated using oneway analysis of variance (ANOVA). Significant differences were determinedamong the
treatment meansusing Tukey test at α=0.05. Sensory data were analyzed using 3-way ANOVA,
with judge, treatment and replication as main effects and all 2-way interactions (judge×treatment,
judge×replication, treatment×replication). These interaction effects were used to evaluate the
consistency and reproducibility of the judges, prior to interpretation of the treatment effects
(Garcia-Juarez, 2005).
An initial screening of the data revealed that a large portion of the data was missing for
one judge; so these data were dropped from further analysis. In addition, preliminary analysis
from another judge indicated that their scores were double that of the other judges and therefore
these data were also dropped. In the end, data from fivejudges wereanalyzed.
Judges evaluated the 11 sensory attributes in the following order: aroma, off-odor, color,
firmness, moistness, fattiness, fish flavor, sweetness, earthiness, off-flavor, and overall
unpleasantness. For the purpose of interpretation, the attributes were grouped into two
categories:desirable (aroma, color, sweetness, firmness, moisture, fattiness) and undesirable (offodor, fish flavor, earthiness, off-flavor, overall unpleasantness) attributes.
25
The mean sensory ratings were determined for the desirable and undesirable attributes
and compared using the Fischer least-significance-difference (LSD) atα=0.05.Since all the
undesirable attributes had an anchor of 1.0, with the exception of fish flavor, sensory scores for
fish flavor were re-scaled (i.e. sensory scores were multiplied by a constant) so that the values
were equivalent to a scale of 1 through 9.
In addition, the inter-correlation of the sensory attributes was determined by calculating
Pearson correlation coefficients (r) from the mean scores. Minitab 16 Version Software (state
College PA, USA) was used to carry out the statistical analysis.
2.3 Results and discussion
2.3.1 Water quality
Water quality parameters were maintained at optimum levels for adequate growth and
survival of shrimp (Table 2.2). However, ammonium (NH4+) levels in the tank holding shrimp
fed ASS-48 presented higher values than all other groups with an average value of 1.52 mg/L.
ASS-48 had the highest protein input (48.34%), which could have contributed toelevated
ammonium levels in the water. According to Kureshy and Davis (2002), protein content and
availability can affect water quality via shrimp nitrogen excretion. No differences existed among
treatments for water quality parameters (P > 0.05).
2.3.2 Growth performance and production parameters
No evidence of disease was observed during the growth trial. Survival was 85-95% and
did not differ among treatments (Table 2.3). The high survival rate during the growth trial
indicated good health conditions of the shrimp. Based on visual observation of feed consumption
using a feeding tray, there were no indicators of feed rejection or reduced palatability. Pacific
26
white shrimp accepted all of the test diets, demonstrating the palatability of the new ingredients.
These observations are consistent with the findings of Ceballos et al. (2005), who reported the
high attractiveness of Spirulina for Litopenaus schmitti.Moreover, our results are in line with the
findings of Silva-Nieto et al.(2012), who reportedthat Spirulina meal is an efficient feeding
attractant for Litopenaeus vannamei.
Production parameters of Pacific white shrimp are presented in Table 2.3. Shrimp weight
increased from 465.7% to 849.9%, relative to initial values. Groups fed FM-37 and COM-36 had
higher final weight and weekly weight gain mean values as opposed to mean values for groups
fed AS-37, ASS-34, and ASS-48. The average weekly growth rate was 1.15 g for group FM-37,
1.02 g for AS-37, 0.77 g for ASS-48, 0.97 g for ASS-34 and 1.31 g for diet COM-36, indicating
that shrimp maintained on the control diets (COM-36) and (FM-37) had the best growth rate.
Diet FM-37 contained FM and FO andthus likely were the most digestibleresulting in the higher
growth performance and nutrient utilization in shrimp. FM is a good source of marine oils that
are rich in highly unsaturated fatty acids (HUFA)(Turchini et al., 2010). Lipid content and the
associated C18 polyusaturated fatty acids (PUFA), linoleic (18: 2n-6) and linolenic (18: 3n-3) as
well as n-3 and n-6 HUFA (EPA, DHA, ARA,), are required in shrimp and other crustacean
feeds for optimal growth performance (Fennuci et al., 1981; Kanazawa et al., 1997). The
minimum requirement of these fatty acids in crustacean feeds is at levels between 5 and 10 g/kg
(Akiyama et al., 1992; Gonzales-Felix et al., 2002).
Shrimp fed diets with substitution of FM with algae protein sources exhibited low final
mean body weight values, and significant differences in weight among the treatments were
observed (Table 2.3). Growth performance of groups fed AS-37, and ASS-34, had the highest
mean weight values of the algae based feeds (Figure 2.1). This latter finding could be attributed
27
to the beneficial properties of DHA from Schizochytrium of AS-37 and the combination of
Spirulina and Schizochytrium of ASS-34. The lowest final weight mean value was found in
shrimp fed the ASS-48 treatment. It is possible that low performance was affected by a low
digestible energy level, which would cause shrimp to utilize protein as a source of energy
(Kureshy and Davis, 2002).These latterauthors studied the protein requirement for maintenance
and maximum weight gain for the Pacific white shrimp by utilizing three practical diets (16%,
32% and 48% dietary protein) and found that the diet containing 48% protein resulted inlower
weight gains in juvenile shrimp. Juvenile and sub-adult shrimp exhibited better growth
performance when fed the 32% protein diet.
Similarly, Hu et al. (2008) investigated the effects of dietary protein to energy ratios on
growth rates of juvenile white shrimp and found that protein efficiencydecreased with increasing
dietary protein from 340 to 420 g/kg, indicating that the surplus protein was degraded rather than
used for growth. Usually, low levels of dietary protein are efficiently used for protein synthesis
by shrimp. In the present study we speculate that there was aninefficient conversion of protein to
growthin the shrimp fed the AM-48 diet due the continued catabolism of protein in the absence
of any protein sparing effect at high protein levels.
The low growth response of shrimp fed ASS-48 may also have been due to reduced
digestibility of the diet.Ammonium needs to be maintained at acceptable levels (below 2.3 mg/L
(0.09 mg/L for NH3; Chen et al., 1990)as build-up in shrimp culture systems hasbeen shown to
reduce growth performance (Shilo and Rimon 1982). Althoughthis research did not measure NH3
in the water directly, we could assume that NH4+ concentrations was less than 2.28 mg/L. Our
NH4+ concentrations for tanks assigned treatmentASS-48 ranged between 0.0025-4.53 mg/L and
had the highest mean value compared to the other treatments (Table 2.2). This may indicate that
28
there was an excess of nitrogen in the diet, which resulted in high nitrogen excretion as shrimp
used excess protein for energy.Research has shown that if protein is in excess relative to energy,
excessive dietary protein will be used for energy, resulting in higher Specific Dynamic Action
(SDA) and more excreted ammonia nitrogen (Cho et al. (1994); Samantaray andMohanty (1997),
Mathis et al. (2003) and Buffordet al. (2004)). Further research on shrimp diets that contain
algae ingredients is needed to investigate nutrient digestibility and establish protein requirements
based on alternative ingredients.
Growth performanceand feed utilization parameters were affected by dietary
treatments(Table 2.3). In the present study FCR ranged between 1.6 and 2.5. Ingested protein
was more efficiently utilized for growth by shrimp fed FM-37orCOM-36 (P< 0.05), which had
the best FCR (Table 2.3). Conversely, high FCR were observed in treatments fed AS-37, ASS-48
and ASS-34. Groups fed ASS-48 exhibited the highest FCR due most likely to the continued
catabolism of protein for energy. Although it is generally accepted that fish and shrimp do not
have a specific requirement for dietary carbohydrates, they are widely used in commercial
aquaculture feeds. Numerous studies have demonstrated that carbohydrates must be supplied in
available form in the diet.
The nutritional composition of diet ASS-48 had the lowest
carbohydrate levels compared to the rest of the diets (Table 2.1). This low carbohydrate level in
diet ASS-48 may have been insufficient to meet the energy requirements of the shrimp. Thus,
shrimp possibly converted part of the dietary protein to energy. In turn, this increased protein
degradation and increased energy expenditure and ammonium release into the water. Using
protein as an energy source is relatively inefficient and reduces the amount of protein available
for tissue deposition (Hochachka, 1991).
29
Overall, the experimental diets met the essential lysine (1.6%) and methionine (0.9%)
requirements on anas fed basis typical for adequate growth of penaeids (Millamena et al., 1996).
Some nutritional requirements of Litopenaeus vannamei are known. However, amino acid
requirements are more widely established for Penaeus monodon.
Millamena et al. (1998) and Xie et al. (2012) claimed that the optimum dietary lysine
requirement is 2.0 % based on specific growth rates. Fox et al. (1995) reported that the optima
lysine requirement for juvenilLitopenaeus vannamei is 1.2-2.3%. Lysine is well known to be an
indispensable amino acid for normal growth of shrimp (Cowey and Forster, 1971; Fox et al.,
1995; Kanazawa and Teshima 1981; Richard et al., 2010). Appropriate lysine content could
reduce the oxidation of other aminoacids by improving the use of other indispensable amino
acids (Kerr and Easter, 1995), by promoting a higher growth rate (Xie et al., 2012).
Lysine, arginine and methionine are considered the most limiting indispensable amino
acids (IAA) for shrimp in commercial feed formulations (Akiyama et al., 1991). In the present
study, lysine levels were below the optimum for the experimental diets AS-37, and ASS-34,
which could represent a dietary deficiency that may have led to lower growth and inferior feed
efficiency. Methionine and arginine levels were somewhat different from the optimum
requirement (Akiyama et al., 1991) (Table 2.4).
Some amino acids (i.e. threonine, arginine, histidine, isoleucine and leucine) were present
in diets at concentrations higher than the AA requirementsfor shrimp proposed by (Millamena et
al., 1999) (Table 2.4). Diet ASS-48 exhibited an excess of the optimum requirement of some
IAA, which in turn may have adversely affected shrimp growth. Generally, weight gains of
shrimp tend to decrease when requiredamino acid levels are exceeded (Millamena et al., 1999).
The reason for the decline in weight gain has not been established, but may be a response to the
30
shrimp’s intolerance to excess amino acids beyond the optimum dietary level (Mertz 1972). The
dietary lipid levels were in the optimum range (5-10%) for adequate growth of white shrimp as
reported by Siccardi (2006). The inclusion of DHA in diet AS-37%produced the best growth
performance in shrimp out of all the algae based diets, demonstrating the high potential of algal
oils as fish oil substitute in aquaculture feeds.
To summarize, shrimp fed the commercial control diet COM-36had the highest weight
gain; whereas, theshrimp fed algae diets had lower weight gains. It is possible that reduced
availability of IAA via leaching could have reduced growth in shrimp fed the algae experimental
diets. According to Millamena (1996), the feeding behavior of shrimp results in these animals
being slow continuous feeders; hence, if feeds are not water stable (i.e. do not retain pellet
physical integrity with minimal disintegration and nutrient leaching), crystalline amino acids are
quickly leached out and may no longer be present when the feed is consumed. However, we
recognize that our laboratory made feeds may not have the same physical properties of
commerciallyextruded manufactured feeds.
2.3.3 Chemical composition of shrimp body
Body chemical composition of shrimp is shown in Table 2.5 and was similar across all
treatments. Fatty acid composition of shrimp tails reflected the fatty acid profile of the
experimental diets qualitatively (Table 2.6). The addition of Schizochitrium and Spirulinato the
experimental diets, in general, did not affect the fatty acid composition of shrimp tails (Table
2.7). Particularly, no differences (P > 0.05) were detected in the content of three essential fatty
acids (DHA, EPA and ARA) in the tail of shrimps fed algae based diet, the control diets.
Therefore, the HUFA contents did not differ between treatment groups. Total n-3 and n-6 ratios
observed in shrimp tissue ranged from 0.61 to 0.72.
31
Given that fish oil was replaced with alternative oils, it is extremely important to ensure
that an animal’s essential fatty acid requirements are met. Our results indicate that the use of
Schizochitrium and Spirulina ingredients contribute to optimal HUFA contents in shrimp tissue,
comparable to that of fish oil. Shrimp tails from groups fed algae based feeds exhibited
comparable HUFA contents, and high n-3/n-6 ratios in relation to shrimp tails from groups fed
fish meal based feeds, which are targeted in seafood production and recommended in the human
diet to promote health. Patnaik et al. (2006) demonstrated that fish oil can be successfully
replaced in diets for Litopenaeus vannamei using cells of heterotrophic algae Schizochytrium sp.
and Mortierella sp. obtained from commercial microbial fermentation.
The results of this study reaffirm that Schizochytrium is a suitable fish oil substitute
(HUFA source) in diets fed toLitopenaeus vannamei. However, our results demonstrate that fatty
acids cannot be the sole consideration in nutritional studies. In spite of the balanced fatty acid
composition of shrimpfed the experimental diets(algae based), the inclusion of algae ingredients
did not result in improvements in growth performance. Digestibility studies are needed in order
to better understand the metabolic processes that might influence the efficacy of including algae
ingredients in shrimp diets. In addition, features of algae feeds, such as water stability, need to be
optimized to prevent nutrientsfrom leaching out that would compromise growth performance.
2.3.4 Sensory evaluation
The results of sensory evaluation of shrimp tails from Litopenaeus vannamei are shown in Table
2.8. The desirable attributes of aroma, color and sweetness differed between treatments (P ≤
0.05). In contrast, the remaining desirable attributes (firmness, moistness, fattiness) did not
exhibit statistical differences (P > 0.05). For the undesirable attributes (0ff-odor, fish flavor,
earthiness, off-flavor and overall unpleasantness)no differences were detected.
32
Judges were a significant source of variation in all cases (P ≤ 0.05) (Table 2.9). Judge
variations are normally attributed to individual taste and scoring differences. Judge-to-judge
variation is accepted in sensory evaluation, however, judge consistency as reflected by the
interaction (Judge x Sample) is considered more critical (Cliff et al., 1996). In this study, there
were differences for the interaction judge x sample for the attributes of aroma and color,
suggesting judge inconsistency. It is possible that these terms were not used in the same way by
all judges. Nonetheless, F-values were recalculated and new values indicated that in spite of
some inconsistency found among the interactions judge x sample for color and aroma, the
samples did differ in these two attributes. On the other hand, the interaction judge x replication
did not exhibitdifferences (P > 0.05) which demonstrates reproducibility. Reproducibility
indicates how an individual agrees on average with the panel as a whole, showing the
homogeneity of the team (Ross, 2001).
Overall the cooked shrimp tail of the groups fed the experimental diets in comparison
with the commercial control had a mild aroma (=2.65-3.55) butno off-odors (=1.30-1.60).
The shrimp muscle was light orange in color (=3.00-4.02). The texture was of medium
firmness (=4.65-5.30), moistness (=5.15-6.05), and fattiness (=2.95-3.65). Flavor wise,
shrimp taste had a mild fish flavor (=4.50-4.70), medium sweetness (=3.80-5.15). It had
almost no earthiness (=2.00-2.40), was considered to be nearly free of other tastes (=1.351.95), and had low scores for overall unpleasantness (=2.95-3.0). Desirable attributes including
fattiness, firmness and moistness were comparable between shrimp fed the algae diets and the
control feeds. These results are somewhat similar to the findings for Litopenaus vannamei of
Forster et al. (2011), whofailed to show differences for these sensory characteristics with dietary
inclusion of plant-derived sources (i.e. SBO). In contrast, the attributes aroma, color and
33
sweetness were different (P < 0.05) between shrimp fed the various diets (Figure2.2). Shrimp fed
FM-37 and COM-36 wereconsidered to be sweeter than AS-34 and ASS-48, which did not differ
from each other. In contrast, shrimp fed AS-37 were less sweet that FM-37, but identical in
sweetness intensity to the shrimp fed the remaining diets (COM-36, ASS-34, ASS-48). Shrimp
fed COM-36 had higher aroma intensity than FM-37 and ASS-34. Also, shrimp fed AS-37
exhibited a stronger aroma than ASS-34. Shrimp fed ASS-48 did not differ from the other
diets.Lastly, shrimp fed AS-37, FM-37 and COM-36 did not differ in color. Shrimp fed AS-37
were also similar in color to ASS-34 and ASS-48, which did not differ.
For the undesirable attributes of off-odor, fish flavor, off flavor, earthiness and overall
unpleasantness, statistical differences were not identified. However, it is noteworthy that shrimp
fed AS-37 exhibited the lowest scores for these attributes, suggesting that judges had a stronger
positive sensory experience with this group of shrimp in relation to groups fed the remaining
diets (Figure 2.3). The correlation coefficients of sensory attributes are shown in Table 2.10. As
expected, firmness and fattiness were highly correlated (r= 0.92). Similarly, perceived fish flavor
and overall unpleasantness were correlated (r=0.97). According to Erickson et al. (2007), shrimp
in general are considered to have a mild but distinctive flavor with a texture described as tender
and delicate. In this study, mean scores revealed that shrimp were perceived as mild in
flavor.Color and sweetness were also correlated (r=0.96), as well as moistness and earthiness
(r=0.93). The undesirable attributes of fish flavor, off-flavor and overall unpleasantness were
negatively correlated with aroma (r=-0.92, -0.95, -0.97),respectively.
Above all, the quality of the shrimp was acceptable based on a reference standard, and
dietary inclusion of algae ingredients did not have a negative influence on most sensory
characteristics of shrimp muscle. Remarkably, shrimp fed AS-37 were perceived at the lowest
34
scores for undesirable attributes. For instance diet AS-37 showed greater promise. Our results
can provide insight into further designs for adequate algae diets to produce shrimp with more
desirable sensory characteristics.
The results of the present study suggest that FM replacement with Spirulina and
Schizochytriumis promising. Although algae diets did notyield improved growth performance in
shrimp compared with the reference diet, they could be a key contributor to reduce the use of FM
in shrimp feeds. Algae diets showed to be a rich source of HUFA, and shrimp were able retain
high levels of HUFA in muscle tissue. Remarkably, diet AS-37 showed great potential as an
alternative feed source for Litopenaeus vannamei, indicating satisfactory shrimp quality. In the
current research, we investigated if Pacific white shrimp can accommodate the algal meals in
their feeds. Our findings show that algae meals did not have a negative influence on most
sensory attributes, hence when considering consumer acceptance, the inclusion of algae
ingredients in shrimp feeds could be commercially viable. Further studies on digestibility of
nutrients should be considered when formulating algae meals for their optimization. Another
important aspect to consider is the feed processing technique and addition of supplements in
order to increase water stability in algae feeds.
2.4 Tables
Table 1.1. General composition of different algae (% of dry matter).
35
Alga
Protein
Carbohydrate
Lipid
Anabaena cylindrica
43-56
25-30
4-7
Aphanizomenon flos-
62
23
3
48
17
21
Chlorella pyrenoidosa 57
26
2
Chlorella vulgaris
51-58
12-17
14-22
Dunaliella salina
57
32
6
Euglena gracilis
39-61
14-18
14-20
Porphyridium
28-39
40-57
9-14
Scenedesmus obliquus
50-56
10-17
12-14
Spirogyra sp.
6-20
33-64
11-21
Arthrospira maxima
60-71
13-16
6-7
Spirulina platensis
46-63
8-14
4-9
Synechococcus sp.
63
15
11
aquae
Chlanydomonas
rheinhardii
cruentum
Table 2.1. Nutritional composition of practical diets for Litopenaeus vannamei.KJ:
Kilojoules/100g.FM-37 (Fish meal based feed, main protein source: fish meal at 37%); AS-37
36
(Algae based feed, main protein sources: Spirulinaat 37%); ASS-48 (Algae based feed, main
protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein source: fish meal at 36%).
Component
Diet
FM-37
AS-37
ASS-48
ASS-34
COM-36
kj
18.72
17.92
19.16
18.38
17.91
Carbohydrate %
37.40
38.30
28.10
42.58
41.50
Protein
%
37.26
37.13
48.34
34.10
35.65
Lipid
%
8.60
8.00
8.40
8.33
5.96
Moisture
%
9.00
10.00
6.30
8.40
9.80
Ash
%
7.80
6.50
8.80
6.50
6.78
Energy
37
Table 2.2. Average of water quality parameters during the 10-week culture period for
Litopenaeus vannamei. Values are means ± SD.FM-37 (Fish meal based feed, main protein
source: fish meal at 37%); AS-37 (Algae based feed, main protein sources: Spirulinaat 37%);
ASS-48 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 48%); ASS-34
(Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%) and COM -36
(Fish meal based feed, main protein source: fish meal at 36%).
Value of Water Quality Parameter
Diet
FM-37
AS-37
ASS-48
ASS-34
COM-36
am 28.83 ± 0.76
28.87 ± 0.77
28.82 ± 0.74
28.77 ± 0.80
28.77 ± 0.77
pm 30.62 ± 0.99
30.57 ± 1.08
30.53 ± 1.03
30.44 ± 1.01
30.46 ± 0.95
am 5.71 ± 0.62
5.69 ± 0.62
5.68 ± 0.60
5.69 ± 0.61
5.69 ± 0.67
pm 5.64 ± 0.62
5.62 ± 0.56
5.60 ± 0.56
5.63 ± 0.55
5.65 ± 0.52
am 8.17 ± 0.19
8.18 ± 0.19
8.17 ± 0.20
8.18 ± 0.19
8.16 ± 0.20
38.6 ± 1.45
38.67 ± 1.47
38.70 ± 1.48
38.71 ± 1.48
38.67 ± 1.47
0.63 ± 0.07
1.03 ± 1.03
1.52 ± 1.19
0.78 ± 0.95
0.87 ± 0.86
Temperature (°C)
Oxygen (mg/L)
pH
Salinity (ppt)
Ammonium
(mg/L)
38
Table 2.3. Average production parameters at the end of a 10-week culture period for
LitopenaeusVannamei fed practical diets with varying levels of algae meal and fish meal diets1.
Values are means ± SD.FM-37 (Fish meal based feed, main protein source: fish meal at 37%);
AS-37 (Algae based feed, main protein sources: Spirulinaat 37%); ASS-48 (Algae based feed,
main protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main
protein sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed,
main protein source: fish meal at 36%).
Parameter
Diet
FM-37
AS-37
ASS-48
ASS-34
COM-36
P-Value
1.72±0.03
1.65±0.07
1.60±0.10
1.6±0.04
1.71±0.09
P> 0.05
13.40±0.31b
12.03±0.77c
9.41±0.50d
11.47±0.25c
14.95±0.67a
P≤0.05
1.15±0.03b
1.02±0.08c
0.77±0.05d
0.97±0.0c
1.31±0.07a
P≤0.05
1.67±0.05c
2.04±0.17b
2.50±0.13a
2.04±0.07b
1.60±0.06c
P≤0.05
95
87
85
89
85
P> 0.05
Initial
weight (g)
Final
weight (g)
Weekly
weight
gain (g)
FCR
Survival
(%)
1
Means within rows that do not share the same letter are significantly different (Tukey, alpha= 0.05).FCR, feed
conversion ratio.
39
Table2.4. Amino acid composition of practical diets for Litopenaeus.vannamei(%)1.FM-37 (Fish
meal based feed, main protein source: fish meal at 37%); AS-37 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%); ASS-48 (Algae based feed, main protein sources:
Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein sources:
Spirulina and Schizochitrium at 34%).
Diet
FM-37
Indispensable
AS-37
Recommended
ASS-48
ASS-34 Level2
amino
acids
Valine
1.33
1.71
2.19
1.56
-
Threonine
1.23
1.61
2.26
1.46
1.40
Methionine
0.68
0.62
0.95
0.58
0.90
Isoleucine
1.06
1.49
1.92
1.32
1.00
Leucine
2.23
2.74
3.59
2.52
1.70
Phenylalanine
1.37
1.64
2.01
1.48
1.40
Hisitidine
1.04
0.96
1.22
0.90
0.80
Lysine
1.84
1.57
2.03
1.46
2.10
Arginine
1.77
2.19
2.86
2.07
1.80
Tryptophan
0.29
0.36
0.49
0.38
0.2
40
Diet
Recommended
FM-37
AS-37
ASS -48
ASS-34
Level2
Glycine
1.61
1.67
2.24
1.52
-
Alanine
1.51
2.04
2.97
1.87
-
Cysteine
0.41
0.37
0.45
0.38
-
Tyrosine
0.90
1.28
1.75
1.14
-
Proline
2.36
2.35
2.54
2.24
-
Serine
1.53
1.86
2.39
1.76
-
Glutamic acid
6.56
6.64
7.22
6.77
-
Non-indispensable
aminoacids
1
Amino acid composition of diets were analyzed by a commercial laboratory (Maxxam, Canada)
2
Values recommended by Millanema et al. (1999)
-
Values not reported
41
Table 2.5. Whole body chemical composition of Litopenaeus vannamei fed various diets1.KJ:
Kilojoules/100g.FM-37 (Fish meal based feed, main protein source: fish meal at 37%); AS-37
(Algae based feed, main protein sources: Spirulina at 37%); ASS-48 (Algae based feed, main
protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein source: fish meal at 36%).
Component
Diet
FM-37
AS-37
ASS-48
ASS-34
COM-36
Energy
kj
18.19
15.20
16.76
16.53
17.27
Carbohydrate
%
0.20
-2
-
-
0.30
Protein
%
22.21
22.65
22.70
23.25
23.11
Lipid
%
0.63
0.57
0.39
0.82
0.80
Moisture
%
75.10
75.00
75.10
74.4
74.0
Ash
%
1.90
1.90
2.00
1.90
1.80
1
Whole body chemical composition of shrimp analyzed by a commercial laboratory (Maxxam, Canada)
2
Not detected
42
Table 2.6. Fatty acid composition (% of FAME) of diets1.FM-37 (Fish meal based feed, main
protein source: fish meal at 37%); AS-37 (Algae based feed, main protein sources: Spirulina at
37%); ASS-48 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 48%);
ASS-34 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%) and
COM -36 (Fish meal based feed, main protein source: fish meal at 36%).
Selected FA
FM-37
AS-37
ASS- 48
ASS-34
COM-36
14:0
7.60
3.13
6.37
9.51
7.92
16:0
29.18
40.32
43.82
41.69
30.44
16:1
6.86
2.93
3.37
1.82
4.63
18:0
3.50
1.86
1.35
1.39
3.77
18:1n-9
15.32
9.37
4.53
5.14
11.87
18:2n-6
8.08
10.32
7.05
7.49
11.12
18:3n-3
3.87
1.77
0.98
1.50
3.13
20:3n-3
0.63
-2
0.18
0.32
0.44
20:3n-6
0.77
-
0.25
0.44
0.10
20:4n-6
0.30
0.08
0.15
0.30
0.14
20:5n-3
0.12
0.20
0.12
0.23
0.10
22:0
0.24
0.29
0.23
0.30
0.32
22:6n-3
4.38
2.62
4.31
8.76
2.86
24:0
4.64
0.20
0.32
0.61
4.41
Saturates3
45.15
45.8
52.10
53.51
46.86
Monounstaturates4
22.18
12.3
7.90
6.96
16.50
PUFA5
13.34
12.09
8.47
9.75
14.79
43
Selected FA
FM-37
AS-37
ASS-48
ASS-34
COM-36
HUFA6
4.80
2.90
4.58
9.29
3.10
Total n-3
9.00
4.59
5.59
10.80
6.53
Total n-6
9.14
10.40
7.45
8.23
11.36
1
2
Values represent averages of duplicate samples.
Not detected.
3
Saturates: 14:0, 16:0, 18:0, 22:0, 24:0.
4
Monosaturates: 16:1, 18:1.
5
PUFA: 18:2, 18:3, 20:3.
6
HUFA: 20:4, 20:5, 22:6.
7
Total n-3: 18:3n-3, 20:3n-3, 20:5n-3, 22:6n-3.
8
Total n-6: 18:2n-6, 20:3n-6, 20:4n-6
44
Table 2.7. Fatty acid composition (% FAME) in shrimp muscle tissue 1.FM-37 (Fish meal based
feed, main protein source: fish meal at 37%); AS-37 (Algae based feed, main protein sources:
Spirulina at 37%); ASS-48 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 48%); ASS-34 (Algae based feed, main protein sources:
Spirulina and
Schizochitrium at 34%) and COM -36 (Fish meal based feed, main protein source: fish meal at
36%).
Selected FA
FM-37
AS-37
ASS- 48
ASS-34
COM-36
14:0
3.16
2.47
2.96
2.09
2.41
16:0
23.66
26.56
27.08
22.73
24.14
16:1
1.70
1.01
1.69
1.92
1.51
18:0
9.69
8.00
8.53
14.05
11.19
18:1n-9
10.78a
7.32bc
5.35c
1.89d
9.73ab
18:2n-6
14.11ab
14.57ab
16.94a
13.2ab
11.19b
18:3n-3
1.27a
0.51c
0.58bc
0.46c
0.91b
20:3n-3
2.0b
3.47ab
4.06a
2.87ab
1.98b
20:3n-6
0.13
0.10
-2
0.06
0.11
20:4n-6
0.14
0.13
0.14
0.22
0.14
20:5n-3
0.11
0.09
0.14
0.14
0.09
22:0
0.53
0.43
0.60
0.77
0.45
22:6n-3
6.99
4.88
5.70
5.28
5.36
24:0
8.72ab
3.83c
4.48bc
3.61c
9.67a
Saturates3
45.77
41.29
43.65
43.26
47.85
Monounstaturates4
12.48a
8.33bc
7.05cd
3.81d
11.24ab
45
Selected FA
FM-37
AS-37
ASS-48
ASS-34
COM-36
PUFA5
17.50ab
18.65ab
21.59a
13.59ab
14.19b
HUFA6
7.25
5.25
5.99
5.64
5.59
Total n-37
10.37
9.11
10.49
8.75
8.34
Total n-68
14.37
14.8
17.09
13.48
11.44
2-8
1
See footnotes in Table 6.
Values represent averages of duplicate samples. Means within rows that do not share letter are significantly
different (Tukey, alpha= 0.05).
46
Table 2.8. Mean sensory characteristics (n=20; 5 judge ×4 replications) of shrimp at the end of
the feeding trial. Values are means ± SE mean. R represents the standard expected value for each
attribute. FM-37 (Fish meal based feed, main protein source: fish meal at 37%); AS-37 (Algae
based feed, main protein sources: Spirulina at 37%); ASS-48 (Algae based feed, main protein
sources:
Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein
sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based feed, main
protein source: fish meal at 36%).
Sensory Attribute
(Numericalanchors
on scale),
Diet
FM-37
AS-37
ASS-48
ASS-34
COM-36
2.84 ± 0.23
3.50 ± 0.23
2.65 ± 0.23
3.05 ± 0.23
4.55 ± 0.23
1.250 ± 0.23
1.45 ± 0.23
1.60 ± 0.23
1.45 ± 0.23
1.30 ± 0.23
4.05 ± 0.26
3.44 ± 0.26
3.00 ± 0.26
3.05 ± 0.26
4.02 ± 0.26
5.16 ± 0.24
4.65 ± 0.24
5.25 ± 0.24
5.20 ± 0.24
5.30 ± 0.24
6.01 ± 0.28
5.15 ± 0.28
6.05 ± 0.28
5.80 ± 0.28
5.65 ± 0.28
reference anchor (R)
Aroma
(1: mild, 9: strong)
R: 3
Off-odor
(1: none, 9: strong)
R: 1
Color
(1: light, 9: dark)
R: 3.5
Firmness
(1: tender, 9: firm)
R: 6
Moistness
(1: dry,9: moist)
R: 6
47
Sensory Attribute
(Numericalanchors
on scale),
Diet
FM-37
AS-37
ASS-48
ASS-34
COM-36
3.49 ± 0.18
2.95 ± 0.18
3.65 ± 0.18
3.30 ± 0.18
3.65 ± 0.18
4.47 ± 0.24
4.05 ± 0.24
4.70 ± 0.24
4.25 ± 0.24
4.20 ± 0.24
5.15 ± 0.23
4.25 ± 0.23
4.05 ± 0.23
3.80 ± 0.23
4.85 ± 0.23
2.40 ± 0.18
2.00 ± 0.18
2.40 ± 0.18
2.15 ± 0.18
2.20 ± 0.18
1.95 ± 0.22
1.40 ± 0.22
1.95 ± 0.22
1.55 ± 0.22
1.35 ± 0.22
3.15 ± 0.27
2.95 ± 0.27
3.25 ± 0.27
3.10 ± 0.27
3.00 ± 0.27
reference anchor (R)
Fattiness
(1: lean,9: fatty)
R: 4
Fish Flavor
(1: mild, 6: strong)
R: 4
Sweetness
(1: none,9: strong)
R: 5
Earthiness
(1: none, 9: earthy)
R: 1
Off-flavor
(1: none,9: strong)
R: 1
Overall unpleasantness
(1: none,9: strong)
R:1
48
Table 2.9. Analysis of variance of sensory characteristics ratings (5 judges): degrees of freedom
(df), F-ratios, and error mean squares (MSE). J*S: Judge and sample interaction. J*R: Judge and
replicate interaction. S*R: Sample and replicate interaction.
F-ratios
Attribute
Judges
Sample
Replicates J*S
J*R
S*R
MSE
(J)
(S)
(R)
Aroma
6.23*
2.94*
0.71
3.59*
1.64
0.82
1.07
Off-odor
8.89*
0.36
0.97
0.43
0.97
1.16
1.08
Color
11.24*
3.59*
1.42
2.35*
1.14
1.07
1.44
Firmness
22.2*
1.20
1.62
1.83
1.16
0.63
1.15
Moistness
6.94*
1.64
0.62
1.08
1.08
1.48
1.60
Fattiness
13.97*
2.44
0.63
1.46
0.36
0.76
0.70
Fish flavor
2.72*
1.08
0.84
2.28
0.91
1.10
1.20
Sweetness
10.17*
5.76*
0.55
1.83
1.32
1.36
1.10
Earthiness
157.52*
0.86
0.05
1.46
0.05
1.17
0.68
Off-flavor
10.64*
1.74
0.43
0.75
0.73
0.62
0.98
Overall
73.1*
0.18
0.38
1.38
0.27
1.29
1.55
4
4
3
16
12
12
48
unpleasantness
Df
* Values within each row that are significantly different at P≤0.05
49
Table 2.10. Correlation matrix among the sensory attributes (df=3)
Attribute
1
2
3
4
5
6
7
8
9
10
1.Aroma
1.00
2.Off-odor
-0.36
1.00
3.Color
0.39
-0.93*
1.00
4.Firmness
-0.43
-0.12
0.08
1.00
5.Moistness
-0.88
-0.02
-0.06
0.83
1.00
6.Fattiness
-0.43
-0.10
0.21
0.92*
0.79
1.00
7.Fish flavor
-0.92*
0.33
-0.23
0.58
0.88*
0.69
1.00
8.Sweetness
0.15
-0.85
0.96*
0.15
0.13
0.33
0.02
1.00
9.Earthiness
-0.81
-0.04
0.12
0.69
0.93*
0.80
0.93*
0.35
1.00
10.Off-flavor
-0.95*
0.17
-0.14
0.34
0.81
0.44
0.91*
0.13
0.88
1.00
11.Overall
-0.97*
0.39
-0.37
0.59
0.91*
0.61
0.97*
-0.14
0.87
0.91*
11
1.00
unpleasantness
* Values represent correlation at p ≤ 0.05
50
2.5 Figures
17
Body mass (g)
15
13
FM-37
11
AS-37
9
ASS-48
7
ASS-34
5
COM-38
3
1
0
9
24
38
Culture day
51
71
Figure 2.1. Final body weight of Pacific white shrimp fed two control (FM-37; COM-36) and,
three experimental (AS-37, ASS-48, ASS-34) diets reared under a green water system.FM-37
(Fish meal based feed, main protein source: fish meal at 37%); AS-37 (Algae based feed, main
protein sources: Spirulina at 37%); ASS-48 (Algae based feed, main protein sources: Spirulina
and Schizochitrium at 48%); ASS-34 (Algae based feed, main protein sources: Spirulina and
Schizochitrium at 34%) and COM -36 (Fish meal based feed, main protein source: fish meal at
36%). Error bars represent SD.
51
6
a
a
5
a
a
4
b
a
b
b
ab ab
Score
bc
abc b
b
c
3
Aroma
Color
Sweetness
2
1
0
FM-37
COM-36
AS-37
Diet
ASS-48
ASS-34
Figure 2.2. Mean scores for the sensory attributes aroma, color and sweetness. Bars represent
aroma, color and sweetness of the various treatments. FM-37 (Fish meal based feed, main
protein source: fish meal at 37%); AS-37 (Algae based feed, main protein sources: Spirulina at
37%); ASS-48 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 48%);
ASS-34 (Algae based feed, main protein sources: Spirulina and Schizochitrium at 34%) and
COM -36 (Fish meal based feed, main protein source: fish meal at 36%). Treatments that do not
share the same letter are significantly different at P ≤ 0.05. Error bars represent SE (standard
error).
52
Off-Odor
3,5
Overall
unpleasantness
3
FM-37
2,5
AS-37
2
Fish flavor
1,5
ASS-48
ASS-34
1
COM-38
Off-Flavor
Earthness
Figure 2.3. Cobweb with descriptive profiles of undesirable attributes for Litopenaeus vannamei
indicating the results of sensory tests where judges evaluated shrimp that had been reared on
experimental and control diets. FM-37 (Fish meal based feed, main protein source: fish meal at
37%); AS-37 (Algae based feed, main protein sources: Spirulinaat 37%); ASS-48 (Algae based
feed, main protein sources: Spirulina and Schizochitrium at 48%); ASS-34 (Algae based feed,
main protein sources: Spirulina and Schizochitrium at 34%) and COM -36 (Fish meal based
feed, main protein source: fish meal at 36%).The center of the figure designates low intensity.
Intensity of each attribute increases with distance.
53
3 General discussion, limitations and future directions
3.1 Research contributions
This researchundertaken in this dissertation shows that Pacific white shrimp can
accommodate algae meals in their diets without compromising survival, fatty acid composition
of muscle and final product quality.
My results indicate that the use of Schizochitrium and Spirulina ingredients contribute to
optimal highly unsaturated fatty acid contents in shrimp tissue, key nutrients in human health.
Therefore, when considering alternative ingredients for aquaculture feeds, highly unsaturated
fatty acid levels in the tissue of farmed species needs to be as good or even better than levels
obtained from species that are maintained on a fish oil diet. The results of this study support the
findings of Patnaik et al. (2006) who reported that fish oil could be successfully replaced in the
diet for Litopenaeus vannamei using cells of heterotrophic algae Schizochytriumsp. This research
also adds to the understanding of how to effectively evaluate the effects of feed formulations on
the organoleptic properties of shrimp by using a comprehensive statistical approach (3-factor
ANOVA). This is a test that would be highly recommended for sensory evaluations of
aquaculture products in order to gain insight into consumer acceptability and possible success in
the market.
Ultimately, this study provides further information to the body of literature on alternative
ingredients for aquaculture feeds. Microalgae has the potential to help reduce and replace the use
of fish meal and fish oil in aquaculture feeds.
54
3.2 Approaches for the evaluation of sensory characteristics in aquaculture products using
a three factor Analysis of Variance
This research also adds to the sensory evaluation field of aquaculture products. This study uses a
3-factor ANOVA, which is a more useful approach to interpret panel performance and gain
insight into the sensory quality of aquaculture products.
Sensory assessments of aquaculture products have become a key component in studies
where new feed formulations are used to evaluate ingredient alternatives that can replace
traditional ones. Currently, the aquaculture feed industry is focused on identifying alternative
protein and lipid sources (Kiron et al., 2012); key drivers being that sources are cost-effective,
eco-friendly and yield excellent growth performance in farmed species. There is also a growing
interest to understand the effects of feed formulation on the organoleptic properties of
aquaculture products to ensure consumer acceptability and their success in the market.
There is a suite ofmethods available to describe sensory characteristics in food items
(International Organization for Standardization, 2009). Historically this field of science has made
use of one-way ANOVA tests to differentiate means for experimental treatments (Garduno-Lugo
et al.,2007; Ganesan et al., 2005; Allan and Rowland, 2005; Diler and Gokoglu, 2004). While
this approach is typically satisfactory for analytical/compositional variables, it does not provide
insight into panel performance.
Another test frequently observed in the literature describing taste test results is the tstudent test (Liang et al.,2008; Goulas et al., 2005; Hernandez et al., 2001). When this test is
applied to more than two treatments, testing multiple pairs of means inflate the probability of
committing a type I error (Whitlock and Schluter, 2009). Thus, in my study use ofa 3-way
ANOVA approachcan be argued to be more applicable for sensory analysis as it allows us
55
todemonstrate panel performance (panel consistency and reproducibility). To our knowledge this
is one of the first studies to show how a 3-way ANOVA can be used to interpret panel
performance thus allowing for a better understanding of sensory differences of aquaculture
products. As such the work could provide researchers, farm managers and extension agents, the
skill to critically evaluate research and make appropriate decisions regarding the sensory quality
of new and existing aquaculture products for release into the marketplace.
3.3 Research limitations and future directions
Some of the shortcomings encountered in this study were with regards to feed quality.
Generally, the most important propertiesin feed quality are: feed digestibility, palatability, and
water stability. These properties were not measured in this research, and these data could have
been useful in explaining performance growth in shrimp.Feed digestibility is a term used to
describe only the portion of the feed that is absorbed by the organism (Siccardi, 2006).
According to Cordova-Mureta and Garcia-Carreno (2002), feed digestibility is affected by not
only the source of the protein, but also the quantity in the diet. Although this property was not
measured in this study, one could speculate that the digestibility (i.e. nutrient availability) of the
proposed new ingredients, Schizochitrium and Spirulina, could have affected the quality of the
feed, thereby influencing shrimp growth performance. Future studies of ingredient digestibility
are encouraged as they can provide valuable information to optimize feed formulations and to
better understand the future success or failure of incorporating algae ingredients in the practical
diets of Pacific white shrimp. Moreover, the optimization of feed formulations can reduce costs
and pollutant levels.
Ingredient palatability is defined as acceptable to the taste or sufficiently agreeable in
flavor to be eaten (Glencross et al., 2007). While it was difficult to accurately determine the feed
56
intake ratein this study, owing to the green water system conditions, it is certainly possible that
the palatability could have affected feed intake. If non-fish meal ingredients reduce feed intake, it
would be of limited use in a feed formulation. Nonetheless, there are strategies to incorporate
attractants to avert or resolve palatability issues of feed ingredients using ingredient processing
or feeding stimulants (Glencross et al., 2007). Low food attractability is another factor that can
lead to decreased feed consumption. Identifying the chemosensory stimuli,which shrimp
normally find palatable may assist in improving ingestion of formulated feeds (Holland and
Borski, 1993). Finding ways of increasing the rate of feeding in shrimp should also minimize
leaching of feed nutrients caused by the animal’s slow feeding behavior (Peñaflorida and
Virtanen, 1996). Artificial diets for crustaceans must be chemically attractive to induce their
location and feeding, and theaddition of small amounts of fish mealchemostimulants might
increase ingestion rates and improve growth, survival, and food conversion (Carr, 1998). Future
studies that involve testing alternative ingredients should take into account the level of
attractability in order to ensure improved feed intake and food conversion.
Pellet water stability is an important quality parameter in the manufacture of aquaculture
diets, especially for shrimp, due to their benthic, slow feeding habits, which make sinking water
stable feed a necessity for shrimp. High pellet water stability is defined as the retention of pellet
physical integrity with minimal disintegration and nutrient leaching while in the water until
consumed by the animal. Pellets that break up into small particles and quickly leach nutrients
could reduce water quality of the culture environment and lead to poor animal growth, inefficient
feed conversion, and low survival (Obaldo et al., 2002). Water stability of experimental diets
was not evaluated in this study and is therefore recommended for further investigation.
57
Lastly, the final quality of feeds depends on variables in the manufacturing process.In
this study, the shortcomings of the feed manufacturing (laboratory made feeds) could have been
a constraint. Pellet size was bigger than the control COM-36 and not homogeneous. Moreover,
based on visual observation, the final product lacked water stability in comparison to the control
COM-36. Individually (or collectively) these factors may have influenced the availability of
nutrients in feeds via leaching, which could have led to lower growth performance. The
pelletizationprocess usedin this study represented a challenge in terms of final feed quality.
Laboratory made feeds commonly lack the manufacturing and extrusion advantages provided by
commercial feeds (Amaya et al., 2007).Extrusion, due to its positive effects on the overall
nutritional quality and water stability of replacement feeds (Davis and Arnold, 2000; Hernandez
et al., 2004; Samocha et al., 2004), is a more appropriate and sophisticated method that is now
widely adopted when producing aquaculture feeds (Carver et al., 1989).
Clearly, an important consideration for future research on alternative ingredients for
aquaculture feeds is to give special attention to the engineering involved in feed processing that
can lead to selecting the right equipment. This could produce high quality experimental diets,
which would be more appropriate to compare to commercial references and therefore accurately
evaluate the success of new ingredients such as algae.
3.4 Analysis of thesis work in future research directions
Replacing fish meal and fish oil with algae ingredients in shrimp feeds is promising. This
research demonstrates that algae meals can be used in the diets of Pacific white shrimpwithout
affectingtheir nutritional value. In addition, this study provides insight into evaluatingthe effect
of alternative algae ingredients on the sensory attributes and overall quality of shrimp.
As aquaculture continues to expand, there will be an increasing need to use alternative
58
and sustainable raw materials in aquaculture feeds. These raw materials will be expected to be
competitive in price, abundant in availability and of great nutrition quality, particularly with high
n-3 highly unsaturated fatty acid contents. The use of microalgae in aquafeeds may be a suitable
alternative since it is rich in n-3 HUFA, yielding high levels of n-3 HUFA in farmed animal’s
tissue. This in turn would allow for desirable quality of aquaculture products for human
consumption. However, other components of alternative ingredients such as digestibility,
palatability, and nutrient utilization need to be further evaluated in order to establish the viability
of algae ingredients in aquaculture feeds. Another extremely important aspect to consider in
future research is the manufacturing of test diets. This should be given priority as processing
techniques play a critical role in the final quality of feeds.
Researches often use laboratory made feeds that may lack the manufacturing and
extrusion benefits supplied by commercial feeds (Amaya et al., 2007). Therefore, special
attention should be paid to the manufacturing process of experimental feeds in order to obtain
high quality feeds and more accurately measure their impact on animal’s growth performance.
Defining the focus and objectives of aquaculture research and development is a priority. The
focus should be on achieving feed formulations that are cost-effective, eco-friendly and that meet
consumer demands.
59
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