A defatted microalgae (Haematococcus pluvialis) meal as a protein ingredient to

partially replace shmeal in diets of Pacic white shrimp (Litopenaeus vannamei,

Boone, 1931)
Zhi Yong Ju , Dong-Fang Deng, Warren Dominy
Aquatic Feeds and Nutrition Department, Oceanic Institute, 41202 Kalanianaole Hwy, Waimanalo, Hawaii 96795, USA
a b s t r a c t a r t i c l e i n f o
Article history:
Received 21 July 2011
Received in revised form 5 April 2012
Accepted 10 April 2012
Available online 2 May 2012
Keywords:
Microalgae
Fishmeal replacement
Shrimp
Growth
Pigmentation
This trial evaluated the effects of partial replacement of shmeal protein by a defatted microalgae meal
(DMM) in a shrimp diet. The DMM was a by-product of astaxanthin production from Haematococcus pluvialis
and contained 40.3% crude protein (CP) and 0.9% crude lipid (CL). Test diets were prepared by using DMM (3,
6, 9 and 12% in a diet) to replace 12.5%, 25%, 37.5% or 50% of a shmeal protein in a control diet (32.3% CP &
8.9% CL). All test diets had similar protein and lipid levels. Each diet was randomly assigned to four tanks (12
juvenile shrimp per tank) in an indoor ow through seawater laboratory. After an 8-week feeding trial,
shrimp fed the diet with 12.5% of the shmeal protein replaced showed a signicantly higher growth rate
and lower feed conversion ratio than the shrimp fed the control diet (Pb0.05). The other three DMM-
added diets had a similar effect on the growth of shrimp as the control diet (P>0.05). Shrimp fed the four
DMM-added diets appeared redder and contained higher free and esteried astaxanthins than shrimp fed
the control diet. The results indicated that DMM could be a valuable alternative protein and pigmentation
ingredient in shrimp feed.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Many species of microalgae have been used to produce nutraceutical
or biofuel products (Acien-Fernandez et al., 2003; Mata et al., 2010; Singh
and Gu, 2010). For example, Haematococcus pluvialis (astaxanthin),
Dunaliella salina (-carotene) and Muriellopsis sphaerica (lutein) have
been used for carotenoid production; Odeontella aurita, Phaedactylum
tricomutum and Isochrysis galbana have been used for algae oil or
omega-fatty acid production; and Chlorella, Nannochloropsis, Chaetoceros
and Dunaliella species have been used for biodiesel production or
development (Acien-Fernandez et al., 2003; Jin and Melis 2003;
Mata et al., 2010). These production activities can result in huge
amounts of defatted microalgae by-products. The defatted microalgae
meal (DMM) may contain many valuable nutrients for animal feed,
such as proteins, carbohydrates, minerals, water-soluble vitamins, and
bioactive compounds even though most of the lipid and lipid-soluble
nutrients have been removed (Ju et al., 2009). Researchers (Cuzon
et al., 1981; Hanel et al., 2007; Ju et al., 2009) found that microalgae
proteins could be promising substitutes for shmeal protein or as a
valuable additive in aquafeeds.
Application of many species of microalgae in shrimp feed is still in
the research stage and only a few have been tested in shrimp trials
(Cuzon et al., 1981; Hasan and Chakrabarti, 2009; Ju et al., 2009).
The feeding trials on shrimp revealed that some microalgae had
enhancing growth effects. Cuzon et al. (1981) reported that lipid
free fraction of Spirulina meal promoted growth of Penaeus japonicas
and suggested that the microalgae might contain an unknown growth
factor. Nakagawa and Gomez-Diaz (1995) found that whole Spirulina
meal signicantly improved growth, survival and feed utilization in
giant freshwater shrimp (Macrobrachium rosenbergii). They suggested
that the improved growth and feed utilization was probably due to
the enhancement of protein assimilation. Ju et al. (2009) reported
enhanced shrimp growth by inclusion of 9% whole microalgae meal of
Thalassiosira weissogii or of Nannochloropsis in the control diet. The
same study found that it was the defatted meal fraction not the lipid
fraction fromboth microalgae that resulted in enhanced shrimp growth.
The enhanced shrimp growth effects were not attributed to the
contribution of macronutrients from whole or defatted microalgae, as
their dietary inclusions decreased crude proteinandcrude lipidcontents
and gross energy value (Ju et al., 2008, 2009). It is unclear what factors
or compounds contained in the microalgae played a role in improving
shrimp growth. In feeding trials with sh, however, microalgae have
been found to increase sh digestibility, physiological activity, stress
response, starvation tolerance, disease resistance, and carcass quality
(Becker, 2004; Mustafa and Nakagawa, 1995).
Shrimp are omnivorous organisms and microalgae are their
natural foods (Moss et al., 2006; Otoshi et al., 2001; Takeuchi et al.,
2002). H. pluvialis meal or its DMM, has never been investigated as
Aquaculture 354355 (2012) 5055
Corresponding author. Tel.: +1 808 259 3128; fax: +1 808 259 7936.
E-mail address: zyju@oceanicinstitute.org (Z.Y. Ju).
0044-8486/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2012.04.028
Contents lists available at SciVerse ScienceDirect
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j our nal homepage: www. el sevi er . com/ l ocat e/ aqua- onl i ne
a protein ingredient in shrimp feed. However, H. pluvialis meal has
frequently beenaddedindiets to test for pigmentationof shrimp, salmon
and trout (Chien and Shiau, 2005). Astaxanthin production from this
microalga has reached industrial scale worldwide (Krichnavaruk et al.,
2008). A company in Hawaii annually produces 70 tons of H. pluvialis
meal. The H. pluvialis meal is usually treated with supercritical CO
2
to
extract the fat-soluble astaxanthin for humannutraceutical andcosmetic
applications due to its strong antioxidant properties (Guerin et al.,
2003). The remaining DMM (>80% biomass) has been treated as
a waste by-product (Krichnavaruk et al., 2008). The extraction
method using CO
2
does not contaminate the by-product and no
toxic chemicals or toxic effects have been reported for the H. pluvialis
algae in animal applications (Satoh et al., 2009). Therefore, DMM
may have potential as an alternative protein ingredient to replace
shmeal in shrimp feed. Additionally, the left-over astaxanthin in
DMM may have a pigmentation effect on shrimp. The objective of this
study is to determine the effects of partial replacement of shmeal
with the defatted H. pluvialis meal on growth performance, nutritional
composition and pigmentation of juvenile shrimp.
2. Materials and methods
2.1. Dry microalgae meal (DMM) and chemicals
The DMM was obtained from Cyanotech Corporation (Kona,
Hawaii, USA). It is an industrial by-product from dried H. pluvialis
after extraction by super-critical CO
2
for astaxanthin. The organic
solvents, including acetone, acetonitrile, and methanol were purchased
from Fisher Scientic (Boston, Massachusetts, USA). The analytical
chemicals triethylamine (TEA) and hydrochloric acid for amino
acid analyses were obtained from Fisher Scientic; and phenyl
isothiocyanate (PITC) were obtained from Sigma-Aldrich Chemical Co.
(St. Louis, Missouri, USA). The DMM was nely ground and stored at
4 C before use.
2.2. Diet formulation and preparation
A control test diet was formulated to contain 32% crude protein
(CP) and 8.9% crude lipid (CL) notated as D-0% (Table 1). The other
four test diets were formulated by adding DMM to replace 12.5%,
25.0%, 37.5%, and 50.0% of the shmeal protein in the control diet,
corresponding inclusion amounts of DMM were 3.0%, 6.0%, 9.0% and
12.0% of diet biomass, respectively. These four test diets were
named: D-12.5%, D-25.0%, D-37.5% and D-50.0% (Table 1). Wheat
starch and menhaden sh oil were adjusted to balance the diets.
The test diets were prepared according to methods described by Ju
et al. (2008). A commercial shrimp feed (Rangen, Inc., Buhl, Idaho,
USA) with 40% CP and 5% squid meal was included in the trial as a
commercial control diet. Finished pellets were then stored in plastic
bins at 1920 C until fed within 3 months.
2.3. Feeding trial
The growth trial was carried out in an indoor ow through system
with 24 glass aquaria (52-L; 76 cm31 cm31 cm). The tanks were
aerated and received a constant supply of seawater (1 L min
1
).
Each aquarium was stocked with juvenile shrimp (~1.0 g), at a density
of 12 shrimp per aquarium (50 shrimp m
2
). Each diet (one control,
four DMM-added, and one commercial feed) was randomly assigned
to four tanks. All shrimp were fed by hand 4 times daily at 09:30,
11:30, 14:00, and 16:15 h for 8 weeks. The shrimp were initially fed
12% and 18% of the initial stocked weight in week 1 and week 2,
respectively. The feeding rate was adjusted weekly in a range of 6% to
10% based on body weight obtained bi-weekly or by using an estimated
growth rate established in the laboratory. Uneaten feed, feces, and
molts were removed by siphoning the aquaria immediately prior to
the morning feeding. Water temperature (~25 C) was monitored
daily. Dissolved oxygen (DO; 6.306.85 mg L
1
), water pH (7.667.86)
and salinity (32.4832.84 ppt) were monitored weekly. Total ammonia
nitrogen level was measured weekly lower than the detectable level
(b0.08 mg L
1
) by the automated analysis method of Solorzano (1969).
Feed conversion ratio (FCR), percent weight gain (PWG), and
specic growth rate (SGR) were calculated as follows:
PWG % W
2
W
1
=W
1
X100;
SGR %=day ln W
2
=W
1
=tX100;
FCR Total dried feed g fed= W
2
W
1
:
where the W
2
is nal weight (g) of shrimp, W
1
is initial weight (g)
and t is the number of days for the growth trial. All surviving shrimp
in each tank were collected after the 8-week feeding and samples
were used for analysis of proximate composition and astaxanthin
content. Whole shrimp body samples were freeze-dried using a 5-L
freeze-drier (EC Apparatus Inc, Holbrook, New York, USA). The dried
samples were nely groundusinga grinder (IKAWorks Inc, Wilmington,
North Carolina, USA), and stored at 20 C until used for analysis.
2.4. Sample analysis
Proximate composition was analyzed following the methods by
AOAC(2000). Moisture was determinedby drying a 2 g of representative
sample in an oven with air circulation at 105 C for 1624 h. Crude
protein (CP) was determined from total nitrogen by a LECO's FP-528
Nitrogen/Protein Determinator (LECO Cooperation, St. Joseph, MI, USA)
using a multiplication factor of 6.25. Total crude lipid (CL) was
determined by ethyl-ether extraction using an Accelerated Solvent
Extractor (Dionex Corporation, Bannockburn, Illinois, USA). Ash was
determined by incineration of a representative 0.5 g sample in an
oven at 550 C for 6 h. Mineral content was analyzed by an inductively
coupled plasma atomic emission spectroscopy (Model Atomscan 16,
Thermo Jarrel Ash, Franklin, Massachusetts, USA). Gross energy was
determined using bomb calorimetry (Parr 1261 Calorimeter, Parr Inst.
Table 1
Formulation (%) of test diets with graded levels of defatted microalgae meal
(Haematococcus pluvialis) replacing shmeal in the control diet.
Diet ingredient Diet with replacement level of shmeal protein
D-0% D-12.5% D-25.0% D-37.5% D-50.0%
Menhaden shmeal
a
15.00 13.12 11.24 9.36 7.48
Defatted microalgae meal
b
0.00 3.00 6.00 9.00 12.00
Whole wheat (hard red winter)
c
37.80 36.25 34.71 33.16 31.61
Soybean meal (47%)
d
25.00 25.00 25.00 25.00 25.00
Squid meal
e
6.00 6.00 6.00 6.00 6.00
Dicalcium phosphate
f
4.50 4.50 4.50 4.50 4.50
Soy lecithin (liquid)
g
2.00 2.00 2.00 2.00 2.00
Cholesterol
f
0.12 0.12 0.12 0.12 0.12
Potassium chloride
f
2.50 2.50 2.50 2.50 2.50
Calcium carbonate
f
1.00 1.00 1.00 1.00 1.00
Magnesium oxide
f
1.60 1.60 1.60 1.60 1.60
Mineral/vitamin premix #1
h
0.23 0.23 0.23 0.23 0.23
Mineral/Vitamin Premix #2
h
0.21 0.21 0.21 0.21 0.21
Stay C-35
i
0.04 0.04 0.04 0.04 0.04
Menhaden oil
j
2.00 2.18 2.35 2.53 2.71
Soybean oil
k
2.00 2.00 2.00 2.00 2.00
a
Kodiak Fishmeal Company, Kodiak, Alaska, USA.
b
Cyanotech Corporation, Kona, Hawaii, USA.
c
Hawaiian Flour Mill, 703 N Nimitz Hwy, Honolulu, HI 96817, USA.
d
Land-o-Lakes, Seattle, WA, USA.
e
Inual, Santiago, Chile.
f
Sigma Aldrich, 3050 Spruce St., St. Louis, Mo 63103, USA.
g
Central Soya Company Inc, Fort Wayne, Indiana, USA.
h
Industrias de Nata, Aguadulce, Panama.
i
DSM Nutritional Products, Inc., 45 Waterview Boulevard, Parsippany, NJ 07054, USA.
j
Omega Protein Inc., Reedville, Virginia, USA.
k
ConAgra Foods Inc., Omaha, Nebraska, USA.
51 Z.Y. Ju et al. / Aquaculture 354355 (2012) 5055
Co., Moline, Illinois, USA). Amino acid (AA) proles of the samples were
analyzed using an Agilent 1200 HPLC equipped with Agilent 1200
Series diode array detectors (Santa Clara, California, USA), following
the methoddescribedby Ju et al. (2008). Free andesteriedastaxanthins
in shrimp samples were analyzed using a modication of the HPLC
method described by Lin et al. (2005).
2.5. Statistical analysis
Data was analyzed by using one way analysis of variance (ANOVA),
to determine if signicant (Pb0.05) differences existed among
treatment means. Means separations were performed using Fishers
Protected Least Signicant Difference Test. All statistical analyses
were carried out using computer software (SigmaStat for Windows
v3.5, Systat Software, Inc., San Jose, California, USA).
3. Results and discussion
3.1. Growth performance and nutritional compositions of shrimp
The DMM contained 40.3% of CP and 12.8% of ash, but a very low
content of CL (0.9%; Table 2). As formulated, the ve test diets were
similar in nutrient compositions (Table 2). Pellet water stability
showed only minor changes (92.7% to 94.1%; P>0.05); suggesting
that the DMM inclusions did not signicantly affect the pellet water
stability.
After the 8-week feeding trial, the test diets and the commercial feed
resulted in a high survival (87.5% to 100%; P>0.05; Table 3). Shrimp
fed the D-12.5% showed a signicantly (Pb0.05) higher growth rate
(1.25 g/wk) than that (1.11 g/wk) of the shrimp fed the D-0% (control
diet). Shrimp fed the remaining three DMM-added diets (D-25.0%,
D-37.5% and D-50.0%) had similar growth rates (1.13 to 1.22 g/wk)
to shrimp fed the control diet. Effects of the test diets on PWG and
SGR followed a similar trend as the results above. These results suggest
that DMM could replace 50% of shmeal protein in the control diet
without negatively affecting shrimp growth. A low inclusion level of
DMM in the control diet actually improved the shrimp growth.
Previous research has documented that inclusion of more than
20% microalgae into a sh feed resulted in poor palatability and
thus affected growth (Hasan and Chakrabarti, 2009). However, for
the current study no test was performed to examine if the high
inclusion of microalgae affected the palatability of shrimp. Based on
feeding observations and results of the similar growth and FCR for
shrimp fed the ve test diets in this study, the inclusion levels (3%
to 12%) did not seem to affect the shrimp palatability. Conversely, a
higher replacement of shmeal by the algae meal may result in reduced
feed intake of shrimp. This will need to be conrmed in future research.
Furthermore, the ve test diets in this study resulted in signicantly
higher growth rates than the commercial feed (0.74 g/wk; Table 3)
even though the commercial feed had a higher CP content (39.9%)
than the CP content (31.3 to 32.3%) in the ve test diets (Table 2). The
ve test diets also showed a better feed conversion ratio (2.13 to
2.28:1) than the commercial feed (2.63:1; Pb0.05; Table 3). Based on
the results of this study, we do not have a clear explanation on why
the performance of shrimp fed the test diets was better than the
shrimp fed the commercial diet. The essential amino acid proles
are similar between the control diet and the commercial diet except
that the commercial diet had relatively lower histidine (0.85%) than
the test diets (1.11.25%; Table 4). The commercial feed contained
lower potassium (0.91%) and magnesium (0.20%) levels than the
potassium and magnesium content (2.032.24%; 1.171.25%) in the
ve test diets. There is very limited information available for the
nutritional requirements of histidine and the minerals by Pacic white
shrimp (Kuhn et al., 2010). These warrant further investigation.
The DMMproteinshowed a similar essential AAprole to menhaden
shmeal protein (Table 4). As a result, the replacement of the shmeal
protein with DMM protein did not cause a signicant change in
essential AA composition for the test diets (Table 4). This may be
the reason that no adverse effect was observed on the shrimp growth
when shrimp were fed the diets containing different levels of DMM.
The low replacement of shmeal (12.5%) by DMM or low inclusion
(3%) of DMM in the control diet signicantly enhanced shrimp growth.
This may be due to some bioactive compounds in the DMM (Cuzon et
al., 1981; Hanel et al., 2007; Ju et al., 2009) or from some of the health
benets of DMM (Hayashi and Hayashi, 1996; Hayashi and Katoh,
1994; Jensen et al., 2001). These bioactive compounds in the microalgae
could be growth hormones or insulin-like growth factors (Guillaume et
al., 1989), micronutrients such as vitamins and minerals (Brown, 2002;
Spolaorea et al., 2006); and compounds inducing gene expression, such
as free amino acids and free fatty acids (Clarke et al., 2002; Distel et al.,
1992; Fafournoux et al., 2000). The benecial effects from microalgae
biomass could enhance the immune system, resulting in anti-viral, anti-
inammatory, phagocytosis, and anti-stress effects in aquatic animals
(Hayashi and Hayashi, 1996; Hayashi and Katoh, 1994; Jensen et al.,
Table 2
Proximate composition (dried sample, n=1) of the ve test diets, a commercial feed, and a defatted microalgae meal (DMM; Haematococcus pluvialis).
Composition Test Diets DMM
D-0% D-12.5% D-25.0% D-37.5% D-50.0% Commercial feed
Proximate (%)
Dry matter 92.8 92.1 92.6 92.7 93.3 89.8 94.5
Ash 14.9 14.9 14.7 14.5 14.4 9.1 12.8
Crude protein 32.3 32.2 31.4 31.7 31.3 39.9 40.3
Crude lipid 8.9 9.1 9.3 9.4 9.3 8.5 0.9
Crude ber 1.5 1.8 2.1 2.4 2.8 2.7 9.6
Gross energy (cal/g) 4045 4042 4097 4112 4162 4349 4082
Macro minerals (%)
Phosphorus 1.71 1.63 1.49 1.44 1.41 1.40 0.95
Potassium 2.09 2.06 2.05 2.03 2.24 0.91 0.43
Calcium 2.49 2.30 2.13 2.01 1.92 2.33 0.48
Magnesium 1.15 1.17 1.19 1.23 1.25 0.20 0.82
Sodium 0.12 0.11 0.11 0.11 0.11 0.40 0.20
Micro-minerals (ppm)
Boron 15 16 16 17 17 10 23
Copper 32 35 29 47 15 24 11
Iron 557 577 594 672 797 259 1287
Manganese 62 72 70 59 65 142 87
Zinc 106 113 132 137 146 120 396
52 Z.Y. Ju et al. / Aquaculture 354355 (2012) 5055
2001; Mustafa and Nakagawa, 1995). The improvement seen in the
growth rates might also be associated with physiological conditions
such as increasing protein assimilation, lipid metabolism, and liver
function (Nakagawa, 2011). However, high microalgae inclusion
increased the ber content in aquafeed (Table 2). High ber content
in diets could increase passage rate of feed in the gut, reducing the
availability of nutrients, decrease feed digestibility, and reduce animal
growth (Sudaryono et al., 1996). Thus, the increased ber content
from high DMM inclusion might counteract the microalgae benecial
effects (Table 2). This may explain why only 3% DMM inclusion
achieved a signicantly enhanced shrimp growth effect, and suggests
that feed manufacturers may consider using the DMM by-product as
a feed additive for better shrimp growth performance. Previous growth
studies on sh found that the growth performance of sh progressively
decreased when diets were incorporated with algal meal (1520%),
and total replacement of shmeal by algal meal generally showed
very poor growth responses (Hasan and Chakrabarti, 2009).
Using DMMto replace shmeal protein did not affect the proximate
and mineral composition of the shrimp product (Table 5). But shrimp
fed the commercial diet had signicantly lower CL than shrimp fed
the ve test diets. This indicated less efciency of nutrient digestibility
or retention for the shrimp fed the commercial feed.
3.2. Pigmentation of shrimp
The harvested raw Pacic white shrimp turned to a pink or red
color after freeze-drying at 50 C. It was observed that the shrimp
increased in redness with elevated DMM inclusions in the diets. HPLC
analysis exhibited signicantly increased contents of free and esteried
astaxanthin in shrimp fed the diet with the levels of DMM inclusion
(Fig. 1). Specically esteriedastaxanthincontents were muchincreased
in shrimp fed the high DMM-added diets in comparison to shrimp fed
the control diet. Therefore, the DMM from H. pluvialis used as an
ingredient for shrimp feed can improve the quality of shrimp.
In general, whole H. pluvialis dried meal contained approximately
15 to 20% lipid and 2% astaxanthin (Lorenz and Cysewski, 2000). The
DMM was determined to contain 0.9% lipid (Table 2) and 0.05%
astaxanthin. Therefore, the four test diets with the elevated levels of
DMM inclusion were estimated to be supplemented at 15, 30, 45 and
60 mg of astaxanthin per kg of diet. These amounts of astaxanthin
supplementation fromDMMresulted in signicant pigmentation effects
for shrimp as shown in Fig. 1. The control diet was not supplemented
with astaxanthin; however the vitamin and mineral mix ingredients
contained -carotene (Table 1). Previous tests (Chien and Shiau, 2005;
Ju et al., 2011; Pan et al., 2001) revealed that the optimumsupplemental
amount of astaxanthin in shrimp diets were from 75 to 100 mg/kg diet
for enhanced color. Currently many diets or commercial feeds for
shrimp and sh are being formulated containing 50 to 60 mg/kg of
synthetic astaxanthin. The synthetic astaxanthin is free astaxanthin
and not preferred by consumers (Rodriguez et al., 2010). The natural
astaxanthins from H. pluvialis meal are mainly composed of esteried
astaxanthins (~95%), and generally are the accepted pigments in animal
products (Barbosa et al., 1999; Coral-Hinostroza and Bjerkeng, 2002;
Lorenz and Cysewski, 2000).
4. Conclusion
In a summary, this study demonstrated that 1) the DMMcan be used
as a feed additive (3% in a diet) to stimulate shrimpgrowth andimprove
feed utilization; 2) replacement of 50% shmeal protein by the DMM
does not have any adverse effect on shrimp based on the growth
performance and nutritional composition of shrimp; 3) the DMM can
also improve shrimp quality by enriched accumulation of astaxanthin,
which is benecial to animal and human health. The results from this
study provide important informationregarding the potential application
of defatted microalgae as a valuable alternative protein and natural
pigment source for shrimp culture. More studies will be needed to
test this by-product at higher replacement levels for shmeal and its
effects on shrimp palatability and digestibility.
Table 3
Effects of replacing shmeal with a defatted microalgae meal (Haematococcus pluvialis) in the control diet on growth and survival of juvenile shrimp, Litopenaeus vannamei (n=4).
Diet Initial weight Final weight Growth rate Survival FCR
1
PER
1
TFF
1
PWG
1
SGR
1
(g) (g) (g/wk) (%) (g/g) (g/g) (g) (%) (%/day)
D-0% 1.12
a2
10.04
b
1.11
b
91.7
a
2.28
c
1.36
b
880.1
b
796.4
b
3.91
b
D-12.5% 1.06
a
11.06
c
1.25
c
95.8
a
2.13
a
1.40
c
974.6
c
943.4
c
4.11
c
D-25.0% 1.09
a
10.12
b
1.13
b
97.9
a
2.21
b
1.44
c
932.7
b
828.4
b
3.93
b
D-37.5% 1.10
a
10.88
bc
1.22
bc
87.5
a
2.27
c
1.39
b
922.6
bc
889.1
bc
4.07
c
D-50.0% 1.08
a
10.28
b
1.15
b
95.8
a
2.22
b
1.44
c
929.9
b
851.9
b
3.96
b
Commercial feed 1.08
a
7.03
a
0.74
a
100.0
a
2.63
a
0.95
a
748.1
a
550.9
a
3.18
a
SEM 0.05 0.61 0.08 3.1 0.14 0.07 37.1 31.8 0.18
1
FCR=feed conversion ratio, PER=protein efciency ratio, TFF=total feed fed, PWG=percent weight gain, and SGR=specic growth rate.
2
Means not sharing a superscript in the same column are signicantly different (Pb0.05, n=4).
Table 4
Amino acid proles (n=1) of the shmeal, a defatted microalgae meal (Haematococcus
pluvialis), test diets and a commercial feed.
Amino
acids
Men.
meal
1
DMM
1
D-0% D-12.5% D-25.0% D-37.5% D-50.0% Com.
feed
1
(g/100 g sample)
No-essential AA
Ala 4.71 3.61 1.68 1.69 1.74 1.70 1.68 2.18
Asp+ASN 5.81 3.32 3.95 4.03 4.04 3.99 4.04 3.53
Cys 0.87 0.34 0.78 0.76 0.74 0.66 0.71 1.22
Glu+Gln 6.68 3.64 4.06 3.58 3.25 3.34 3.55 3.82
Gly 4.93 2.78 1.83 2.17 2.22 2.18 2.42 2.95
Pro 2.69 1.76 1.48 1.68 1.72 1.77 1.81 2.29
Ser 2.17 1.62 1.14 1.26 1.38 1.46 1.54 1.66
Tyr 2.12 1.45 0.99 1.09 1.12 1.16 1.14 1.22
Subtotal 29.98 18.51 15.91 16.25 16.22 16.26 16.90 18.85
Essential AA
Arg 5.57 2.49 2.38 2.32 2.28 2.22 2.21 2.99
His 1.78 0.96 1.25 1.23 1.17 1.14 1.10 0.85
Ile 2.82 1.99 1.43 1.41 1.38 1.31 1.15 1.68
Leu 4.55 3.06 2.23 2.02 1.98 1.97 1.82 2.55
Lys 5.83 2.22 1.85 1.88 1.89 1.82 1.75 2.25
Met 1.08 0.60 0.64 0.69 0.68 0.67 0.67 0.77
Phe 2.98 1.84 1.60 1.57 1.54 1.52 1.38 1.71
Thr 2.86 2.01 1.00 1.00 0.96 0.96 0.97 1.47
Val 3.53 2.69 2.17 2.20 2.23 2.16 2.17 2.84
Subtotal 31.01 17.86 14.56 14.32 14.11 13.77 13.22 17.10
Total 60.99 36.37 30.47 30.57 30.32 30.03 30.12 35.95
1
Men. meal =menhaden meal; DMM=defatted microalgae meal, Com.
feed=commercial feed.
53 Z.Y. Ju et al. / Aquaculture 354355 (2012) 5055
Acknowledgments
Support for this research, through grant agreement No. 59-5320-
2-712 from the United States Department of Agriculture, Agricultural
Research Service, is gratefully acknowledged. The authors also wish
to thank Valerie L. Harmon, Director of Cultivation of Cyanotech in
Kona of Hawaii for providing the defatted microalgae meal for this
study. Ward Kashiwa, Gavin Nagaue, Richard Lee, David Anderson,
and Jarryd Maeda are acknowledged for their technical assistance
with the feeding trial and sample analyses and Lindon Hansink for
her editorial work on the manuscript are also appreciated.
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Table 5
Proximate composition (dry matter, n=4) of whole shrimp body fed commercial feed and ve test diets (D-0% to D-50.0%) with graded levels of a defatted microalgae meal
replacing shmeal (0 to 50%) in the control diet.
Composition Content in whole shrimp body (MeanSD)
D-0% D-12.5% D-25.0% D-37.5% D-50.0% Com. feed
1
Proximate (%)
Dry matter 93.30.9
a2
93.50.5
a
94.70.2
a
93.70.9
a
94.40.3
a
93.90.4
a
Ash 10.30.5
a
10.50.3
a
10.70.4
a
10.50.3
a
10.40.5
a
10.40.2
a
Crude protein 71.80.6
a
72.11.4
a
71.41.3
a
70.51.0
a
71.60.3
a
71.71.6
a
Crude lipid 6.70.4
b
6.90.2
b
6.80.1
b
6.70.4
b
6.90.4
b
6.00.2
a
Macro-minerals (%)
Phosphorus 1.10.0
b
1.10.0
b
1.10.0
b
1.10.0
b
1.20.1
b
0.90.0
a
Potassium 1.30.0
a
1.30.0
a
1.20.0
a
1.20.0
a
1.30.1
a
1.20.0
a
Calcium 2.70.4
a
2.40.5
a
2.40.5
a
2.80.5
a
2.60.6
a
2.80.1
a
Magnesium 0.30.0
a
0.30.0
a
0.20.0
a
0.30.0
a
0.30.0
a
0.20.0
a
Sodium 1.00.0
a
1.00.0
a
1.00.0
a
0.90.0
a
1.00.1
a
1.00.0
a
Micro-minerals (ppm)
Boron 2.40.5
a
2.40.5
a
2.40.3
a
2.30.1
a
2.70.3
a
2.50.3
a
Copper 102.34.9
a
103.25.3
a
102.46.4
a
99.84.8
a
100.76.7
a
100.24.4
a
Iron 16.45.1
a
14.51.6
a
16.35.6
a
15.11.7
a
16.91.8
a
15.92.7
a
Manganese 1.30.1
a
1.50.1
b
1.40.1
ab
1.40.1
ab
1.50.1
b
1.50.1
b
Zinc 52.312.7
a
46.95.4
a
44.91.5
a
47.65.1
a
53.815.3
a
45.81.7
a
1
Com. feed=commercial feed.
2
Means not sharing a superscript in the same row are signicantly different (Pb0.05, n=4).
0
10
20
30
40
50
60
D-0% D-12.5% D-25.0% D-37.5% D-50.0%
A
s
t
a
x
a
n
t
h
i
n

c
o
n
t
e
n
t

(
m
g
/
k
g
)
Fed diet
Free Esterified
a
a
a
c
b
c
b
d
d
e
d
Fig. 1. Free and esteried astaxanthin content (n=4) in freeze-dried shrimp body after
feeding the ve test diets (D-0% to D-50.0%) with graded levels of a defatted microalgae
meal replacing shmeal (0 to 50%) in the control diet. Means not sharing a common
superscript are signicantly different (Pb0.05).
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