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Aquaculture 245 (
The use of inert artificial commercial food sources as replacements
of traditional live food items in the culture of larval shrimp,
Farfantepenaeus aztecus
C.B. Robinsona,b, T.M. Samochaa,b,*, J.M. Foxb, R.L. Gandya, D.A. McKeeb
aTexas Agricultural Experiment Station–Shrimp Mariculture Research Facility, Texas A&M University System, 4301 Waldron Rd.,
Corpus Christi, TX 78418, USAbCenter for the Sciences, Room 251, Texas A&M University–Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412, USA
Received 10 May 2004; received in revised form 24 August 2004; accepted 30 November 2004
Abstract
Replacement of live feeds with alternate food sources is of major importance in commercial shrimp hatcheries. In this study,
partial and complete replacement of live microalgae and Artemia nauplii with microalgae pastes and inert feeds is reported in
the larval rearing of the brown shrimp Farfantepenaeus aztecus. Five different experimental feeds were used for each live food
replacement study. Partial replacement of live microalgae using Chaetoceros 1000 bPremium FreshQ Instant Algaek paste and
Liqualifek liquid larval feed showed survival rates similar to a control feed, however, postlarvae mean dry weight and length
were significantly less than the control. Use of other replacement feeds, Epifeedk liquid larval feed, Zeiglerk E-Z Larvae
liquid feed, and Zeiglerk Z-Plus feed, yielded inferior results compared to the control feed. Statistical analysis of results from
the live Artemia nauplii replacement study indicated that larval shrimp fed a control feed had significantly greater survival,
mean PL length, and mean PL dry weight. The only exception was the partial replacement of Artemia nauplii using Liqualifekin which there was no significant difference in survival between the control and this treatment. Results indicated that although
Liqualifek, Epifeedk, Zeiglerk E-Z Larvae, Zeiglerk Z-Plus and Zeiglerk E-Z Artemia feeds could serve as partial
replacement of newly hatched Artemia nauplii in production of F. aztecus postlarvae. Significantly improved results can be
expected when larvae are fed newly hatched Artemia nauplii (control diet) with no supplement.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Shrimp; Larvae; Algae; Artemia
1. Introduction
0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2004.11.051
* Corresponding author. Texas Agricultural Experiment
Station–Shrimp Mariculture Research Facility, 4301 Waldron
Rd., Corpus Christi, TX 78418 USA. Tel.: +1 361 937 22 68;
fax: +1 361 937 64 70.
E-mail address: [email protected] (T.M. Samocha).
Development of cost-effective, nutritionally com-
plete inert larval feeds would be of tremendous benefit
to commercial hatchery operations. Despite extensive
research, nutrient requirements of aquatic suspension
2005) 135–147
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147136
feeders have not been well understood (Villamar and
Langdon, 1993). Sorgeloos and Leger (1992)
described finfish and crustacean larviculture diets for
the early first feeding stages as being the major
bottleneck for complete replacement of live larval
feeds.
In the wild, the diet of larval shrimp typically
consists of diverse phytoplankton and zooplankton
species of various sizes and biochemical composition.
For that reason, traditional feeding techniques used in
commercial shrimp hatcheries still rely on complex
regimes of live food items. Larval rearing diets are
typically composed of different species of microalgae
and newly hatched Artemia nauplii (New, 1976;
Kumlu and Jones, 1995) and, as such, are difficult
to maintain (Persoone and Claus, 1980). Assurance of
adequate concentrations of live food items requires
careful and frequent monitoring and can result in
increased operating costs, variable nutritional quality
(Watanabe et al., 1983) and could serve as vectors for
shrimp pathogens (Kurmaly et al., 1989). These
limitations underscore the importance of identifying
alternative inert feeds as substitutes for live food
items.
The determination of nutrient essentiality to larval
penaeid shrimp is also complicated by a life cycle
consisting of a series of planktotrophic stages result-
ing in substantial trophic level change (Jones et al.,
1993). Of the small amount of information available,
most has been derived from three species endemic to
the Gulf of Mexico: Litopenaeus setiferus, Farfante-
penaeus aztecus, Farfantepenaeus duorarum and
various Pacific species: Marsupenaeus japonicus,
Litopenaeus vannamei and Penaeus monodon.
Microparticulate feeds in microencapsulated,
microbound and microcoated forms have met with
various levels of success in commercial hatcheries as
partial replacements for live foods (Langdon et al.,
1985) and their use often results in deterioration of
water quality (Muir et al., 1991). Degradation and
leaching of nutrients from inert feeds not only
impairs shrimp growth and survival but also depre-
dates the quality of culture water via proliferation of
bacteria and general fouling (Jones et al., 1987; Muir
et al., 1991). Furthermore, the use of inert dry feeds
typically requires increased water exchange and
supplemental aeration to maintain food particles in
suspension. Constant monitoring of culture water is
also required to avoid significant bio-fouling or
decline in water quality due to residual, uneaten
feed (Muir and Sutton, 1994). Studies comparing
liquid to dry inert feeds have shown increased
benefits of liquid products, including reduced tank
fouling and increased suspension time in the water
column (Jones et al., 1987). Liquid feeds also appear
to allow for a reduction in both water exchange and
aeration (Jones et al., 1987). Ultimately, the develop-
ment of nutritionally stable inert feeds could reduce
operational costs for penaeid shrimp hatcheries by
minimizing reliance on live foods with variable
nutrient composition.
Our study addressed live feed replacement in the
larval rearing of F. aztecus, a species indigenous to the
Gulf of Mexico. F. aztecus was selected to increase
the knowledge and understanding of how various
formulated diets as substitutes for live feeds affect the
larviculture of this species as well as to provide
insight into its commercialization for the live bait-
shrimp industry.
The objectives of this study were to (1) evaluate
the partial and complete replacement of live micro-
algae and newly hatched Artemia nauplii by various
commercially available inert larval feeds and (2)
comment on their application to larviculture of F.
aztecus.
2. Materials and methods
2.1. Source of shrimp and stocking
This study was conducted at the Texas Agricultural
Experiment Station (TAES) Shrimp Mariculture
Research Facility (SMRF), Corpus Christi, TX.
Shrimp larvae (F. aztecus) were provided by Lone
Star Farm (Austin, TX) from certified viral-pathogen-
free captive broodstock. Nauplii from a single spawn
were first acclimated to artificial seawater (35 ppt;
Instant Ocean Artificial Salt Mix, Aquarium Systems,
Mentor, OH) for a period of 60 min and then
transferred to a common holding tank and maintained
at a density of 100 nauplii L�1 until the initiation of
the study. Larvae (N5) from this holding tank were
used to stock experimental vessels for a microalgae
replacement study (Study 1). For the Artemia replace-
ment study (Study 2), the larvae were maintained in
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147 137
this tank until Z3 and then stocked into cones. Larvae
in substages Z1 and Z2 were fed live diatoms
(Chaetoceros muelleri) at a density of 2.0�105 cells
mL�1. Each experimental tank (1 L Imhoff cone) was
stocked with 100 hand-counted larvae and filled with
1 L distilled artificial seawater.
2.2. Water source and monitoring
Artificial seawater was prepared daily to a salinity
of 35 ppt, 24 h in advance of use and completely
replaced in all culture vessels for both studies. In order
to chelate trace metals, EDTA was added to culture
water at 10 mg L�1. Dissolved oxygen (DO), pH,
salinity, temperature and ammonium–nitrogen con-
centrations were determined on a daily basis accord-
ing to Standard Methods (Clesceri et al., 1998).
Ammonium–nitrogen levels were subsequently used
to evaluate feed water stability.
2.3. Experimental system
The culture system used in Study 1 and Study 2
was a modification of the one developed by Wilken-
feld et al. (1984) and consisted of clear, 1-L plastic
Imhoff settling cones inserted into a common frame
and partially submerged in a thermally regulated
(29F1 C) water bath. Water circulation within the
bath was provided by a small submersible pump.
Aeration was supplied via a manifold system with
individual airlines connected to plastic pipettes
secured to the bottom of each cone. Rate of aeration
was limited to a slow trickle of bubbles which
maintained uniform distribution of larvae and food
particles within cones. A wooden-frame lid with a
clear plastic polyethylene cover was positioned on top
of the water bath to minimize evaporation and
introduction of airborne contaminants into the culture
medium. Continuous overhead illumination was
provided via two 60-W fluorescent light fixtures
suspended 1.5 m above the bath.
2.4. Daily monitoring and maintenance of experimen-
tal system
Survival and stage of metamorphosis were deter-
mined on a daily basis for all cones in both studies.
Larvae were also examined daily for extent of bio-
fouling and fullness of gut. Daily seawater replace-
ment in each cone was accomplished by first pouring
seawater through a sieve to concentrate larvae. Larvae
retained on sieves remained submerged for the
duration of the process. Filtrate from each exper-
imental cone was collected for subsequent ammo-
nium–nitrogen determination. Cones were then
refilled with newly cured artificial seawater. At this
point, microalgae, Artemia nauplii or inert feeds were
added to cones according to assigned treatments.
Larvae were then backwashed from collection sieves
into receiving cones.
2.5. Experimental feeds and feeding
Five experimental feeds (microalgal pastes, liquid
suspensions and dry powders) were used to evaluate
both 50% replacement and 100% replacement of
both, live microalga (C. muelleri; Study 1) and
Artemia nauplii (Study 2). A sevenfold level of
replication was used to evaluate a total of 11 dietary
treatments, including live control treatments of
microalgae and Artemia nauplii. Replacement feeds
for live microalgae included Chaetoceros 1000
bPremium FreshQ Instant Algaek (Reed Mariculture,
San Jose, CA), Liqualifek (Cargill, Inc., Minneap-
olis, MN), Epifeedk Liquid Larval Hatchery Feed
(Epicore Bionetworks, East Hampton, NJ), ZeiglerkZ-Plus (Zeigler Bros, Gardners, PA) and ZeiglerkE-Z Larvae (Zeigler Bros, Gardners, PA). Table 1
provides particle sizes and proximate analysis of inert
feeds. For Study 2, replacement of Artemia nauplii
was accomplished using the aforementioned inert
feeds. The only exception was replacement of Instant
Algaek, with E-Z Artemia (Zeigler Bros, Gardners,
PA). Preparation of inert larval feeds and final feed
concentrations for Study 1 and Study 2 followed
manufacturers’ instructions (Tables 2a and 2b,
respectively).
2.6. Termination of study and data collection
All cones within a dietary treatment were har-
vested when a mean of N80% of the larvae
metamorphosed to postlarva. Cones were harvested
by gently pouring contents onto a 500-Am sieve and
rinsing with fresh filtered artificial seawater. At this
point, 10 postlarvae were gently removed and
Table 1
Particle size, proximate analysis and marine fatty acid content of experimental feeds used in larval feeding trials with Farfantepenaeus aztecus
Experimental
feed
Particle
sizea (Am)
Crude
protein (%)bCrude
fat (%)bMoisture
(%)
Ash
(%)bDHAc
(%)
EPAd
(%)
E-Z Artemia 250–600 36.0 6.0 75.0 12.0
E-Z Larvae 10–100, 100–250 6.5 3.5 80.0 3.0 16.00 8.48
Z-Plus b100 50.0
100–150 12.0 8.0 12.0 6.07 7.55
150–250
Liqualifek 13–150 3.0
25–300 2.0 66.0 N/A 10.80 9.50
75–600
Epifeedk 4–44 14.0 15.0 43.0 5.0
4–124 14.0 15.0 39.0 5.0 8.32 8.65
30–400 19.0 16.0 36.0 6.0
Live microalgae 4–6 30.0 16.0 12.0 3.0 3.70 20.80
Live Artemia nauplii 400–500 50.0 19.0 6.1 8.0 TRe 1.0
a Particle size shown is that stated by manufacturer.b Dry matter concentration.c Docosohexaenoic acid; values are for one observation only.d Eicosohexaenoic acid; values are for one observation only.e Trace.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147138
subjected to a salinity stress-test similar to the one
described by Samocha et al. (1998). Postlarvae (PL)
were transferred into a 100-mL beaker filled with
fresh 35 ppt artificial seawater using dissecting scope
and a large-mouth eyedropper. From this beaker PL
were transferred into a separate cone filled with 17
ppt artificial seawater supplemented with gentle
aeration. After a 2-h exposure, PL were concentrated
on a sieve and survival was determined using a
dissecting scope. Following separation of the 10 PL
Table 2a
Feeding regimes and feed concentrations used in a larval feed study with
Treatment feed
(replacement level)
Feed concentrationa
Instant Algaek (50%) 0.5–1.0�105 cells mL�1
Instant Algaek (100%) 1.0–2.0�105 cells mL�1
Liqualifek (50%) 0.005–0.025 mL L�1 day�1
Liqualifek (100%) 0.010–0.050 mL L�1 day�1
Epifeedk (50%) 0.005–0.038 mL L�1 day�1
Epifeedk (100%) 0.010–0.075 mL L�1 day�1
Z-Plusk (50%) 0.005–0.080 mL L�1 day�1
Z-Plusk (100%) 0.010–0.160 mL L�1 day�1
E-Z Larvaek (50%) 0.009–0.043 mL L�1 day�1
E-Z Larvaek (100%) 0.018–0.086 mL L�1 day�1
Live microalgae Na
a Offered from Z1-PL1.b Offered from Z3-PL1.
for the stress-test, remaining animals on the sieve
were transferred into a labeled petri dish in 25 mL of
35 ppt fresh artificial seawater and placed in a
refrigerator for several hours to immobilize the
animals. At that point a dissecting scope was used
for counting, determination of larval developmental
stage and length measurement of 20 randomly
selected PL. Length measurements were taken from
the tip of the telson to the tip of the rostrum.
Harvested animals from each cone were rinsed with
Farfantepenaeus aztecus (Study 1)
Live algae concentration
(cells mL�1)aArtemia concentration
(nauplii mL�1)b
0.5–1.0�105 0.25–8.0
0 0.25–8.0
0.5–1.0�105 0.25–8.0
0 0.25–8.0
0.5–1.0�105 0.25–8.0
0 0.25–8.0
0.5–1.0�105 0.25–8.0
0 0.25–8.0
0.5–1.0�105 0.25–8.0
0 0.25–8.0
1.0–2.0�105 0.25–8.0
Table 2b
Feeding regimes and feed concentrations used in a larval feed study
with Farfantepenaeus aztecus (Study 2)
Treatment feed
(replacement
level)
Feed
concentration
(mL L�1 day�1)a
Artemia
concentration
(nauplii mL�1)a
Live
microalgae
(cells mL�1)b
E-Z Artemiak(50%)
0.016–0.050 0.13–4.0 0.25–8.0
E-Z Artemiak(100%)
0.032–0.092 0 0.25–8.0
Liqualifek(50%)
0.017–0.027 0.13–4.0 0.25–8.0
Liqualifek(100%)
0.034–0.054 0 0.25–8.0
Epifeedk(50%)
0.010–0.040 0.13–4.0 0.25–8.0
Epifeedk(100%)
0.020–0.080 0 0.25–8.0
Z-Plusk(50%)
0.012–0.090 0.13–4.0 0.25–8.0
Z-Plusk(100%)
0.024–0.150 0 0.25–8.0
E-Z Larvaek(50%)
0.016–0.050 0.13–4.0 0.25–8.0
E-Z Larvaek(100%)
0.032–0.092 0 0.25–8.0
Live Artemia
nauplii
na 0.25–8.0 0.25–8.0
a Offered from Z3-PL1.b Offered from Z1-PL1.
Table 3
Survival, final length and final weight of Farfantepenaeus aztecus
postlarvae in a study where larvae were offered inert feeds at the
50% and 100% replacement levels (Study 1)*
Treatment feed Survival (%) Mean length
(mm)
Mean weight
(mg)
50% replacement
Instant Algaek 85.00F4.55a 5.68F0.06c 0.239F0.01b
Liqualifek 88.14F2.04a 5.37F0.10b 0.201F0.01c
Epifeedk 79.43F8.99a 5.21F0.13b 0.197F0.01c
Z-Plus 71.00F20.53b 5.42F0.13b 0.212F0.02c
E-Z Larvae 69.14F23.79b 5.39F0.28b 0.208F0.02c
Live microalage 90.86F3.19a 6.55F0.24a 0.259F0.01a
100% replacement
Instant Algaek 0.00 (Day 4) na** na**
Liqualifek 10.00F6.16b 3.93F0.25b 0.153F0.02b
Epifeedk 2.00 2.58c 3.53F2.41b 0.139F0.10b
Z-Plus 10.14F9.23b 3.83F1.71b 0.147F0.07b
E-Z Larvae 14.86F14.35b 4.13F0.31b 0.160F0.03b
Live microalgae 90.86F3.19a 6.55F0.24a 0.259F0.01a
* Values are meansFS.D. Means within columns and having a
similar superscript are similar.
** na=not applicable; due to total mortality of larvae.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147 139
deionized water, blotted dry on absorbent paper and
placed on a marked pre-weighed microscope slide.
Slides were placed in an oven at 60 8C for 12 h.
Dried animals were group weighed, and individual
weight (reported in micrograms) was determined
based on the total number of animals on each slide.
2.7. Statistical analyses
Paired-samples Student’s t-test (SPSS; Chicago, IL)
was used to determine statistical differences in per-
formance factors (mean survival, final length, final dry
weight and stress-test survival) between level of
replacement of microalgae/Artemia nauplii and con-
trols for each experimental feed. Least Significant
Difference (LSD) and one-way ANOVAwere used at a
significance level of a=0.05 to identify statistical
differences among treatment means (SPSS; Chicago,
IL). All data were evaluated for normality and
homogeneity prior to analysis. Survival and stress-test
data were arcsine transformed prior to analysis.
3. Results
3.1. Replacement of live microalgae (Study 1)
3.1.1. Water quality analyses
Mean water temperature, salinity, dissolved oxygen
(DO) and pH in treatment cones was 29.1–29.9 C, 35
ppt, 5.15–6.08 mg L�1 and 7.93–8.40, respectively.
Ammonium–nitrogen ranged from 0.00 to 0.25 mg
L�1, except in experimental cones receiving ZieglerkZ-Plus at both the partial and complete-replacement
levels.
3.1.2. Growth and survival
Survival and final length and weight of shrimp
offered feeds as either partial or complete replacement
of live microalgae are shown in Table 3. Larvae offered
inert feeds at the 50% replacement level showed
increased survival, final length and final weight
compared to those fed at the 100% level (Pb0.05).
Analysis of survival, mean length and weight among
shrimp offered feeds at the 50% replacement level
showed a significant difference among treatments
(Pb0.02). Survival of larvae ranged from 69.1% to
88.1% (Table 3). Larvae fed Instant Algaek,
Liqualifek and Epifeedk demonstrated similar sur-
Table 4
Percentage survival by developmental substages of Farfantepe-
naeus aztecus larvae fed partial and complete replacement of live
microalgae (Study 1)*
Treatment Feed Z3 (%) M3 (%) PL1 (%)
50% replacement
Instant Algaek 97.86F0.69a 93.86F0.69b 85.00F4.55c
Liqualifek 98.00F0.82a 93.14F0.69b 88.14F2.04c
Epifeedk 97.71F0.76a 89.71F0.95b 79.43F8.99c
Z-Plus 97.43F0.98 a 95.14F1.22b 71.00F20.53c
E-Z Larvae 93.71F1.38 a 78.43F14.73b 69.14F23.79c
Live microalgae 97.86F0.69 a 96.29F1.11b 90.86F3.19c
100% replacement
Instant Algaek 88.71F1.11b 0.00 na**
Liqualifek 95.71F0.76b 35.43F3.55b 10.00F6.16b
Epifeedk 95.57F0.79b 26.29F2.81b 2.00F2.58b
Z-Plus 94.43F0.98b 31.00F3.92b 10.14F9.22b
E-Z Larvae 92.00F1.29b 28.43F3.10b 14.86F14.35b
Live microalgae 97.86F0.69a 96.29F1.11a 90.86F3.19a
* Values are meansFS.D. Means within columns and having a
similar superscript are similar.
** na=not applicable; due to total mortality of larvae at the Z3-
M1 molt.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147140
vival to those offered live microalgae (PN0.05). Other
inert feeds (Zeigler Z-Plusk and Ziegler E-Z
Larvaek) showed greatly reduced survival (Pb0.01).
Mean final length and weight of shrimp offered inert
feeds at the 50% replacement level ranged from 5.2 to
5.7 mm and 0.20 to 0.24 mg, respectively, with
Chaetoceros 1000 bPremium FreshQ Instant Algaekproducing the best performance (Pb0.01).
Significantly lower survival (Pb0.00) and final
mean length and weight (Pb0.02) were shown by
larvae offered inert feeds at the 100% replacement
level than those fed live microalgae. Survival of
shrimp offered inert feeds at the 100% replacement
level ranged from 0.00% to 14.9%, with the highest
numerical survival observed by larvae fed the Zeigler
E-Z Larvaek (Table 3). Shrimp fed the Zeigler E-Z
Larvaek, Liqualifek and Zeigler Z-Plusk feeds had
no significant differences in survival rates. However,
survival in these treatments was significantly better
than the shrimp fed the Epifeedk. Complete mortality
occurred within 4 days post-stocking for larvae
offered Instant Algaek. Final length and weight of
shrimp offered inert feeds at the 100% replacement
level ranged from 3.5–4.1 mm and 0.14–0.16 mg,
respectively, with Zeiglerk E-Z Larvae exhibiting the
best numeric performance, though not statistically
different from Liqualifek and Z-Plusk feeds.
In conclusion, except for similar survivals of the
Instant Algaek, Liqualifek and Epifeedk treat-
ments at the 50% replacement level of live algae, final
mean length and dry weight of larvae offered the
control feed were significantly higher than animals in
all other inert feed treatments (Pb0.009).
3.1.3. Survival, by developmental stage
Mean percent survival of larvae offered inert feeds
by developmental stage is presented in Table 4.
Survival through Zoea was better than through Mysis
at both levels of microalgae replacement (P=0.009). A
similar relationship was shown in terms of survival
through M3 compared to PL1 (P=0.030). The same
relationship was shown for shrimp fed the live micro-
algae control. Highest mortality of larvae offered inert
feeds (50% replacement) and live algae occurred
during metamorphosis from M3 to PL1 (Table 4).
Survival through M3 decreased as much as 24% for
shrimp offered inert feeds at the 50% replacement level
versus 5.4% for shrimp offered live microalgae. Shrimp
offered inert feeds at the 100% replacement level
experienced the greatest mortality during metamor-
phosis from Zoea to Mysis, at which point survival
decreased as much as 64%.
3.1.4. Marine fatty acid content and survival
Concentrations of DHA among the inert feeds and
the microalga control ranged from 3.7% to 16.0% and
showed no apparent trends for the 50% and the 100%
replacement levels (Table 5). Live microalgae, receiv-
ing the lowest concentration of DHA (3.7%), elicited
highest survival (90.9%).
Concentrations of EPA in inert feeds and the
microalga control ranged from 7.6% to 20.8% and
appeared to be positively correlated with survival
(Table 5). Shrimp offered live microalgae (highest
EPA concentration, 20.8%) had the highest percentage
survival (90.9%) among both treatments. These
results suggest that this level of EPA concentration
does not adversely affect survival.
3.1.5. Ammonium–nitrogen
Day 10 ammonium–nitrogen concentrations in
experimental cones receiving 50% replacement of live
microalgae were similar to those at the 100% level
(PN0.05). Analysis of day 10 ammonium–nitrogen in
Table 6
Day 10 ammonium–nitrogen concentration of treatment cones at the
50% and 100% replacement levels of live microalgae (Study 1)
Treatment feed Mean ammonium–nitrogen
concentration (mg L�1)*
50 % replacement
Instant Algaek 0.125F0.10a
Liqualifek 0.161F0.04a
Epifeedk 0.241F0.07a
Z-Plus 1.889F0.44b
E-Z Larvae 0.215F0.12a
Live microalgae 0.154F0.09a
100% replacement
Instant Algaek 0.197F0.02ac
Liqualifek 0.088F0.06a
Epifeedk 0.021F0.03ad
Z-Plus 2.139F0.34b
E-Z Larvae 0.139F0.05a
Live microalgae 0.154F0.09a
* Means with similar superscripts are not significantly different
( PN0.05).
Table 5
Comparison of Farfantepenaeus aztecus postlarvae survival and
fatty acid content of inert feeds offered at two different rates
Treatment feed Survival (%) % EPAa % DHAb
50% replacement
Liqualifek 88.14 9.50 10.80
Epifeedk 79.43 8.65 8.32
Z-Plus 71.00 7.55 6.97
E-Z Larvae 69.14 8.48 16.00
Live microalgae 90.86 20.80 3.70
100% replacement
Liqualifek 10.00 9.50 10.80
Epifeedk 2.00 8.65 8.32
Z-Plus 10.14 7.55 6.97
E-Z Larvae 14.86 8.48 16.00
Live microalgae 90.86 20.80 3.70
a Eicosopenaenoic acid.b Docosohexaenoic acid.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147 141
experimental cones receiving 50% replacement of live
microalgae showed a significant difference among
treatments (Pb0.00), with concentrations ranging from
0.125 to 1.889 mg L�1 (Table 6). Cones receiving
Zeiglerk Z-Plus elicited a significantly higher ammo-
nium–nitrogen at day 10 than all other algal replace-
ment feeds (Pb0.00), including live microalgae.
Analysis of day 10 ammonium–nitrogen in exper-
imental cones receiving 100% replacement of live
microalgae showed a significant difference among
treatments (Pb0.00), with concentrations ranging from
0.088 to 2.139 mg L�1 (Table 6). Cones receiving
Zeiglerk Z-Plus had a significantly higher day 10
ammonium–nitrogen concentration than all other rep-
lacement feeds (Pb0.00), including live microalgae.
Furthermore, with the exception of Instant Algaekand Z-Plus, it appears that ammonium–nitrogen levels
were lower in the 100% replacement in terms of
reduced water quality degradation. Nevertheless, even
if the stability of some of the inert feeds means a slight
improvement in water quality, the dominating factor is
the poor survival and the growth of the larvae on these
feeds.
3.2. Replacement of live Artemia nauplii (Study 2)
3.2.1. Water quality analyses
Mean water temperature, salinity, dissolved oxygen
and pH in treatment cones was 28.8–29.5 8C, 35 ppt,
5.4–6.1 mg L�1 and 8.1–8.3, respectively. Ammo-
nium–nitrogen ranged from 0.00 to 0.55 mg L�1,
except in cones fed Zieglerk Z-Plus at both the 50%
and 100% replacement levels where maximum con-
centrations of 1.94 and 3.56 mg L�1, respectively.
3.2.2. Growth and survival
Survival, final length, final weight and stress-test
survival of shrimp offered inert feeds at the 50%
replacement level of live Artemia (Table 7) were
greater than corresponding values for the 100%
replacement level (Pb0.04, Table 7). There was a
significant difference in survival, final length and final
weight of shrimp fed the 50% replacement feeds
(Pb0.01). Shrimp offered live Artemia exhibited
significantly higher survival rates than those fed inert
replacement feeds (Pb0.05), with the exception of
those fed Liqualifek (P=0.22). Overall survival of
shrimp fed 50% replacement feeds ranged from 87.1%
to 92.1%, with the highest numeric survival seen in
those offered the Liqualifek. Shrimp offered
Liqualifek exhibited significantly higher survival than
all others fed 50% replacement diets. Stress-test
survival was similar among all shrimp fed the 50%
replacement feeds (P=0.01). Stress-test survival of
shrimp offered 50% replacement feeds ranged from
7.1% to 12.9%, with the highest numeric survival
shown by those fed Zeiglerk E-Z Larvae. Shrimp
Table 7
Survival, final length, final weight and stress-test survival of postlarvae when larvae were offered inert feeds at the 50% and 100% live Artemia
nauplii replacement levels (Study 2)*
Treatment feed Survival (%) Mean length
(mm)
Mean weight
(mg)
Stress-test
survival (%)
50% replacement
E-Z Artemia 89.71F4.39bc 5.55F0.08cd 0.287F0.01c 7.14F7.56b
Liqualifek 92.14F1.95a 5.91F0.16b 0.304F0.01b 10.00F8.17b
Epifeedk 90.43F1.72bc 5.71F0.18bc 0.294F0.01bc 10.00F8.17b
Z-Plus 87.14F3.02c 5.87F0.12b 0.300F0.01b 8.57F6.90b
E-Z Larvae 90.86F1.77b 5.51F0.12de 0.281F0.01cd 12.86F7.56b
Live Artemia nauplii 93.43F4.32a 6.95F0.22a 0.356F0.01a 18.57F6.90a
100% replacement
E-Z Artemia 81.14F1.95bc 4.26F0.13e 0.217F0.01e 1.43F3.78b
Liqualifek 80.14F1.35c 5.40F0.15b 0.278F0.01b 1.43F3.78b
Epifeedk 83.14F1.86b 5.18F0.21c 0.263F0.02c 1.43F3.78b
Z-Plus 81.14F2.27b 4.57F0.20d 0.235F0.01d 2.86F4.88b
E-Z Larvae 84.29F3.90b 5.32F0.09b 0.273F0.01b 2.86F4.88b
Live Artemia nauplii 93.43F4.32a 6.95F0.22a 0.356F0.01a 18.57F6.90a
* Values are meansFS.D. Means within columns and having a common superscript are similar.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147142
offered the live Artemia diet exhibited significantly
higher survival in the stress-test than seen in with all
other Artemia replacement feeds. Shrimp offered inert
replacement feeds showed significantly lower final
length and weight than those fed live Artemia
(Pb0.000). Final length and weight of shrimp fed
50% replacement feeds ranged from 5.5 to 5.9 mm and
0.281 to 0.304 mg, respectively. Shrimp fed
Liqualifek exhibited significantly greater final length
and weight compared to those fed Zeiglerk E-Z
Artemia ( P=0.001) or Zeiglerk E-Z Larvae
(Pb0.000). Shrimp fed Liqualifek exhibited similar
final length and weight gains compared to those fed
Epifeedk (P=0.06) and Zeiglerk Z-Plus (P=0.44).
Shrimp offered the live Artemia control had
significantly higher survival, mean length, mean
weight and survival in stress-test than those offered
inert feeds at the 100% replacement level (Pb0.00). A
significant difference in survival, final length, final
weight and stress-test survival was also shown among
shrimp offered inert feeds (Pb0.000). Survival of
shrimp fed at the 100% replacement level ranged from
80.1% to 84.3%, with the highest numeric survival
shown by those offered Zeiglerk E-Z Larvae. Shrimp
fed this feed had significantly greater survival than
those fed Liqualifek (P=0.04), but not those fed
Zeiglerk E-Z Artemia (P=0.10), Epifeedk (P=0.5)
or Zeiglerk Z-Plus (P=0.10).
Shrimp fed inert feeds at the 100% replacement
level showed significantly lower final length and
weight than those offered live Artemia nauplii
(Pb0.00). Mean final length and weight of shrimp
fed 100% replacement feeds ranged from 4.3 to 5.4
mm and 0.217 to 0.278 mg, respectively, with
Liqualifek showing the best results. Shrimp fed
Liqualifek had significantly greater final length and
weight than those fed Zeiglerk E-Z Artemia ,
Epifeedk and Zeiglerk Z-plus (Pb0.004), but not
those fed Zeiglerk E-Z Larvae.
Shrimp fed live Artemia nauplii had significantly
higher stress-test survival than those fed any of the
replacement feeds (Pb0.00). Stress-test survival of
shrimp fed inert feeds at the 100% replacement level
ranged from 1.4% to 2.9%, with the highest numeric
survival seen in those fed Zeiglerk Z-Plus and E-Z
Larvae. No significant difference in stress-test survival
was shown among shrimp fed inert feeds (P=0.50).
3.2.3. Percentage survival by developmental stage
Survival of shrimp through metamorphosis to
Postlarva is presented in Table 8. Survival through
M3 exceeded that through PL1 for all types of inert
feeds at all levels of replacement (P=0.003). Survival
of shrimp fed live Artemia nauplii through M3
exceeded that through PL1 (P=0.038). Survival from
M3 through PL1 decreased as much as 15% for shrimp
Table 8
Percentage survival of Farfantepenaeus aztecus developmental
substages when inert feeds were fed at the 50% and 100%
replacement levels of live Artemia nauplii (Study 2)*
Treatment feed M3 PL1
50% replacement
E-Z Artemia 96.43F1.13 89.71F4.39
Liqualifek 97.14F1.22 92.14F1.95
Epifeedk 96.00F1.16 90.43F1.72
Z-Plus 95.86F1.07 87.14F3.02
E-Z Larvae 95.86F0.90 90.86F1.77
Live Artemia nauplii 97.00F1.00 93.43F4.32
100% replacement
E-Z Artemia 94.86F1.35 81.14F1.95
Liqualifek 95.00F1.41 80.14F1.35
Epifeedk 94.86F0.90 83.14F1.86
Z-Plus 95.71F1.38 81.14F2.27
E-Z Larvae 94.57F0.98 84.29F3.90
Live Artemia nauplii 97.00F1.00 93.43F4.32
* Values are meansFS.D. None of the values shown, by
substage of metamorphosis, were significantly different ( Pb0.050).
Table 9
Survival, stress-test survival of Farfantepenaeus aztecus postlarvae
and fatty acid concentrations of experimental feeds offered at the
50% and 100% replacement levels (Study 2)
Treatment feed Survival
(%)
Stress-test
survival (%)
% EPAa % DHAb
50% replacement
E-Z Artemiak 89.71 7.14 5.21 4.84
Liqualifek 92.14 10.00 9.50 10.80
Epifeedk 90.43 10.00 8.65 8.32
Z-Plus 87.14 8.57 7.55 6.97
E-Z Larvae 90.86 12.86 8.48 16.00
Live Artemia nauplii 93.43 18.57 1.00 TRc
100% replacement
E-Z Artemiak 81.14 1.43 5.21 4.84
Liqualifek 80.14 1.43 9.50 10.80
Epifeedk 83.14 1.43 8.65 8.32
Z-Plus 81.14 2.86 7.55 6.97
E-Z Larvae 84.29 2.86 8.48 16.00
Live Artemia nauplii 93.43 18.57 1.00 TR
a Eicosopentaenoic acid; values shown constitute one observa-
tion only.b Docosohexaenoic acid; values shown constitute one observa-
tion only.c Trace.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147 143
offered inert feeds at the 100% replacement level. On
the other hand, this decrease did not exceed 8% for
shrimp fed at the 50% replacement level. Shrimp fed
live Artemia nauplii experienced a decrease in
survival of only 4% for the same period (e.g.,
metamorphosis from M3 to PL1).
3.2.4. Marine fatty acid content and survival
Comparison of concentrations of EPA and DHA in
inert feeds and live Artemia nauplii to overall survival
and stress-test survival suggest some trends (Table 9).
At the 50% replacement level, shrimp offered live
Artemia nauplii (lowest EPA concentration) exhibited
the highest overall survival (93.4%) and stress-test
survival (18.6%). Concentration of EPA among inert
feeds ranged from 5.21% to 9.50% and showed no
apparent influence on overall survival at the 50%
replacement level. Among shrimp offered inert feeds,
those offered Liqualifek exhibited the highest EPA
concentration (9.50%) and percent survival (92.1%).
Shrimp offered E-Z Larvae (EPA=8.5%) demonstrated
the highest stress-test survival (12.9%). There were no
apparent trends in EPA concentration and overall
survival or stress-test survival at the 100% replacement
level.
Shrimp offered live Artemia nauplii (trace amounts
of DHA) exhibited the highest overall survival
(93.43%) and stress-test survival (18.6; Table 9).
Among the inert feeds, shrimp offered E-Z Larvae
(highest DHA concentration of 16.00%) at the 50%
replacement level had the highest stress-test survival
(12.9%). Shrimp offered E-Z Larvae (DHA=16.00%)
at the 100% replacement level showed the highest
overall survival (84.3%) and stress-test survival
(2.9%).
3.2.5. Ammonium–nitrogen
Day 7 ammonium–nitrogen concentrations in
100% Artemia replacement cones were significantly
different from those offered inert feeds at the 50%
replacement level (Pb0.04), with the exception of the
Zeiglerk E-Z Larvae treatment (P=0.32). A signifi-
cant difference (Pb0.00) was shown in day 7
ammonium–nitrogen concentrations, which ranged
from 0.001 to 1.938 mg L�1 among inert feeds
offered at the 50% replacement level (Table 10). The
highest concentration was shown in cones containing
Zeiglerk Z-Plus (1.938 mg L�1). This feed yielded a
significantly higher ammonium–nitrogen concentra-
tion (Pb0.00) than all other replacement feeds,
including live Artemia nauplii.
Table 10
Day 7 ammonium–nitrogen concentration of treatment cones at the
50% and 100% replacement levels of live Artemia nauplii (Study 2)
Treatment feed Mean ammonium–nitrogen
concentration (mg L�1)*
50% replacement
E-Z Artemia 0.039F0.05b
Liqualifek 0.001F0.02b
Epifeedk 0.424F0.07b
Z-Plus 1.938F0.13a
E-Z Larvae 0.087F0.05b
Live Artemia nauplii 0.127F0.07b
100% replacement
E-Z Artemia 0.356F0.08b
Liqualifek 0.113F0.12b
Epifeedk 0.548F0.18b
Z-Plus 3.563F0.12a
E-Z Larvae 0.061F0.04b
Live Artemia nauplii 0.127F0.07b
* Means with similar superscripts are not significantly different
( PN0.05).
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147144
A significant difference (Pb0.00) was seen in day
7 ammonium–nitrogen concentration of cones receiv-
ing inert feeds at the 100% replacement level (Table
10): concentrations ranged from 0.061 to 3.563 mg
L�1, with the highest occurring in the Zeiglerk Z-
Plus cones. This feed elicited a significantly higher
ammonium–nitrogen concentration than all other
replacement feeds (Pb0.00), including live Artemia
nauplii (Pb0.00).
4. Discussion
Replacement of live microalgae with inert feeds
appeared to be marginally successful at the 50%
replacement level; however, complete replacement
could not be achieved without a significant negative
effect on final weight and length and survival of
shrimp. Complete mortality of shrimp fed Instant
Algaek (a paste) could have been associated with
nutritional deficiencies associated with inappropriate
preparation procedures, as recommended by the
manufacturer. Microscopic evaluation of this paste-
like product showed substantial lysed and small-
diameter cells compared to that of the live microalga
control. This observation suggests that shrimp offered
this feed may have not received adequate nutrition.
Overall performance of shrimp offered inert feeds
at the 100% replacement level was poor with respect
to survival and final length and weight. Other studies
with different inert feeds have reported good survival
but only moderate growth of larval shrimp offered
these feeds (Kanazawa et al., 1982; Jones et al., 1987;
Smith and Lawrence, 1987; Kurmaly et al., 1989;
Kumlu and Jones, 1995). Although no explanation for
their results was provided, these studies suggested
further investigation to determine whether inert feeds
were nutritionally imbalanced, less digestible or of a
non-suitable particle size. It was further suggested that
it may be necessary to evaluate and characterize the
potentially toxic conditions caused by the increased
release of toxic nitrogenous compounds via ammoni-
fication and/or nitrification.
Jones et al. (1979) demonstrated that the use of
non-encapsulated microparticulate feeds warranted
strict monitoring of feed levels and quality to avoid
deterioration of water quality and associated adverse
effects on growth and survival of M. japonicus.
Nitrogenous products including ammonium, nitrite
and nitrate were reported to be toxic to several marine
organisms at extremely low concentrations (Spotte,
1979; Muir et al., 1991). Muir et al. (1991) reported
that degradation of microencapsulated feeds yielded
an increase in dissolved ammonium–nitrogen within
1.5 h of addition to culture water, which surpassed a
two- to threefold increase within 48 h of the addition
of microcapsules. These concentrations were not
considered as toxic until the final 2 days of the trial.
In the present studies, sub-lethal ammonium–nitrogen
concentrations were detected within a short time after-
feeding (24 h) and steadily increased until the end of
the trial. Cones receiving the Zeiglerk Z-Plus had
ammonium–nitrogen concentrations as high as 2.14
mg L�1 in the live algae study and 3.56 mg L�1 in the
live Artemia study. This level is substantially higher
than the 0.13 mg L�1 considered toxic to shrimp
(Chin and Chen, 1987). Chin and Chen (1987)
reported ammonium–nitrogen 24- and 96-h LC50s
of 5.71 and 1.26 mg L�1, respectively, for postlarval
P. monodon. The present studies failed to elucidate a
direct relationship between sub-lethal concentrations
of ammonium–nitrogen and reduced growth and/or
survival.
Shrimp excretion of NH3–N probably contributed
to overall ammonium–nitrogen concentrations in the
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147 145
present studies, but was tempered by survival.
Wajsbrot et al. (1989) reported excretion rates for
various sizes of Penaeus semisulcatus were highest
between 4 and 8 h post-feeding. The dominant waste
product in their study was NH3–N (61–83%).
Although larval shrimp were not included in that
study, a trend of decreasing NH3–N excretion per unit
body weight was seen as shrimp weight increased. In
the present studies, higher levels of ammonium–
nitrogen were observed with the Zieglerk Z-Plus
treatments in both the live algae and live Artemia
replacement studies. In the live algae study, with the
exception of Instant Algaek and Z-Plus, it appears
that ammonium–nitrogen levels were lower in the
100% replacement treatments in terms of reduced
water quality degradation. These results are not what
would be expected when increasing substitution levels
from 50% to 100%. A high level of shrimp mortality
was recorded early on in the study with the 100%
replacement treatments. The fall in larval density in
these cones means a decrease in excretion rates. It also
means fewer animals are feeding on and breaking
down the particles. The stability of the test feeds
would be intake for a longer period of time. Break-
down of the feed particles would take longer as would
the rise in ammonium–nitrogen levels. The result
would probably be a decrease in ammonium–nitrogen
concentrations. The live Artemia study supports these
findings. Ammonium–nitrogen levels were higher in
the 100% replacement treatments. Shrimp survival
throughout the study was higher which means an
increase in larval excretion rates and a more rapid
breakdown of feed particle stability due to feeding.
Even if the stability of some of the inert feeds means a
slight improvement in water quality, the dominating
factor is the poor survival and the growth of the larvae
on these feeds. Nevertheless, no clear trend of
decreased survival was seen as a result of these
higher ammonium–nitrogen levels. Although the 50%
replacement of live algae and live Artemia out-
performed the 100% replacement levels, feeding the
larvae live algae and live Artemia provided signifi-
cantly improved survival, growth and performance in
stress-tests than all inert feeds tested in the present
study.
Feed particle diameter could have reduced growth
and survival of larval shrimp. Feed particles must be
of an appropriate size in order to assure maximum
rate of ingestion. Jones et al. (1979) reported that the
optimum particle size for Zoea stage M. japonicus
was approximately 10 Am. Those same authors
observed a trend towards selection of larger particles
at M2–M3 and suggested an optimum particle
diameter approximating 28 Am. They measured the
median particle diameter (Md) of capsules prior to
and post-feedings. In the majority of replicates, Md
increased for subsequent feedings, suggesting larvae
were selecting for more suitable-diameter particles
and rejecting larger diameter particles. In the present
study, which focused on larval feeding of F. aztecus,
it is possible that feed particle size, especially during
early Zoea stage, did not possess the proper Md.
Guts of shrimp offered inert feeds at the 100%
replacement level during Zoea and early Mysis
stages were seldom observed to be full, whereas
larvae offered inert feeds at the 50% replacement
level typically revealed full guts throughout the
duration of the study. As shown in Table 1, inert
feeds had minimum particle sizes which were of
similar, smaller or slightly larger diameter than that
recommended by Jones et al. (1979). The only
exception was Ziegler Z-Plus, which had a minimum
particle size of bb100 Am.Q The maximum particle
size for all inert feeds evaluate was substantially
outside this optimal range, especially with respect to
the Zoea stage. Frequency distribution of particles
sizes of inert feeds was not evaluated; however, it is
postulated that a high proportion were inappropriate
and thus contributed to higher overall survival in
Study 2. Most mortality was experienced during the
metamorphosis from Zoea to Mysis stages, especially
for shrimp fed the 100% replacement treatments
(Table 4).
According to Jones et al. (1979), to ensure high
growth and survival rates, particle size and feed rates
must be closely controlled, implying a need for
increased frequency of feeding of an appropriate
particle size. Unfortunately, with the inert feeds tested
in Study 1 (microalgae replacement) this recommen-
dation would have ultimately resulted in substantial
waste of expensive feed and a reduced cost/benefit to
commercial hatcheries. Probably the most efficient
means of improving microparticle feed efficiency lies
in reducing variance in particle Md and could
ultimately be derived from improvements in feed
particle manufacturing technology.
C.B. Robinson et al. / Aquaculture 245 (2005) 135–147146
The replacement of Artemia nauplii with inert test
feeds appeared marginally successful. Larvae offered
the live control diet in this study performed
significantly better in terms of final length, and
weight and performance in stress-tests than those fed
any of the inert feeds. The only exception was that
of similar survival associated with replacement of
Artemia nauplii with Liqualifek at the 50% level.
The significantly better performance of the control
diet suggests that survival by itself should not be
used as the sole criteria to select inert diets for larval
shrimp. Potential for replacement of live feed at
lower rates than those tested in the present study
(e.g., 10–25%) should be evaluated to determine
whether reduction in live feed consumption is
feasible without compromising shrimp growth and
performance in stress-tests. The results from Study 1
suggest that further investigation is necessary to
determine whether inert feeds offers a less nutrition-
ally balanced diet, a less digestible diet, or inad-
equate particle sizes that are rejected by larval
shrimp. Additional research is also required to study
the long-term effects of inert feeds on growth and
survival of the shrimp in the nursery and grow-out
phases.
5. Conclusions
Partial and complete replacement of live micro-
algae at the 50% and 100% level showed inferior
performance to the live microalgae control diet.
Similarly, Study 2 suggests that significantly better
results were obtained when the larvae were fed live
Artemia (control diet) than with any of the inert feeds.
No clear conclusion can be made concerning the
correlation between the concentrations of EPA and
DHA in the inert feeds and the performance of the
larvae. More in-depth studies are needed to clarify this
issue.
Acknowledgments
Funding for this research was provided by Lone
Star Farm Inc. and Texas Agricultural Experiment
Station-Shrimp Mariculture Research Facility. Spe-
cial thanks to the Lone Star Farm staff for the help
provided. Special thanks also to Reed Mariculture,
Inc., Cargill, Inc., Epicore Bionetworks, Inc. and
Zeigler Bros. Inc. for donating the feeds used in
these studies. Finally, we thank Mrs. Linda Smith
Lemmon for her help in improving the quality of this
manuscript.
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