Aquaculture Nutrition 2002 8;121^137

..............................................................................................

Effect of culture system on the nutrition and growth

performance of Pacific white shrimp Litopenaeus
vannamei (Boone) fed different diets
A.G.J. TACON1, J.J. CODY2, L.D. CONQUEST2, S. DIVAKARAN2, I.P. FORSTER2 &
O.E. DECAMP2
1
Halliday Place, Kaneohe, HI, USA; 2The Oceanic Institute, Waimanalo, HI, USA

Introduction
Abstract
Of the estimated 375 913 shrimp farms reportedly in
Two 8-week feeding trials were conducted with juvenile Pacific
existence in the world in 1999, 54% used extensive pond-
white shrimp, Litopenaeus vannamei (Boone) to compare the
based growout culture systems (stocking density below
growth and performance of animals fed a series of experimen-
2.5 m–2, shrimp production 50–500 kg ha–1 year–1, produc-
tal and commercial pelleted shrimp and fish feeds and dietary
tion costs US$1–3 kg–1 live shrimp), 28% used semi-intensive
feeding regimes within an indoor running-water culture system
pond-based growout culture systems (stocking density below
and an outdoor zero-water-exchange culture system. The best
30 m–2, shrimp production 500–5000 kg ha–1 year–1, produc-
overall shrimp growth performance was observed for animals
tion costs US$2–6 kg–1 live shrimp), and 18% used intensive
fed the experimental shrimp diet and all-day feeding regime
pond-based growout culture systems (stocking density above
under outdoor zero-water-exchange culture conditions. Final
30 m–2, shrimp production 5000–20 000 kg ha–1 year–1, pro-
body weight and average weekly growth rate under these
duction costs US$4–8 kg–1 live shrimp; Rosenberry 1999).
conditions were 2.8 and 3.4 times greater, respectively, than
Moreover, although over 1.1 million tonnes of marine
animals of similar size fed with the same diet under indoor
shrimp (valued at US$6.8 billion) were produced in 1998
running-water culture conditions. Although direct compar-
(FAO 2000), little or no information exists concerning the
ison between indoor and outdoor culture systems is difficult
optimum dietary nutrient levels for rearing these species
because of the lower indoor water temperatures, and conse-
under practical pond-based culture systems (Lawrence 1996;
quently lower mean daily feed intake of animals, it is believed
Tacon 1996).
that the higher growth and feed performance of animals reared
As a result of the pressure faced by the shrimp farming
under outdoor ‘green-water’ culture conditions was primarily
community for increased biosecurity, and disease and effluent
due to their ability to obtain additional nutrients from food
control (Bullis & Pruder 1999), there has been a trend within
organisms endogenously produced within the zero-water-
many countries towards the development of biosecure closed
exchange culture system. The most promising features of zero-
shrimp production systems, including zero-water-exchange
water-exchange culture systems are that they offer increased
or recirculating culture systems employing in situ (McIntosh
biosecurity, reduced feed costs and water use for the farmer,
1999; Avnimelech 2000; McNeil 2000) or external biofiltra-
and by doing so provide a potential avenue of moving the
tion techniques (Reid & Arnold 1992; Moss et al. 1998; Ogle
shrimp culture industry along a path of greater sustainability
& Lotz 2000; Van Wyk 2000). Trials of zero water exchange
and environmental compatibility.
systems were conducted in Tahiti during the 1980s with
Litopenaeus vannamei and Penaeus monodon and with yields
KEY WORDS: diet, feeding regime, Litopenaeus vannamei,
of approximately 20 000 kg ha–1 year–1 (AQUACOP, perso-
methodology, Pacific white shrimp, zero water exchange
nal communication). This paper describes two feeding trials
Received 22 February 2001, accepted 15 August 2001 conducted from July to September 1999 at the Oceanic
Correspondence: I.P. Forster, The Oceanic Institute, 41-202 Kalanianaole Institute (OI), Hawaii, USA. The objective was to compare
Highway, Waimanalo, HI 96795, USA. E-mail: iforster@oceanicinsitute. the growth and performance of juvenile Pacific white shrimp
org

..............................................................................................

2002 Blackwell Science Ltd 121

122 A.G.J. Tacon et al.
L. vannamei (Boone) fed a series of different practical shrimp
feeds and dietary feeding regimes within an indoor
running-water culture system and an outdoor zero-water-
exchange culture system.
Materials and methods
Shrimp and experimental culture conditions
Pacific white shrimp L. vannamei (Boone) were obtained
from the Oceanic Institute shrimp hatchery (industry pro-
duction run, N-99–3 strain, Moss et al. 2001a) and fed ini-
tially with a 520-g kg–1 protein commercial larval shrimp diet
(Higashimaru Co. Ltd, Kagoshima, Japan), and later a
350–400 g kg–1 protein commercial nursery shrimp diet
(Rangen, Inc., Buhl, ID, USA) prior to the start of the two
56-day feeding trials.
In the indoor feeding trial, juvenile shrimp of mean initial
weight 1.58 (0.05 standard deviation) g were stocked within
indoor rectangular glass aquaria (0.76 · 0.31 · 0.31 m; 52-L
water volume) at an initial stocking density of 24 shrimp
aquaria–1 (equivalent to a shrimp density of 100 m–2 bottom
surface area or 461 m–3 water volume), with three aquaria
allotted per dietary treatment [laboratory studies conducted
at OI using these culture systems showed no difference in the
growth or survival of shrimp reared at densities of 50 m–2 or
100 m–2 (unpublished data)]. A seawater flow-through sys-
tem with a water exchange rate of 100% hour–1 was
employed for the duration of the experiment (water tem-
perature ranged from 26 to 27 C). The aquaria were cleaned
every morning before first feeding by siphoning out any
uneaten feed, faeces, moults, or dead shrimp that were
present. A 12-h photoperiod was maintained within the
indoor laboratory using fluorescent lighting (daylight hours
from 06.00 to 18.00 hours).
In the outdoor feeding trial, juvenile shrimp (of the same
strain and size as above) were stocked within outdoor free-
standing 1500 L cylindrical black-coated fibreglass micro-
cosm tanks (1.52 m dia with a conical bottom) at an initial
Table 1 Environmental conditions of culture containers in 8-week outdoor trial
Open cover
Parameter Mean ! SD Minimum^Maximum
Temperature (AM) 28.2 ! 1.0 25.3)30.4
Temperature (PM) 31.3 ! 1.3 27.1)34.0
Dissolved oxygen (mg L)1) 6.0 ! 0.5 4.7)8.2
pH 7.8 ! 0.4 6.7)9.2
Salinity (g L)1) 33.9 ! 1.1 31)38
stocking density of 100 shrimp tank–1 (equivalent to a shrimp
density of 51 m–2 cone surface area, 55 m–2 flat bottom
surface area or 71 m–3 water volume), with three tanks
allotted per dietary treatment. Water within the microcosms
was continuously mixed and aerated using six air lift tubes
(to keep all particulate matter in suspension) and a zero-
water-exchange ‘green water’ management system operated
within the tanks for the duration of the 56-day culture trial
(for tank configuration and operation see Freeman & Duerr
1991). Air was continuously supplied to all experimental
tanks with an EG&G Rotron 5 HP regenerative blower
(Saugerties, NY, USA). Freshwater was used as required to
replace evaporative losses. Diurnal water temperature, dis-
solved oxygen, pH and salinity measurements throughout the
study were recorded (Table 1).
Diets and feeding protocols
Tanks were randomly assigned one of four diets in both
feeding trials: a sinking pelleted shrimp diet (OI shrimp diet)
formulated to contain 350 g kg–1 protein and 25 g kg–1 squid
meal (Tables 2 and 3); a commercially available sinking
pelleted shrimp diet formulated to contain 350 g kg–1 protein
and 25 g kg–1 squid meal; and a commercially available
pelleted catfish diet in two forms (pelleted-floating, and
crumbled-sinking) formulated to contain 370 g kg–1 protein.
The OI shrimp diet was prepared by first mixing all the
major dry feed ingredients (previously ground in a hammer
mill to pass through a 60-mesh or 0.25 mm screen) for
15 min in a Hobart food mixer (Model D-300, Hobart
Manufacturing Corporation, Troy, OH, USA). A warm
(approximately 60 C) aqueous solution of sodium phos-
phate, potassium phosphate, choline chloride, and trace
element premix, was then added to the dry ingredient mix, to
bring the moisture content of the resulting mash to approxi-
mately 34–35%. The mash was then blended for a further
15 min. Half the supplemental oil and lecithin and all the
cholesterol were blended in a KitchenAid mixer (Model
K5SS, KitchenAid, St Joseph, MI, USA), added to the mash
Clear plastic cover
Mean ! SD Maximum^Minimum
29.6 ! 1.0 25.6)31.5
32.6 ! 1.2 29.7)35.1
5.8 ! 0.5 4.6)7.1
7.7 ! 0.4 7.2)8.8
34.0 ! 1.1 31)36
..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137
 Diet, culture and shrimp growth 123

Table 2 Formulation of the Oceanic Institute (OI) experimental cabinet using an air blower at 38 C until the moisture level
sinking pelleted shrimp diet used in the 8-week feeding trial was below 10%. The vitamin premix and vitamin C source
Ingredient [crude protein (%)/crude lipid (%); OI shrimp diet (Table 2) were then emulsified with the remaining oil and
ingredient cost – US$ kg^1] (g kg ^1 dry weight) lecithin in a KitchenAid mixer and this mixture was added to
Fishmeal ^ LT 94 (71.83/11.14; 1.28)1 245.0 the dry cooled pellets by top coating using a Hobart D300
Squid meal (58.94/4.19; 2.85)2 25.0 food mixer with a whisk beater. The finished pellets were
Soya bean meal, dehulled, solvent 95.0 then stored in plastic bins at 19–20 C until used.
extracted (43.84/1.69; 0.16)3
Wheat, whole hard red winter 469.4
(13.88/1.76; 0.12)4
Wheat gluten meal (72.97/1.06; 1.00)4 40.0
Indoor protocols
Brewers yeast (40.30/0.29; 0.62)5 30.0
Krill hydrolysate (59.38/10.45; 8.30)6 20.0
Four diets were tested with four different feeding regimes and
Soya lecithin, liquid (0.88)7 20.0 two pellet forms as follows:
Marine fish oil, Menhaden (0.96)8 30.0
Cholesterol-FG (60%; potency 64%) (22.00)9 2.34 DFF OI shrimp diet; sinking pellet; fixed ration; day
OI mineral premix LV99.1 (64.75)10 0.6 feeding with feeders.
Potassium phosphate, dibasic 5.6 NFF OI shrimp diet; sinking pellet; fixed ration; night
(17.78% P, 44.9% K; 2.40)11
Calcium phosphate, monobasic 5.6 feeding with feeders.
(26.46% P, 17.12% Ca; 2.40)11 ADF OI shrimp diet; sinking pellet; fixed ration; day and
Sodium phosphate, dibasic (21.82% 5.6 night feeding with feeders.
P, 32.39% Na; 2.40)11
DFS OI shrimp diet; sinking pellet; fed to satiation; day
OI vitamin premix ^ LV99.1 (47.74)12 4.0
Choline chloride (60%; 52% potency) (1.21)13 1.154 feeding by hand.
Vitamin C (35% ascorbic acid potency) (15.00)14 0.714 CSS Commercial shrimp diet; sinking pellet; fed to
1
SSF Sildolje-og Sildemelindustriens Forskningsinstitut, Norway. satiation; day feeding by hand.
2
Agribrands Purina Mexico, S.A. de C.V., Mexico (by courtesy of). CCFS Commercial catfish diet; floating pellet; fed to
3
Land-o-Lakes, Seattle, WA, USA. satiation; day feeding by hand.
4
Hawaii Flour Mills, Honolulu, HI, USA.
5
Williams Bio-Products, Decatur, IL, USA (by courtesy of). CCCS Commercial catfish diet; sinking crumble; fed to
6
Specialty Marine Products,West Vancouver, BC, Canada (by courtesy of). satiation; day feeding by hand.
7
Central Soya Company Inc, Fort Wayne, IN, USA (by courtesy of).
8 Day feedings. four times daily (08.00, 11.00, 14.00,
Omega Protein Inc., Reedville,VA, USA.
9
Solvay Pharmaceuticals B.V.,Veenendaal,The Netherlands. 17.00 hours), Night feedings: four times nightly (20.00,
10
OI mineral premix LV99.1 ^ to supply the following elements (mg kg)1 23.00, 02.00, 05.00 hours), All-day feedings: eight times
diet): zinc (Zn, as sulphate) 72 mg, iron (Fe, as sulphate) 36 mg,
during the day and night (at 08.00, 11.00, 14.00, 17.00,
manganese (Mn, as sulphate) 12 mg, copper (Cu, as sulphate) 24 mg,
cobalt (Co, as chloride) 0.6 mg, iodine (I, as iodate) 1.2 mg, chromium 20.00, 23.00, 02.00, and 05.00 hours) using battery operated
(Cr, trivalent, as chloride) 0.8 mg, selenium (Se, as selenate) 0.2 mg, and Aquarium Fish Feeders (Fish Mate F14, Pet Mate Ltd,
molybdenum (Mo, as molybdate) 0.2 mg. Hersham, Surrey, UK). The fixed feeding ration employed
11
ICN Biomedicals, Inc., Aurora, OH, USA.
12
OI vitamin premix LV99.1 ^ to supply the following vitamins (mg or was based on a shrimp daily feeding guide developed at the
IU kg ^1 diet): thiamine 40 mg, ribo£avin 60 mg, pyridoxine 60 mg, Oceanic Institute (Table 4).
pantothenic acid 180 mg, niacin 80 mg, biotin 0.6 mg, inositol 400 mg, In the case of satiation feeding animals were fed to satiation
folic acid 6 mg, cyanocobalamine 0.10 mg, vitamin A 6000 IU, vitamin D3
four times daily (08.00, 11.00, 14.00, 17.00 hours); latex
2000 IU, vitamin E 250 mg, vitamin K 40 mg, and astaxanthin 60 mg
(premix prepared for OI by Roche V|tamins Inc, Parsippany, NJ, USA, by gloves were used for all feedings and handling of feed. All
courtesy of). experimental animals were weighed individually at bi-weekly
13
Choline 60% ^ to supply 600 mg of active choline kg ^1 diet (Roche
intervals for the duration of the experiment, and feeding rates
V|tamins Inc., Parsippany NJ; by courtesy of).
14
Stay-C 35% ^ to supply 250 mg of active vitamin C kg ^1 diet (Roche adjusted weekly; animals blotted with an absorbent towel and
V|tamins Inc, Parsippany NJ; by courtesy of). weighed on a Mettler Toledo PB 3002 (Mettler-Toledo Inc.,
Hightstown, NJ, USA) electronic balance.

and mixed for a further 15 min. The resulting mash was then Outdoor protocols
passed through a Hobart grinder fitted with a 3-mm diameter
die. The pellet temperature at the die was below 70 C. The Four diets were tested with four different feeding regimes and
resulting moist pellets were then dried overnight in a drying two pellet forms as follows:

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

124 A.G.J. Tacon et al.

Table 3 Chemical composition (g kg^1 dry weight) of the test diets

Diets1
Composition (g kg ^1 as feed basis
except as noted) DFF^DFS CSS CCFS CCCS

Proximate composition
Moisture 61.1 64.6 47.2 46.8
Crude protein (N · 6.25) 351.7 349.5 374.7 376.0
Crude lipid 82.4 91.1 54.3 49.6
Cholesterol 2.7 1.46 0.97 0.97
Ash 62.8 102.8 103.4 104.9
Gross energy (MJ kg ^1) 19.00 18.28 18.45 18.29
Amino acid composition
Aspartic acid 29.74 34.96 37.07 37.07
Serine 15.44 17.62 17.65 17.65
Glutamic acid 69.94 52.49 59.97 59.97
Glycine 17.40 20.32 29.36 29.36
Alanine 18.53 20.83 24.01 24.01
Taurine 1.96 1.12 0.98 0.98
Cystine 4.73 5.79 4.73 4.73
Tyrosine 12.15 (A /E)2 11.20 (A /E) 11.51 (A /E) 11.51 (A /E)
Isoleucine 14.70 (86) 14.63 (85) 12.72 (69) 12.72 (69)
Leucine 31.03 (183) 31.81 (186) 34.18 (185) 34.18 (185)
Methionine 8.64 (79)3 8.28 (82) 7.83 (68) 7.83 (68)
Phenylalanine 14.82 (159)4 14.98 (153) 17.07 (155) 17.07 (155)
Histidine 7.82 (46) 7.33 (43) 10.45 (57) 10.45 (57)
Threonine 13.41 (79) 14.29 (83) 14.72 (80) 14.72 (80)
Lysine 20.51 (121) 19.95 (116) 23.78 (129) 23.78 (129)
Valine 17.25 (101) 18.22 (106) 20.45 (111) 20.45 (111)
Arginine 21.17 (125) 21.39 (125) 23.39 (127) 23.39 (127)
Tryptophan 3.70 (22) 3.34 (19) 3.78 (20) 3.78 (20)
Mineral composition
Phosphorus (P, g kg ^1) 4.912 4.071 6.121 6.121
Potassium (K, g kg ^1) 8.09 4.986 7.186 7.186
Calcium (Ca, g kg ^1) 5.733 19.84 25.23 25.23
Magnesium (Mg, g kg ^1) 1.635 2.385 2.182 2.182
Sodium (Na, g kg ^1) 4.103 2.292 2.544 2.544
Manganese (Mn, mg kg ^1) 32.88 117.1 116.8 116.8
Iron (Fe, mg kg ^1) 135.9 397.3 430.7 430.7
Copper (Cu, mg kg ^1) 27.35 38.65 8.789 8.789
Zinc (Zn, mg kg ^1) 104 138.5 122.8 122.8
Boron (B, mg kg ^1) 1.164 9.252 9.409 9.409
Feed cost5, US$ kg^1 1.04 0.61 0.43 0.43
Fatty acids (% total fatty acids)
C12:0 0.1 0.2 0.1 0.1
C14:0 5.1 8.0 4.6 4.6
C14:1 0.2 0.1 0.2 0.2
C15:0 0.4 0.5 0.4 0.4
C16:0 19.2 20.5 20.3 20.3
C16:1n-7 5.3 9.3 6.1 6.1
C16:2n-4 1 1.4 1 1
C16:3n-4 0.9 1.4 0.9 0.9
C16:4n-1 0.1 nd6 nd nd
C17:0 0.3 0.4 0.4 0.4
C18:0 2.4 3 4.3 4.3
C18:1n-9 15.0 16.4 24 24
C18:1n-7 nd 0.1 nd nd
C18:1n-5 0.2 nd nd nd
C18:2n-6 21.3 16.1 14.2 14.2
C18:2n-4 0.1 0.2 nd nd
C18:3c 0.1 0.2 0.2 0.2
C18:3n-4 0.3 0.4 0.2 0.2

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

 Diet, culture and shrimp growth 125

Table 3 (continued)

Diets1
Composition (g kg ^1 as feed basis
except as noted) DFF^DFS CSS CCFS CCCS

C18:3 n-3 2.5 1.7 1.6 1.6

C18:4 n-3 1.6 1.1 1.1 1.1
C18:4 n-1 0.1 0.2 nd nd
C20:0 0.1 0.1 nd nd
C20:1n-9 2.7 0.7 1.6 1.6
C20:1n-7 0.1 0.1 nd nd
C20:2 n-6 0.1 0.1 0.2 0.2
C20:3 n-6 0.2 0.2 0.3 0.3
C20:3 n-3 0.1 0.1 nd nd
C20:4 n-6 0.5 0.9 0.6 0.6
C20:4 n-3 0.6 0.6 0.9 0.9
C20:5 n-3 6.5 8.0 6.8 6.8
C21:5 n-3 0.1 0.1 0.5 0.5
C22:1n-11 2.2 0.1 1.1 1.1
C22:4 n-6 0.2 0.2 0.6 0.6
C22:5 n-3 1.1 1.4 1.3 1.3
C22:6 n-3 7.6 3.8 5.3 5.3
C23:0 0.3 0.5 0.1 0.1
C24:1 n-9 0.2 nd nd nd
Total n-6 22.3 17.5 15.9 15.9
Total n-3 20.1 16.8 17.5 17.5
Unknown peaks 1.3 2.1 1.0 1.0
1
DFF^DFS = OI shrimp feed; CSS = commercial shrimp feed; CCFS = commercial cat¢sh feed (pelleted-£oating form); CCCS = commercial cat¢sh feed
(crumbled-sinking form).
2
A /E ratio is de¢ned as [(each essential amino acid content/total essential amino acid content including cystine and tyrosine) · 1000].
3
Methionine + cystine.
4
Phenylalanine + tyrosine.
5
Feed costs: OI feed ^ US$ 1.04 kg ^1 diet ingredient costs only, excludes manufacturing costs; all other costs are from Rangen price list, F.O.B. Buhl, Idaho;
FTL: full truck load quantities (5 July 1999).
6
nd = Not detected or value lower than 0.05%.

Table 4 Feeding rates for shrimp in 8-week feeding trials DFFP OI shrimp diet; sinking pellet; fixed ration; day
Percentage of estimated shrimp biomass
feeding by hand; plastic tank cover.
CCFF Commercial catfish diet; floating pellet; fixed ration;
Shrimp
day feeding by hand.
body weight (g) 21^24 C 24^28 C 28^32 C
CCCF Commercial catfish diet; sinking crumble; fixed
1^3 8 6 7
ration; day feeding by hand.
3^5 7 5 6
5^7 6.5 4.5 5.5 CSF Commercial shrimp diet; sinking pellet; fixed ration;
7^9 6 4 5 day feeding by hand.
9^11 5.5 3.5 4.5
11^13 5 3 4 Day feedings. four times daily (08.00, 11.00, 14.00,
13^15 4.5 2.5 3.5 17.00 hours); Night feedings: four times nightly (20.00,
15^17 4 2.5 3 23.00, 02.00, 05.00 hours); All-day feedings: eight times
17^30 3 2 2.5
during the day and night (at 08.00, 11.00, 14.00, 17.00,
20.00, 23.00, 02.00, and 05.00 hours) fed manually by hand
application. The fixed feeding ration employed was based on
DFF OI shrimp diet; sinking pellet; fixed ration; day the shrimp daily feeding guide described above (Table 4);
feeding by hand. allotted daily feed allocations were equally divided into four
NFF OI shrimp diet; sinking pellet; fixed ration; night or eight portions per day as required. Sinking feed was
feeding by hand. applied using a feeding tube directly onto a 0.12-m–2
ADF OI shrimp diet; sinking pellet; fixed ration; day and submersible feeding tray placed 0.6 m below the water
night feeding by hand. surface on one side of the tank. During the last 2 weeks of

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

126 A.G.J. Tacon et al.
the trial the feeding rates were reduced from the highest to
the lowest temperature range feeding regime (Table 4) to
avert the possible crash of the microbial community within
the experimental tanks as a result of the high biomass loading
brought on by the exceptional growth rates observed in some
of the treatments. All experimental animals were weighed
individually at the start and end of the 56-day feeding trial.
At least 10% of the estimated remaining population of each
tank was sampled bi-weekly using a net or minnow trap and
this data was used to adjust feeding rates weekly.
Chemical analyses
Water quality. Routine water quality testing was performed
during each feeding trial. In the indoor trial, water tempera-
ture was measured daily (at about 08.00 hours) within all
experimental tanks using a handheld mercury thermometer.
All other water quality parameters were measured on a
weekly basis, and included pH (using a Model 1001 Sentron
pH meter, Sentron Inc., Gig Harbor, WA, USA), dissolved
oxygen (using a Model 55 Yellow Springs Instrument oxygen
meter), salinity (using a temperature compensated refrac-
tometer, Aquatic Eco-Systems Inc., Apopka, FL, USA), and
total ammonia nitrogen (TAN) determined by the automated
analysis method of Solorzano (1969) using a Technicon
Auto-Analyzer II. In the outdoor feeding trial, water
temperature and dissolved oxygen were measured twice daily
(at about 08.00 and 16.00 hours), and pH, salinity and TAN
twice weekly (Monday and Thursday at 13.00 hours), as
described above. In addition, nutrient analyses were per-
formed twice weekly, including chlorophyll a [following the
method of Strickland & Parsons (1972) using a Turner
Designs Fluorometer], total phosphorus and orthophosphate
(total phosphorus – Grasshoff et al. 1983; orthophosphate –
Murphy & Riley 1962), total nitrogen (D’Elia et al. 1977),
and nitrate and nitrite using a Technicon Auto-Analyzer II
(Nitrate–Nitrite in water and seawater; Industrial method no.
158-71 W, December 1972; Technicon Industry Systems,
Tarrytown, NY, USA).
Diets, shrimp tissue and suspended particulate matter. Shrimp
were collected at the start of the experiments (from a
representative population sample) and from each tank at the
end of the feeding trials (10 shrimp per indoor and outdoor
tank) and frozen for subsequent analysis on an individual
tank basis. In the case of the large animals harvested at the
end of the feeding trials, shrimp were macerated, freeze-dried
to a constant weight, and then ground prior to chemical
analysis. Samples of the suspended particulate matter (SPM)
present within the water column of the outdoor microcosm
tanks were collected at the end of the 8-week feeding trial. A
stirred water sample (8 L) was collected from each tank and
vacuum filtered through Whatman No. 1 hardened filter
paper using a 20-cm Buchner funnel, and the filtrate then
freeze-dried to constant weight using a Freezemobile 12
freeze-drier (Virtis Inc., Gardiner, NY, USA). Chemical
analyses, including moisture, total nitrogen, crude lipid, and
ash, were conducted in duplicate as described previously
(Divakaran 1999). The gross caloric content of experimental
diets and SPM were determined using a Parr 1261 Isoperibol
Bomb Calorimeter (Parr Instrument Co, St Moline, IL,
USA) using benzoic acid as the standard. Mineral analysis of
diets, shrimp tissue, and SPM was undertaken by Inductively
Coupled Plasma Atomic Emission Spectroscopy using a
Model Atomscan 16 radial configuration instrument (Ther-
mo Jarrel Ash, TJA Solutions, Franklin, MA, USA), after
first ashing the samples at 600 C for 6 h and then dissolving
the ash in 3 N HCl prior to analysis (AOAC 1990a). Amino
acids in freeze-dried SPM and shrimp tissue were analysed
using a Beckman System 6300 Amino Acid Analyzer
following hydrolysis in 6 N HCl for 20 h at 115 C (using
norleucine as an internal standard) following the method of
Hamilton (1963). For cystine/2 analysis, samples were
oxidized at 50 C for 15 min with performic acid prior to
hydrolysis, following the method of Hirs (1967). For tryp-
tophan analysis, samples were hydrolysed in 4.2 N NaOH at
135 C for 48 h prior to neutralization and analysis (Hugli &
Moore 1972). The crude lipid content of freeze-dried SPM
was determined using the method of Hara & Radin (1978)
with the following modifications: samples were homogenized
with a solution of 0.01 M MgCl2 and extracted with a
chloroform:isopropanol 2:1 (v/v) mixture and 1 M HCl. The
homogenate was then rinsed with the solvent mixture and
centrifuged to recover the organic layer. The organic layer
was then washed with 0.3 M HCl, and lipid was recovered by
drying over a stream of nitrogen. Fatty acid analysis of
experimental diets and freeze-dried SPM samples was un-
dertaken by a modified direct methylation method (AOAC
1990b) using a Hewlett Packard 5890 Gas Chromatograph
(Hewlett-Packard Co., Palo Alto, CA, USA) with a Flame
Ionization Detector.
Calculations and statistics
Shrimp growth was measured by mean weight gain, weekly
weight gain, and specific growth rate. Feed efficiency was
calculated as the mean weight gain divided by the amount of
diet fed. Nitrogen and phosphorus efficiency were calculated
..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137
 Diet, culture and shrimp growth 127

in the outdoor trial as the accumulation of these elements in
the shrimp whole body as a proportion of the total amount
presented in the diets over the course of the trial. In the
outdoor system, the efficiency parameters (feed, nitrogen and
phosphorus) are referred to as ‘apparent’ efficiency and are of
more practical than biological significance, because actual
consumption of the diets could not be monitored, nor could
the impact of cannibalism and consumption of natural food
production be directly assessed.
Data obtained from the experiments, which had a com-
pletely randomized design with three replicates per treat-
ment, were analysed by one-way analysis of variance to
determine if significant differences existed among treatment
means (Snedecor & Cochran 1967). Arcsin transformation
[sin–1(x0.5)] was applied to the data prior to analysis. Tukey’s
test for mean separation was used to evaluate differences
among treatment means. All statistical analyses were per-
formed in SigmaStat version 2.03 (SPSS Inc., Chicago, IL,
USA – 1997). Differences were considered significant at the
5% level of probability.
animals exhibited a final body weight 13.3 and 12.2%
higher than those animals fed during the day-light and
night-hours, respectively, and 24-h fed animals displayed a
final body weight 16.1% higher than animals fed with the
same diet fed to satiation four times daily (Table 5).
Moreover, there was no significant difference in the growth
of shrimp fed during daylight hours or during the night-
hours. However, survival and feed efficiency were lowest
among shrimp in the treatments fed during night-hours.
Although the overall growth response and final body weight
of shrimp fed with the control OI shrimp feed (DFS) were
higher than those of animals fed with the commercial
shrimp diet (CSS), these differences were not significant. The
poorer growth response and performance of the commercial
shrimp diet (CSS) corresponded to the lower mean volun-
tary feed intake of animals fed with this ration compared
with the OI shrimp diet (DFS). Animals rapidly (within a
few minutes) learned to swim to the water surface to
consume the floating pelleted catfish feed (CCFS), and grew
as well as animals that were with fed the same diet in
crumbled-sinking form.

Results
Indoor feeding trial
Shrimp growth (expressed in terms of final body weight and
weekly growth) was highest in those treatments which fed
eight times daily over a 24-h feeding period; 24-h fed
Outdoor feeding trial
As with the indoor feeding trial, shrimp growth (expressed in
terms of final body weight and weekly growth) was highest in
those treatments fed eight times daily over a 24-h feeding

Table 5 Shrimp growth and feed performance in an indoor flow-through culture system over the 8-week experimental period. Values within a
row sharing a common superscript are not significantly different (Tukey’s test; P < 0.05; n = 3)

Dietary treatment1 DFF NFF ADF DFS CSS CCFS CCCS SEM2

Shrimp weight
Mean initial body weight (g) 1.61a 1.61a 1.59a 1.59a 1.60a 1.57a 1.60a 0.02
Mean final body weight (g) 5.92ab 5.98ab 6.71a 5.78b 5.17b 3.66c 3.67c 0.18
Shrimp feed intake
Mean feed intake (g shrimp ^1 day ^1) 0.21 0.24 0.22 0.17 0.14 0.12 0.12
Shrimp growth response
Total weight gain (%) 268.4ab 272.6ab 323.8a 264.5ab 223.7b 133.4c 130.2c 13.0
Mean weekly weight gain (g week ^1) 0.54ab 0.55ab 0.64a 0.52ab 0.45b 0.26c 0.26c 0.02
Specific growth rate (% day ^1)3 2.33ab 2.34ab 2.57a 2.31ab 2.10b 1.51c 1.48c 0.07
Shrimp feed utilization
Feed efficiency (%)4 35.8ab 28.9bc 40.4a 42.4a 45.6a 26.1bc 21.8c 0.13
Total shrimp production
Shrimp survival (%) 93.1a 77.8a 93.1a 86.1a 92.7a 79.2a 72.2a 4.5
1
DFF = OI shrimp diet; sinking pellet; ¢xed ration; day feeding with feeders; NFF = OI shrimp diet; sinking pellet; ¢xed ration; night feeding with
feeders; ADF = OI shrimp diet; sinking pellet; ¢xed ration; day and night feeding with feeders; DFS = OI shrimp diet; sinking pellet; fed to satiation;
day feeding by hand; CSS = commercial shrimp diet; sinking pellet; fed to satiation; day feeding by hand; CCFS = commercial cat¢sh diet; £oating
pellet; fed to satiation; day feeding by hand; CCCS = commercial cat¢sh diet; sinking crumble; fed to satiation; day feeding by hand.
2
Standard error of means.
3
Speci¢c growth rate = [loge¢nal body weight (g) ) logeinitial body weight (g)]/time (days) · 100.
4
Feed e⁄ciency = [¢nal shrimp biomass (g) ) initial shrimp biomass (g)] · 100/total feed o¡ered (g, as fed basis).

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

128 A.G.J. Tacon et al.

Table 6 Shrimp growth and feed performance in an outdoor zero-water-exchange culture system over the 8-week experimental period. Values
in a row sharing a common superscript are not significantly different (Tukey’s test; P < 0.05; n = 3)

Dietary treatment1 DFF NFF ADF DFFP CCFF CCCF CSF SEM2

Shrimp weight
Mean initial body weight (g) 1.60a 1.56a 1.58a 1.59a 1.60a 1.60a 1.57a 0.03
Mean final body weight (g) 17.20a 17.34a 18.89a 16.78a 11.35b 10.46b 12.94b 0.68
Shrimp feed intake
Mean feed offered (g shrimp)1 day)1) 0.56 0.44 0.53 0.54 1.76 0.54 0.55
Shrimp growth response
Total weight gain (%) 977.4ab 1015.0a 1098.2a 956.6ab 609.5c 554.7c 728.0bc 52.1
Mean weekly weight gain (g week)1) 1.95a 1.97a 2.16a 1.90a 1.22b 1.11b 1.42b 0.09
Specific growth rate (% day)1) 4.24a 4.30a 4.43a 4.20ab 3.50c 3.35c 3.76bc 0.10
Shrimp feed utilization
Apparent feed efficiency (%)3 49.7ab 63.1a 57.7a 47.1ab 3.5c 25.6bc 34.1ab 6.17
Apparent feed nitrogen efficiency (%)4 28.45ab 35.05a 32.01a 26.61ab 1.79c 13.01bc 18.99ab 3.46
Apparent feed phosphorus efficiency (%)5 33.44ab 46.56a 39.42ab 31.19abc 2.06d 12.05cd 23.93bc 4.14
Total shrimp production
Total initial shrimp biomass (kg m)3) 0.11a 0.10a 0.11a 0.11a 0.11a 0.11a 0.10a 0.01
Total final shrimp biomass (kg m)3) 0.75abc 0.92a 0.89ab 0.68abc 0.14 0.39c 0.50bc 0.08
Total biomass increase (kg m)3) 0.64abc 0.82a 0.78ab 0.57abc 0.04 0.29c 0.40bc 0.08
Total feed offered (kg) (as fed basis) 1.93 1.94 2.03 1.82 1.58 1.67 1.76
Shrimp survival (%) 64.7a 79.3a 69.7a 61.0a 19.0b 56.7a 58.3ab 6.2
)1
Feed cost kg shrimp production ($) 2.29 1.67 1.93 2.20 46.74 1.71 1.87
1
DFF = OI shrimp diet; sinking pellet; ¢xed ration; day feeding by hand; NFF = OI shrimp diet; sinking pellet; ¢xed ration; night feeding by hand;
ADF = OI shrimp diet; sinking pellet; ¢xed ration; day and night feeding by hand; DFFP = OI shrimp diet; sinking pellet; ¢xed ration; day hand
feeding; plastic tank cover; CCFF = commercial cat¢sh diet; £oating pellet; ¢xed ration; day feeding by hand; CCCF = commercial cat¢sh diet; sinking
crumble; ¢xed ration; day feeding by hand; CSF = commercial shrimp diet; sinking pellet; ¢xed ration; day feeding by hand.
2
Standard error of means.
3
Apparent feed e⁄ciency = [¢nal shrimp biomass (g) ^ initial shrimp biomass (g)] · 100/total feed o¡ered (g, as feed basis); value excludes the
consumption of natural food organisms present within the culture system.
4
Apparent feed nitrogen e⁄ciency = whole body nitrogen gain · 100/shrimp feed nitrogen o¡ered.
5
Apparent feed phosphorus e⁄ciency = whole body phosphorus gain · 100/shrimp feed phosphorus o¡ered.

period (Table 6; Figs 1 & 2). Animals fed over a 24-h period
exhibited a final body weight 9.8 and 8.9% higher than
animals fed during the daylight and night-hours, respectively,
but these differences were not statistically significant. More-
over, there were no significant differences in the growth, feed
efficiency or survival of shrimp fed during daylight hours or
during the night-hours.
Although shrimp growth and body weight were satisfac-
tory in those tanks with plastic covers and significantly
higher than the commercial control, water temperatures were
high, ranging from a low of 25.6 C (AM) to a high of 35.1 C
(PM) during the experiment – mean 29.6–32.6 C, and were
clearly in excess of or close to the reported lethal tempera-
tures for shrimp. In contrast, tanks without covers displayed
a range of 25.3 C (AM) to a high of 34.0 C (PM) – mean
28.2–31.3 C. Despite this, dissolved oxygen concentrations
in the covered tanks were generally satisfactory, ranging
from 4.5 to 7.1 mg L–1. However, the use of plastic covered
tanks during the colder winter months may be more
beneficial (the current trial was conducted during the hotter
summer months).
Shrimp fed with the OI shrimp diet had significantly higher
final body weight and growth rate than shrimp fed with the
commercial control diet (30–46% higher), although both diet
series had similar proximate composition (35% crude
protein, 9% lipid) and both diets contained 2.5% squid
meal. Calculated feed costs per kg of shrimp production
ranged from $1.87 for the commercial shrimp feed, to $1.67
for the night-time OI feed, to $1.93 for the all-day OI feed
(shrimp within this treatment were 46% larger by weight
than the commercial shrimp feed; Table 6).
The growth, feed efficiency and survival of shrimp fed with
the floating catfish feed were very low, primarily because the
animals were not aware that feed was being administered (the
feed eventually sank), and growth was equivalent to the
crumbled (sinking) version of the same diet. Interestingly,
there were no significant differences between the growth of
the shrimp in the three commercial feed treatments (Table 6).
Dietary mineral concentration had little effect on the final
tissue mineral concentrations of the experimental shrimp
(Table 8), with tissue iron and copper concentrations actually
decreasing over the 8-week period. This was particularly

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137


24
DFF
22 NFF
ADF
DFFP
20 CCFF
CCCF
18 CSF
Shrimp weight (g)
16
14
12
10
8 Within all the microcosm tanks there was a rapid
6 development of a microbial food chain, initially in the visible
4
2 developing later; the latter was visible in the water column as
0 suspended particulate matter or ‘microbial floc’ (‘floc’).
0 2 4 6
Week
Figure 1 Growth response of outdoor shrimp fed with the experi-
mental diets for 8 weeks (mean weight ± SD; n ¼ 3). DFF ¼ OI
shrimp diet; sinking pellet; fixed ration; day feeding by hand;
NFF ¼ OI shrimp diet; sinking pellet; fixed ration; night feeding by
hand; ADF ¼ OI shrimp diet; sinking pellet; fixed ration; day and
night feeding by hand; DFFP ¼ OI shrimp diet; sinking pellet; fixed
ration; day hand feeding; plastic tank cover; CCFF ¼ Commercial
catfish diet; floating pellet; fixed ration; day feeding by hand;
CCCF ¼ Commercial catfish diet; sinking crumble; fixed ration; day
feeding by hand; CSF ¼ Commercial shrimp diet; sinking pellet;
fixed ration; day feeding by hand.
unexpected, considering the initial differences between
dietary treatments in terms of the mineral composition of
the diets fed (the OI diet had reduced levels of calcium,
magnesium, iron and manganese compared with the com-
mercial shrimp and fish rations tested; Table 3). However,
shrimp fed exclusively during night-hours had significantly
lower carcass moisture content and elevated carcass zinc and
phosphorus content than those fed with the commercially
prepared feeds (Tables 7 and 8).
Despite the high feed intake (Fig. 3) and growth (Fig. 4) of
shrimp during the first 6 weeks of the experiment, there was a
progressive reduction in the growth response of shrimp
during the final 2 weeks of the experiment. This correlated
with the progressively deteriorating water quality (elevated
ammonia and nitrite and reduced pH) within all experimental
tanks (Fig. 5) and the consequent need to reduce the daily
feeding rates of all treatments from the higher 28–32 C
feeding rate range to the lower 21–24 C feeding rate range
(Table 4) to avoid tank or system crashes. Mean levels of
TAN were initially low, peaking on day 42 at an average of
..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137
Diet, culture and shrimp growth 129
7.4 mg L–1 (range 6–9 mg L–1). Nitrite values in the system
were below 0.1 mg L–1 until day 43. From day 43 onwards,
nitrite increased concurrently with the decrease in TAN,
reaching a level of 20 mg L–1 by the end of the experiment
(Fig. 5). Total nitrogen and total phosphorus accumulated
steadily within the experimental tanks over the course of the
study, from 2 to 30 mg L–1 and 0.2 to 16 mg L–1, respect-
ively. By contrast, the pH of the system decreased progres-
sively, from a high of 8.4 near the beginning of the
experiment to a low of 7.1 by the end of the study (Fig. 5).
form of a green algal-based autotrophic microbial food web,
with a bacterial-based heterotrophic microbial food web
8
Phytoplankton biomass, as indicated by chlorophyll a,
peaked between day 25 and 32 at an average of 350 lg L–1
(range 200–580 lg L–1). After the peak, chlorophyll a values
declined slightly, averaging between 250 and 300 lg L–1 until
the end of the experiment (Fig. 5).
Chemical analysis of the ‘floc’ taken at the end of the trial
revealed a valuable potential food source for the resident
shrimp. As shown in Tables 9 and 10, ‘floc’ was a valuable
dietary source of amino acids, fatty acids, and minerals. Each
tank was calculated to contain 87–200 mg L–1 of ‘floc’ by the
end of the experiment, or the equivalent of 122–280 g of dry
‘floc’ per 1.40 m3 of water (mean 201 g; Table 9).
Discussion
The observation that in the indoor trial, the shrimp fed
during the day grew as well as, and had better feed efficiency
and survival than, those fed at night, is in agreement with
those of Robertson et al. (1993), who found that day feeding
was as good as, or slightly better than, night feeding in terms
of shrimp (L. vannamei) growth. In the outdoor trial, the
higher feed efficiency and survival among the shrimp fed
during the night compared to day time feeding is in
agreement with the findings of Nunes et al. (1996), who
found no significant difference between diurnal and noctur-
nal food consumption patterns of Farfantepenaeus subtilis,
with animals displaying continuous feeding activity during
day and night. However, Velasco et al. (1999) reported no
beneficial effect of increasing feeding frequency or ration size
on the growth or survival of shrimp (L. vannamei) post larvae
(185 mg body weight) fed with a diet containing 19.5% crude
protein within an experimental zero-water-exchange culture
system.
130 A.G.J. Tacon et al.

Figure 2 Histogram of mean shrimp body weight (g/shrimp) at the end of the outdoor feeding trial. DFF ¼ OI shrimp diet; sinking pellet; fixed
ration; day feeding by hand; NFF ¼ OI shrimp diet; sinking pellet; fixed ration; night feeding by hand; ADF ¼ OI shrimp diet; sinking pellet;
fixed ration; day and night feeding by hand; DFFP ¼ OI shrimp diet; sinking pellet; fixed ration; day hand feeding; plastic tank cover;
CCFF ¼ Commercial catfish diet; floating pellet; fixed ration; day feeding by hand; CCCF ¼ Commercial catfish diet; sinking crumble; fixed
ration; day feeding by hand; CSF ¼ Commercial shrimp diet; sinking pellet; fixed ration; day feeding by hand.

Shrimp fed with the floating pellets indoors had growth
similar to that of shrimp fed with the sinking crumbled feed.
In this trial, a feed designed for catfish was used. If a floating
feed specifically formulated for shrimp was prepared for use
in clear water conditions (where the shrimp can readily sense
the presence of feed particles), perhaps growth rates would
have markedly improved. Apart from the obvious nutritional
benefits of improved carbohydrate digestibility and water
stability, the use of a floating shrimp feed within these
intensive clear water systems would allow the culturist to
more accurately judge the correct amount of feed by
observing the animals feeding at first hand, instead of relying
on feeding tables.
The best overall shrimp growth performance was observed
in animals fed with the OI shrimp diet and all-day feeding
regime under outdoor zero-water exchange culture condi-
tions; final body weight and average weekly growth rate were
2.8 and 3.4 times greater, respectively, than animals of similar

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

 Diet, culture and shrimp growth 131

Table 7 Proximate composition of shrimp carcass (whole body) at the beginning and end of the outdoor, zero-water-exchange 8-week
experiment. Crude protein, crude lipid, ash, and NFE are reported on a shrimp live weight basis. Values within a column that share a common
superscript are not significantly different (Tukey’s test; P < 0.05; n = 3 except initial n = 1)

Treatment1 Moisture (%) Crude protein (%) Crude lipid (%) Ash (%) NFE2 (%)

Initial 75.88 17.41 1.86 2.64 2.22

DFF 74.54ab 19.82a 1.63a 2.62a 1.39
NFF 73.89b 19.19a 1.49a 2.88a 2.61
ADF 74.73ab 19.41a 1.41a 2.69a 1.76
DFFP 74.58ab 19.48a 1.67a 2.61a 1.66
CCFF 76.06a 18.49a 1.51a 2.50a 1.44
CCCF 75.29ab 19.00a 1.53a 2.64a 1.54
CSF 76.65a 17.86a 1.47a 2.51a 1.51
SEM3 0.447 0.406 0.116 0.112
1
DFF = OI shrimp diet; sinking pellet; ¢xed ration; day feeding by hand; NFF = OI shrimp diet; sinking pellet; ¢xed ration; night feeding by hand;
ADF = OI shrimp diet; sinking pellet; ¢xed ration; day and night feeding by hand; DFFP = OI shrimp diet; sinking pellet; ¢xed ration; day hand
feeding; plastic tank cover; CCFF = commercial cat¢sh diet; £oating pellet; ¢xed ration; day feeding by hand; CCCF = commercial cat¢sh diet; sinking
crumble; ¢xed ration; day feeding by hand; CSF = commercial shrimp diet; sinking pellet; ¢xed ration; day feeding by hand.
2
Nitrogen free extract calculated by di¡erence (100% ^ all other components). Includes carbohydrate and chitin.
3
Standard error of the means.

Table 8 Mineral composition of shrimp carcass (whole body) at the beginning and end of the 8-week experiment. Values within a column that
share a common superscript are not significantly different (Tukey’s test; P < 0.05; n = 3 except initial n = 1)

P K Ca Mg Na Mn Fe Cu Zn B
Treatment1 (g kg ^1) (g kg ^1) (g kg ^1) (g kg ^1) (g kg ^1) (mg kg ^1) (mg kg ^1) (mg kg ^1) (mg kg ^1) (mg kg ^1)

Initial 2.50 2.16 6.40 0.73 1.72 1.031 107.302 28.485 16.715 2.116
DFF 3.20ab 2.39a 6.11a 0.75ab 1.65a 0.797a 15.687a 15.091b 18.358bc 1.933a
NFF 3.50a 2.43a 7.05a 0.89a 1.94a 0.906a 16.247a 18.300ab 21.522a 2.218a
ADF 3.24ab 2.31a 6.66a 0.76ab 1.74a 0.916a 22.445a 19.888ab 19.093ab 1.870a
DFFP 3.13bc 2.49a 6.04a 0.80ab 1.72a 0.815a 15.916a 17.263ab 18.161bc 1.649a
CCFF 2.79bc 2.43a 5.92a 0.72ab 1.67a 0.979a 15.004a 16.426ab 17.724bc 2.087a
CCCF 2.78bc 2.40a 6.55a 0.73ab 1.59a 1.259a 23.176a 24.776a 17.776bc 1.787a
CSF 2.78bc 2.58a 5.81a 0.70b 1.76a 1.160a 21.652a 11.641b 15.837c 1.819a
SEM2 0.0787 0.141 0.60 0.040 0.13 0.0148 6.138 1.892 0.6162 0.1433
1
DFF = OI shrimp diet; sinking pellet; ¢xed ration; day feeding by hand; NFF = OI shrimp diet; sinking pellet; ¢xed ration; night feeding by hand;
ADF = OI shrimp diet; sinking pellet; ¢xed ration; day and night feeding by hand; DFFP = OI shrimp diet; sinking pellet; ¢xed ration; day hand
feeding; plastic tank cover; CCFF = commercial cat¢sh diet; £oating pellet; ¢xed ration; day feeding by hand; CCCF = commercial cat¢sh diet; sinking
crumble; ¢xed ration; day feeding by hand; CSF = commercial shrimp diet; sinking pellet; ¢xed ration; day feeding by hand.
2
Standard error of the means.

initial size fed with the same diet under indoor running-water
culture conditions. Although direct comparison between
experiments is not possible because of the lower indoor water
temperatures (26–27 C compared with 28–31 C) and lower
mean daily feed intake of animals (0.22 g shrimp–1 compared
with 0.53 g shrimp–1), it is believed that the higher growth (as
much as three times higher) and performance of animals
reared under outdoor ‘green-water’ culture conditions is due
to their culture environment and ability to obtain additional
nutrients from natural food organisms present within the
water column and/or pond ecosystem (Leber & Pruder 1988;
Moss 1995; Tacon 1996; Moriarty 1997).
It is important to highlight here that outdoor zero-water
exchange culture systems are completely closed farming
systems, with no water exchange for the duration of the
culture cycle other than that added to the system to make up
for evaporative losses. Moreover, shrimp growth is achieved
through the simultaneous consumption of both exogenously
supplied compound aquafeeds (the OI shrimp diet in this
case), and endogenously produced living microbial feeds or
‘microbial floc’ (‘floc’), which is a complex mixture of micro-
organisms and invertebrates. For example, the biological
diversity of the microbial food web within the microcosm
tanks is evidenced by the presence of not only bacteria and
algae (including diatoms), but also flagellates, ciliates,
amoebae, rotifers, nematodes, and gastrotrichs. It is inter-
esting to note the similarity between the organisms associated
with the macro-aggregates or ‘flocs’ from the microcosm

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

132 A.G.J. Tacon et al.

Figure 3 Mean daily feed application in

the outdoor trial. DFF ¼ OI shrimp
diet; sinking pellet; fixed ration; day
feeding by hand; NFF ¼ OI shrimp diet;
sinking pellet; fixed ration; night feeding
by hand; ADF ¼ OI shrimp diet; sink-
ing pellet; fixed ration; day and night
feeding by hand; DFFP ¼ OI shrimp
diet; sinking pellet; fixed ration; day
hand feeding; plastic tank cover;
CCFF ¼ Commercial catfish diet; float-
ing pellet; fixed ration; day feeding by
hand; CCCF ¼ Commercial catfish diet;
sinking crumble; fixed ration; day feed-
ing by hand; CSF ¼ Commercial shrimp
diet; sinking pellet; fixed ration; day
feeding by hand.

3.5
Week 0 2 Week 2 4

3.0 Week 4 6 Week 6 8 Figure 4 Mean weekly shrimp growth

(g week–1) in the outdoor trial.
2.5 DFF ¼ OI shrimp diet; sinking pellet;
fixed ration; day feeding by hand;
Weekly growth (g)

NFF ¼ OI shrimp diet; sinking pellet;

2.0
fixed ration; night feeding by hand;
ADF ¼ OI shrimp diet; sinking pellet;
1.5 fixed ration; day and night feeding by
hand; DFFP ¼ OI shrimp diet; sinking
1.0 pellet; fixed ration; day hand feeding;
plastic tank cover; CCFF ¼ Commercial
catfish diet; floating pellet; fixed ration;
0.5
day feeding by hand; CCCF ¼ Com-
Commercial catfish diet; sinking crum-
0.0 ble; fixed ration; day feeding by hand;
DFF NFF ADF DFFP CCFF CCCF CSF CSF ¼ Commercial shrimp diet; sinking
Treatment pellet; fixed ration; day feeding by hand.

tanks and those normally encountered on marine and lake
snow, and on ‘floc’ from activated sludge (Curds 1992). The
fundamental difference between this culture system and the
traditional ‘open’ or running-water pond-based shrimp
culture system is that the culture target is changed from a
single-stomached animal (the shrimp), where micro-organ-
isms generally play a limited (although important) role in
digestion and nutrient supply, to the equivalent of a
multistomached animal through the provision of an in situ
microbial aerobic digester or bioreactor (the microcosm),
where micro-organisms play a major role in digestion and
nutrient supply, as they do in ruminants (Tacon et al. 1999).
Indeed, recent studies with shrimp (L. vannamei) within zero-
water exchange culture systems have shown the nonessen-
tiality of dietary vitamin and trace mineral supplementation
within exogenously supplied compound aquafeeds (A.G.J.
Tacon, unpublished data; Velasco & Lawrence 2000) and the
ability of totally replacing fishmeal in prepared feeds with
rendered terrestrial animal by-product meals with little or no
loss in growth and feed efficiency (Tacon 2000). Of course,
this ‘floc’ also has important functions in removing and
harnessing potentially toxic faecal wastes and metabolites
(e.g. by nitrification) from the shrimp within the culture
system.

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137


Figure 5 Changes in water quality
within the outdoor microcosm tanks
over the course of the 56-day experi-
mental test period. TN ¼ total nitrogen;
Chl a ¼ Chlorophyll a; NO2 ¼ nitrite;
TP ¼ total phosphorus; TAN ¼ total
ammonia nitrogen.
It is also important to mention here the rapid growth rates
observed for shrimp reared within the outdoor zero-water
exchange culture systems, and in particular for those animals
fed with the OI shrimp diet and all-day feeding regime;
shrimp displayed an average weekly growth rate of
2.16 g week–1, increasing from 1.58 to 18.89 g (market size)
in only 8 weeks (shrimp stocking density 71 m–3 water
volume, Table 6). This growth rate was more than twice
that reported for shrimp (L. vannamei) reared under com-
mercial conditions within intensive zero-water exchange
pond-based culture systems [0.8–1.0 g week–1 at 112–128
animals m–2 or equivalent to 60–69 animals m–3 water
volume (McIntosh & Carpenter 1999)]. Growth rates as high
as 2.7 g week–1 have been reported for juvenile shrimp
(L. vannamei) reared within outdoor microcosm tanks and
fed with a high-quality shrimp diet (containing 52% crude
protein) over an 18-day experimental test period (Freeman &
Duerr 1991). It is interesting to note that during the present
outdoor feeding trial, average weekly growth rates peaked at
3.2 g week–1 in one treatment (Fig. 4).
As stated above, an important factor contributing to the
very high growth rates of shrimp within these zero-exchange
culture systems was likely the endogenous production and
availability of microbial food organisms (‘floc’) for the
resident shrimp. Not surprisingly, nutritional analysis of
the ‘floc’ collected from the experimental tanks at the end of
the feeding trial revealed a composition and nutrient profile
comparable with that of similar ‘flocs’ harvested from
domestic waste water ‘activated sludge’ treatment facilities
(Tacon & Ferns 1978/1979; Tacon 1978/1979). In Tahiti,
experiments were reportedly run utilizing domestic activated
sludge as an inoculum for experimental tanks (AQUACOP,
personal communication). Of particular note was the fact
..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137
Diet, culture and shrimp growth 133
that amino acids constituted over 25% of the ‘floc’ by weight;
compared with the estimated dietary amino acid requirement
profile of shrimp (L. vannamei), the ‘floc’ provided a rich
source of threonine, valine, isoleucine and phenylalanine
(plus tyrosine), although it was deficient in lysine, histidine,
and to a lesser extent, arginine and tryptophan (Table 9).
Lipids constituted only 2.6% of the ‘floc’ by weight, and
fatty acid analyses revealed modest quantities [albeit rather
low relative to levels found in the diets (Table 3)] of n-6 and
n-3 polyunsaturated fatty acids, and in particular the highly
unsaturated fatty acids arachidonic acid (1.65%), eicosapen-
taenoic acid (3.0%), and docosahexaenoic acid (1.35%;
Table 10). The high proportion of unknown peaks (16.3% of
total fatty acids) was probably related to the high number of
branched or odd carbon number fatty acids commonly
present in bacteria (Kharlamenko et al. 1999); the richness of
the ‘floc’ in 16:0, 16:1n-7 and 18:1n-7 fatty acids was similar
to that reported for bacterial-based microbial communities
from biological phosphate removal systems (Liu et al. 2000).
Interestingly, 18:1n-7 (usually present at high levels in
bacteria) was found to be present in the ‘floc’, but was not
present in the experimental test diets (Table 2).
The high ash content of the ‘floc’ was similar to that
reported for ‘activated sludge’ and probably related to the
presence of considerable amounts of acid-insoluble oxides
and mixed silicates (Tacon & Ferns 1978/1979). Despite
having relatively high sodium content (because of the
seawater environment), the ‘floc’ is a good source of essential
minerals and trace elements (Table 9). Moreover, apart from
serving as a direct source of nutrients to the shrimp, there is
evidence that these organisms also exert a positive effect on
the shrimp digestive enzyme activity and gut microflora
(Moss et al. 2001b).
134 A.G.J. Tacon et al.

Table 9 Composition of suspended particulate matter or ‘microbial floc’ (‘floc’) collected from the outdoor shrimp rearing tanks. Values are
ranges and means (freeze-dried basis)

‘Floc’ Diet1 Shrimp2

Range Mean AA /RAA (%) AA /RAA (%) AA /RAA (%)

Nutrient
Suspended microbial floc (mg L ^1) 87.3^200.8 157
Moisture (g kg ^1) 58.6^73.1 66
Crude protein (N · 6.25) (g kg ^1) 292.0^343.3 312
Crude lipid (g kg ^1) 25.7^26.3 26.0
Cholesterol (g kg ^1) 0.47^0.49 0.48
Ash (g kg ^1) 255.5^318.1 282
Gross energy (MJ kg ^1) 10.3^12.8 12
Phosphorus (P) (g kg ^1) 3.6^21.2 13.5
Potassium (K) (g kg ^1) 1.3^8.9 6.4
Calcium (Ca) (g kg ^1) 5.6^28.6 17
Magnesium (Mg), (g kg ^1) 1.2^4.5 2.6
Sodium (Na) (g kg ^1) 4.1^43.1 27.5
Manganese (Mn) (mg kg ^1) 8.9^46.8 28.5
Iron (Fe) (mg kg ^1) 170.8^521.0 320
Copper (Cu) (mg kg ^1) 3.8^88.6 22.8
Zinc (Zn) (mg kg ^1) 78.3^577.9 338
Boron (B) (mg kg ^1) 8.8^45.7 27.3
Amino acid (g kg^1)
Aspartic acid 30.3^31.1 31.1 12.25 9.20 9.85
Serine 12.7^13.8 13.2 5.21 4.78 4.13
Glutamic acid 31.7^34.3 33.0 13.01 21.66 14.67
Proline 12.1^12.8 12.5 4.91 Not analysed 6.76
Glycine 16.8^17.6 17.2 6.78 5.39 8.04
Alanine 17.6^19.4 18.5 7.31 5.74 5.60
Taurine 0.34^0.36 0.35 0.14 0.61 0.75
Cystine 3.9^4.1 4.0 1.57 1.46 1.03
Tyrosine 9.9^10.1 10.0 3.93 (A /E)3 3.76 (A /E) 4.13 (A /E)
Isoleucine 12.1^12.6 12.4 4.88 (97) 4.55 (86) 4.13 (82)
Leucine 17.8^19.7 18.7 7.39 (146) 9.61 (183) 7.13 (142)
Methionine 4.7^5.2 4.9 1.94 (70)4 2.67 (79) 2.13 (63)
Phenylalanine 14.2^15.3 14.8 5.83 (194)5 4.59 (159) 4.97(181)
Histidine 4.3^4.5 4.4 1.73 (34) 2.42 (46) 2.16 (43)
Threonine 14.4^15.0 14.7 5.79 (115) 4.15 (79) 4.00 (80)
Lysine 9.0^9.6 9.3 3.66 (73) 6.35 (121) 5.35 (107)
Valine 16.6^18.0 17.3 6.82 (135) 5.34 (101) 4.57 (91)
Arginine 14.6^16.3 15.4 6.08 (120) 6.55 (125) 9.70 (193)
Tryptophan 1.8^2.2 2.0 0.77 (16) 1.15 (22) 0.91 (18)
Total amino acids (RAA) 245^263 254 100 100 100
E/NE ratio6 50.4:49.6 52.6:47.47 50.2:49.8
1
OI control shrimp diet (Table 2).
2
Estimated dietary amino acid requirement pro¢le of shrimp (Litopenaeus vannamei) calculated according to the method of Ogino (1980) andTacon &
Cowey (1985) upon the daily deposition of amino acids in whole body protein of rapidly growing shrimp (data calculated for outdoor shrimp fed the OI
shrimp diet and all day feeding regime, with animals growing from an initial body weight of1.58 g (whole body tissue containing alanine 0.97%, arginine
1.42%, asparagine 1.70%, cystine 0.16%, glutamic acid 2.58%, glycine 1.20%, histidine 0.39%, isoleucine 0.70%, leucine 1.24%, lysine 0.90%, methionine
0.36%, phenylalanine1.02%, proline1.25%, serine 0.73%, taurine 0.15%, threonine 0.73%, tryptophan 0.16%, tyrosine 0.73% and valine 0.78% by weight) to
a ¢nal body weight of 18.36 g after a 8-week period (whole body tissue containing alanine 1.03%, arginine 1.76%, asparagine 1.81%, cystine 0.19%,
glutamic acid 2.70%, glycine 1.46%, histidine 0.40%, isoleucine 0.76%, leucine 1.31%, lysine 0.98%, methionine 0.39%, phenylalanine 0.93%, proline
1.25%, serine 0.76%, taurine 0.14%, threonine 0.74%, tryptophan 0.17%, tyrosine 0.76% and valine 0.84% by weight).
3
A /E ratio ^ de¢ned by Arai (1981) as [(essential amino acid/total essential amino acids plus cystine and tyrosine) · 1000].
4
Methionine + cystine.
5
Phenylalanine + tyrosine.
6
E/NE ratio ^ total essential amino acids, including cystine and tyrosine/nonessential amino acid ratio.
7
NE value is low compared with others because of the absence of a value for proline (not analysed).

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

 Diet, culture and shrimp growth 135

Table 10 Fatty acid composition of lipids within ‘microbial floc’ growth of shrimp in high-density culture conditions are being
(‘floc’) collected from the outdoor shrimp rearing tanks. Values are undertaken.
expressed as percentage total recovered fatty acids
In view of the fact that zero-water exchange culture systems
Fatty acid Range Mean are usually operated as closed farming systems (with no solids
C6:0 0.0^0.2 0.1 removal or water exchange in the present case), it is not
C8:0 0.1^0.2 0.15 surprising that many essential nutrients will be progressively
C10:0 0.2 0.2 depleted from the water column (including those additional
C12:0 0.6 0.6
C13:0 nd nd
mineral elements required by the ‘floc’ and resident phyto-
C14:0 5.2^7.2 6.2 plankton) and that other digestive/excretory metabolites or
C14:1 4.0^4.7 4.35 feed contaminants (including possible heavy metal contam-
C15:0 1.1^1.2 1.15
inants) could progressively accumulate to toxic levels within
C15:1 nd nd
C16:0 22.7^23.3 23 the culture system with time (McNeil 2000). In the present
C16:1n-7 11.3^13.7 12.5 instance, the last 2 weeks of the outdoor feeding trial saw a
C16:2n-4 0.5^1.0 0.75 progressive deterioration in water quality, as evidenced by a
C16:3n-4 1.6^2.9 2.25
C16:4n-1 0.5 0.5 decrease in pH, a marked increase in nitrite (following a peak
C17:0 0.5^0.7 0.6 in ammonia at week 6), and consequent reduced shrimp
C17:1 nd nd growth (Figs 4 & 5). Clearly, closed zero-water exchange
C18:0 2.1^2.5 2.3
C18:1n-9 5.5^6.2 5.85
culture systems can only biologically support a certain level of
C18:1n-7 4.2^5.5 4.85 nutrient input and shrimp biomass without the system
C18:1n-5 0.2^0.3 0.25 ‘crashing’ and compromising shrimp growth and survival.
C18:2n-6 3.9^4.6 4.25
For example, McIntosh (2000) reports that organic loadings
C18:2n-4 nd nd
C18:3c 0.2^0.3 0.25 could reach as high as 500 kg ha –1 day–1 within zero-water-
C18:3n-4 nd nd exchange shrimp ponds operated in Belize. Interestingly, this
C18:3n-3 3.4^4.5 3.95 is equivalent to a daily loading rate of 50 g feed tank–1 day–1,
C18:4n-3 0.2 0.2
C18:4n-1 nd nd which is similar to that reached during the final weeks of the
C20:0 0.3 0.3 present outdoor feeding trial (Fig. 3). However, considerably
C20:1n-9 0.6^1.3 0.95 higher loading rates and shrimp yields (as high as 8 kg m–2)
C20:1n-7 0.0^0.3 0.15
C20:2n-6 0.1^0.2 0.15
have been reported within experimental indoor zero-water-
C20:3n-6 0.1^0.2 0.15 exchange culture systems in Montana (USA) operated with
C20:3n-3 nd nd continuous illumination, buffer input, cation addition, and
C20:4n-6 1.2^2.1 1.65
solids management (R. McNeil, personal communication,
C20:4n-3 0.0^0.1 0.05
C20:5n-3 2.1^3.9 3 August 2000; McNeil 2000).
C21:5n-3 0.1^0.2 0.15 Despite the encouraging results obtained with zero-water-
C22:0 nd nd exchange culture systems, it is clear from the two feeding
C22:1n-11 0.1^0.7 0.4
C22:4n-6 0.1^0.2 0.15 trials that the nutrition and feeding of shrimp reared under
C22:5n-3 0.6^0.8 0.7 closed culture conditions will be different from that of
C22:6n-3 1.0^1.7 1.35 animals reared under open running water culture conditions.
C23:0 0.1^0.2 0.15
C24:0 nd nd
Apart from the obvious differences in terms of natural food
C24:1n-9 nd nd availability, it is almost impossible to view shrimp feed
Total saturated 34.5^35.0 34.75 consumption or feeding behaviour in zero-water-exchange
Total monounsaturated 28.4^30.2 29.3
culture systems because of ‘floc’ production in the culture
Totaln-6 6.3^6.4 6.35
Totaln-3 9.3^9.4 9.25 tanks. Clearly, the nutrition and feeding of the target species
Unknown peaks 15.9^16.7 16.3 must be studied under conditions which mimic as closely as
nd = Not detected (value lower than 0.05%). possible those of the intended farm production unit and
environment (Tacon 1996). The most promising features of
zero-water-exchange culture systems are that they offer both
As a result of the work carried out in this and other trials, increased biosecurity (Bullis & Pruder 1999) and reduced feed
further studies examining the relationship of dietary nutrient costs and water use (Chamberlain & Hopkins 1994; Boyd
levels, development of ‘floc’ throughout the growth cycle and 2000), and by doing so increase the possibility of moving the

..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

136 A.G.J. Tacon et al.
shrimp culture industry along a path of greater sustainability
and environmental compatibility.
Acknowledgements
The authors appreciate the technical assistance of the
following staff members of the Aquatic Feeds and Nutrition
program at the Oceanic Institute: Michael C. Haring, Eric H.
Beyer, Jesse H. Terpstra, Brent E. Larsen, Kekoa K.
Nakachi, Gary A. Delanoy and William L. Mulherin. This
paper was prepared as part of the activities of the ‘Tropical
Aquaculture Feeds and Culture Technology Development
Project II: Development of Shrimp Feeds’ awarded to the
Oceanic Institute by the US Department of Agriculture,
Agricultural Research Service, under Agreement No.
59-5320-7-989. Mention of trade names or commercial
products in this article is solely for the purpose of providing
specific information and does not imply recommendation or
endorsement by the authors.
References
AOAC (Association of Official Analytical Chemists) (1990a) AOAC
method no. 968.08. In: Official Methods of Analysis of the
Association of Official Analytical Chemists (Heilrich, K. ed.) 15th
edn, p. 84. AOAC, Arlington, VA.
AOAC (Association of Official Analytical Chemists) (1990b) Methyl
esters of fatty acids in oils and fats: gas chromatographic method
963.22. In: Official Methods of Analysis of the Association of
Official Analytical Chemists (Heilrich, K. ed.) 15th edn, p. 964.
AOAC, Arlington, VA.
Arai, S. (1981) A purified test diet for coho salmon, Oncorhynchus
kisutch, fry. Nippon Suisan Gakk., 47, 547–550.
Avnimelech, Y. (2000) Nitrogen control and protein recycling:
activated suspension ponds. Global Aquaculture Advocate, 3,
23–24.
Boyd, C.E. (2000) Water use in aquaculture. Global Aquaculture
Advocate, 3, 12–13.
Bullis, R.A. & Pruder, G.D. eds. (1999) Controlled and Biosecure
Production Systems: Evolution and Integration of Shrimp and
Chicken Models. Proceedings of a Special Session of the World
Aquaculture Society, Sydney, Australia, 27–30 April 1999. 106 pp.
The Oceanic Institute, Waimanalo, HI.
Chamberlain, G.W. & Hopkins, J.S. (1994) Reducing water use and
feed costs in intensive ponds. World Aquaculture, 25, 29–32.
Curds, C.R. (1992) Protozoa and the Water Industry. Cambridge
University Press, Cambridge, UK, 122 pp.
D’Elia, C.F., Steudler, P.A. & Corwin, N. (1977) Determination of
total nitrogen in aqueous samples using persulfate digestion.
Limnol. Oceanog., 22, 760–764.
Divakaran, S.D. (1999) AFIA Laboratory Methods Compendium II,
Vol. 4: Aquaculture. American Feed Industry Association,
Arlington, VA, 109 pp.
FAO (Food and Agriculture Organization of the United Nations)
(2000) Yearbook of Fishery Statistics 1998, Vol. 86/2. Aquaculture
production. FAO Statistics Series No. 154 and Fisheries Series No.
56, FAO, Rome, 182 pp.
Freeman, D.W. & Duerr, E.O. (1991) Design and use of outdoor
microcosm laboratory tanks for the evaluation of shrimp diets.
Aquacult. Engineer., 10, 89–97.
Grasshoff, K., Ehrhardt, M. & Kremling, K. eds. (1983) Methods of
Seawater Analysis. Verlag Chemie, Weinheim, 419 pp.
Hamilton, P.B. (1963) Ion exchange chromatography of amino acids.
Anal. Chem., 35, 2055–2064.
Hara, A. & Radin, N.S. (1978) Lipid extraction of tissues with a low-
toxicity solvent. Anal. Biochem., 90, 420–426.
Hirs, C.H.W. (1967) Determination of cystine as cysteic acid.
In: Methods in Enzymology, Vol. XI, Enzyme Structure (Hirs,
C.H.W. ed.), pp. 59–62. Academic Press, New York.
Hugli, T.E. & Moore, S. (1972) Determination of the tryptophan
content of proteins by ion exchange chromatography of alkaline
hydrolysates. J. Biol. Chem., 247, 2828–2834.
Kharlamenko, V.I., Zhukova, N.V., Khotimchenko, S.V., Svetashev,
V.I. & Kamenev, G.M. (1999) Fatty acids as markers of food
sources in shallow-water hydrothermal ecosystem (Kraternaya
Bight, Yankich Island, Kurile Islands). Mar. Ecol. Prog. Ser., 120,
231–241.
Lawrence, A. (1996) Shrimp feeds: match diet to production systems.
Feed Int., 17, 18–22.
Leber, K.M. & Pruder, G.D. (1988) Using experimental microcosms
in shrimp research: the growth-enhancing effect of shrimp pond
water. J. World Aquacult. Soc., 19, 197–203.
Liu, W.-T., Linning, K.D., Nakamura, K., Mino, T., Matsuo, T. &
Forney, L.J. (2000) Microbial community changes in biological
phosphate-removal systems on altering sludge phosphorus con-
tent. Environ. Microb., 146, 1099–1107.
McIntosh, R.P. (1999) Changing paradigms in shrimp farming. I.
general description. Global Aquaculture Advocate, 2, 42–47.
McIntosh, R.P. (2000) Changing paradigms in shrimp farming. III.
Pond design and operation considerations. Global Aquaculture
Advocate, 3, 42–45.
McIntosh, R.P. & Carpenter, N. (1999) Changing paradigms in
shrimp farming. II. Breeding for performance. Global Aquaculture
Advocate, 2, 36–39.
McNeil, R. (2000) Zero exchange, aerobic, heterotrophic systems:
key considerations. Global Aquaculture Advocate, 3, 72–76.
Moriarty, D.J.W. (1997) The role of microorganisms in aquaculture
ponds. Aquaculture, 151, 333–349.
Moss, S.M. (1995) Production of growth enhancing particles in a
plastic-lined shrimp pond. Aquaculture, 132, 253–260.
Moss, S.M., Reynolds, W.J. & Mahler, L.E. (1998) Design and
economic analysis of a prototype biosecure shrimp growout
facility. In: Proceedings of the US Marine Shrimp Farming
Program Workshop (Moss, S.M. ed.), pp. 3–18. The Oceanic
Institute, Waimanalo, HI.
Moss, S.M., Arce, S.M., Argue, B.J., Otoshi, C.A., Calderon, F.R.O.
& Tacon, A.G.J. (2001a) Greening of the blue revolution: efforts
toward environmentally responsible shrimp culture. In: The New
Wave, Proceedings of the Special Session on Sustainable Shrimp
Culture, Aquaculture 2001 (Browdy, C.L. & Jory, D.E. eds). pp.
1–19. The World Aquaculture Society, Baton Rouge, LA, USA.
Moss. S.M., Divakaran, S. & Kim, B.G. (2001b) Stimulating effects
of pond water on digestive enzyme activity in the Pacific white
shrimp, Litopenaeus vannamei (Boone). Aquaculture Res., 32, 125–
132.
Murphy, J. & Riley, J.P. (1962) A modified simple solution method
for the determination of phosphate in natural waters. Anal. Chim.
Acta, 27, 31–36.
Nunes, A.J.P., Goddard, S. & Gesteira, T.C.V. (1996) Feeding
activity patterns of the Southern brown shrimp Penaeus subtilis
..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137

under semi-intensive culture in North East Brazil. Aquaculture,
144, 371–386.
Ogino, C. (1980) Requirements of carp and rainbow trout for
essential amino acids. Nippon Suisan Gakk., 46, 171–174.
Ogle, J. & Lotz, J. (2000) Closed systems for maturation and grow-
out of shrimp. Global Aquaculture Advocate, 3, 89–90.
Reid, B. & Arnold, C.R. (1992) The intensive culture of the Penaeid
shrimp Penaeus vannamei Boone in a recirculating raceway system.
J. World Aquacult. Soc., 23, 146–153.
Robertson, L., Lawrence, A.L. & Castille, F.L. (1993) Effect of
feeding frequency and feeding time on growth of Penaeus vannamei
(Boone). Aquacult. Fish. Manag., 24, 1–6.
Rosenberry, R. (1999) World Shrimp Farming 1999. Shrimp News
International, San Diego, CA, USA, 320 pp.
Snedecor, G.W. & Cochran, W.G. (1967) Statistical Methods. The
Iowa State University Press, Ames, IA, 593 pp.
Solorzano, L. (1969) Determination of ammonia in natural waters
by the phenolhypochlorite method. Limnol. Oceanogr., 14,
799–801.
Strickland, D.H. & Parsons, T.R. (1972) A practical handbook of
seawater analysis. Bull. Fish. Res. Bd. Canada 167, 311 pp.
Tacon, A.G.J. (1978/1979) Activated sewage sludge, a potential
animal foodstuff II. Nutritional characteristics. Agric. Envir., 4,
271–279.
Tacon, A.G.J. (1996) Nutritional studies in crustaceans and the
problems of applying research findings to practical farming
systems. Aquacult. Nutr., 1, 165–174.
..............................................................................................

2002 Blackwell Science Ltd Aquaculture Nutrition 8;121^137
Diet, culture and shrimp growth 137
Tacon, A.G.J. (2000) Rendered animal by-products: a necessity in
aquafeeds for the new millennium. Global Aquaculture Advocate, 3,
18–19.
Tacon, A.G.J., Conklin, D.E. & Pruder, G.D. (1999) Shrimp feeds
and feeding: at the crossroads of a cultural revolution. In:
Controlled and Biosecure Production Systems: Evolution and
Integration of Shrimp and Chicken Models. Proceedings of a
Special Session of the World Aquaculture Society, Sydney, Austra-
lia, 27–30 April 1999 (Bullis, R.A. & Pruder, G.D. eds), pp. 55–66.
The Oceanic Institute, Waimanalo, HI.
Tacon, A.G.J. & Cowey, C.B. (1985) Protein and amino acid
requirements. In: Fish Energetics: New Perspectives (Tytler, P. &
Calow, P. eds), pp. 155–183. Croom-Helm, London, UK.
Tacon, A.G.J. & Ferns, P.N. (1978/1979) Activated sewage sludge, a
potential animal foodstuff I. Proximate and mineral content:
seasonal variation. Agric. Envir., 4, 257–269.
Van Wyk, P.M. (2000) Culture of Penaeus vannamei in single-phase
and three-phase recirculating aquacultrue systems. Global Aqua-
culture Advocate, 3, 41–43.
Velasco, M. & Lawrence, A. (2000) Initial evaluation of shrimp
vitamin requirements in laboratory tanks without water exchange.
Global Aquaculture Advocate, 3, 23.
Velasco, M., Lawrence, A.L. & Castille, F.L. (1999) Effect of
variations in daily feeding frequency and ration size on growth of
shrimp, Litopenaeus vannamei (Boone), in zero-water exchange
culture tanks. Aquaculture, 179, 141–148.