Effect of culture system on the nutrition and growthperformance of Pacific white shrimp Litopenaeusvannamei (Boone) fed different diets

A.G.J. TACON1, J.J. CODY2, L.D. CONQUEST2, S. DIVAKARAN2, I.P. FORSTER2 &

O.E. DECAMP21Halliday Place, Kaneohe, HI, USA; 2The Oceanic Institute, Waimanalo, HI, USA

Abstract

Two 8-week feeding trials were conducted with juvenile Pacific

white shrimp, Litopenaeus vannamei (Boone) to compare the

growth and performance of animals fed a series of experimen-

tal and commercial pelleted shrimp and fish feeds and dietary

feeding regimeswithin an indoor running-water culture system

and an outdoor zero-water-exchange culture system. The best

overall shrimp growth performance was observed for animals

fed the experimental shrimp diet and all-day feeding regime

under outdoor zero-water-exchange culture conditions. Final

body weight and average weekly growth rate under these

conditions were 2.8 and 3.4 times greater, respectively, than

animals of similar size fed with the same diet under indoor

running-water culture conditions. Although direct compar-

ison between indoor and outdoor culture systems is difficult

because of the lower indoor water temperatures, and conse-

quently lower mean daily feed intake of animals, it is believed

that the higher growth and feed performance of animals reared

under outdoor ‘green-water’ culture conditions was primarily

due to their ability to obtain additional nutrients from food

organisms endogenously produced within the zero-water-

exchange culture system. Themost promising features of zero-

water-exchange culture systems are that they offer increased

biosecurity, reduced feed costs and water use for the farmer,

and by doing so provide a potential avenue of moving the

shrimp culture industry along a path of greater sustainability

and environmental compatibility.

KEY WORDSKEY WORDS: diet, feeding regime, Litopenaeus vannamei,

methodology, Pacific white shrimp, zero water exchange

Introduction

Of the estimated 375 913 shrimp farms reportedly in

existence in the world in 1999, 54% used extensive pond-

based growout culture systems (stocking density below

2.5 m–2, shrimp production 50–500 kg ha–1 year–1, produc-

tion costs US$1–3 kg–1 live shrimp), 28% used semi-intensive

pond-based growout culture systems (stocking density below

30 m–2, shrimp production 500–5000 kg ha–1 year–1, produc-

tion costs US$2–6 kg–1 live shrimp), and 18% used intensive

pond-based growout culture systems (stocking density above

30 m–2, shrimp production 5000–20 000 kg ha–1 year–1, pro-

duction costs US$4–8 kg–1 live shrimp; Rosenberry 1999).

Moreover, although over 1.1 million tonnes of marine

shrimp (valued at US$6.8 billion) were produced in 1998

(FAO 2000), little or no information exists concerning the

optimum dietary nutrient levels for rearing these species

under practical pond-based culture systems (Lawrence 1996;

Tacon 1996).

As a result of the pressure faced by the shrimp farming

community for increased biosecurity, and disease and effluent

control (Bullis & Pruder 1999), there has been a trend within

many countries towards the development of biosecure closed

shrimp production systems, including zero-water-exchange

or recirculating culture systems employing in situ (McIntosh

1999; Avnimelech 2000; McNeil 2000) or external biofiltra-

tion techniques (Reid & Arnold 1992; Moss et al. 1998; Ogle

& Lotz 2000; Van Wyk 2000). Trials of zero water exchange

systems were conducted in Tahiti during the 1980s with

Litopenaeus vannamei and Penaeus monodon and with yields

of approximately 20 000 kg ha–1 year–1 (AQUACOP, perso-

nal communication). This paper describes two feeding trials

conducted from July to September 1999 at the Oceanic

Institute (OI), Hawaii, USA. The objective was to compare

the growth and performance of juvenile Pacific white shrimp

121

Received 22 February 2001, accepted 15 August 2001

Correspondence: I.P. Forster, The Oceanic Institute, 41-202 Kalanianaole

Highway, Waimanalo, HI 96795, USA. E-mail: iforster@oceanicinsitute.

org

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� 2002 Blackwell Science Ltd

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-Lwater 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 cleanedevery 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

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

Table 1 Environmental conditions of culture containers in 8-week outdoor trial

Open cover Clear plastic cover

Parameter Mean ! SD Minimum^Maximum Mean ! SD Maximum^Minimum

Temperature (AM) 28.2 ! 1.0 25.3)30.4 29.6 ! 1.0 25.6)31.5Temperature (PM) 31.3 ! 1.3 27.1)34.0 32.6 ! 1.2 29.7)35.1Dissolved oxygen (mg L)1) 6.0 ! 0.5 4.7)8.2 5.8 ! 0.5 4.6)7.1pH 7.8 ! 0.4 6.7)9.2 7.7 ! 0.4 7.2)8.8Salinity (g L)1) 33.9 ! 1.1 31)38 34.0 ! 1.1 31)36

A.G.J. Tacon et al.

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and mixed for a further 15 min. The resulting mash was then

passed through a Hobart grinder fitted with a 3-mm diameter

die. The pellet temperature at the die was below 70 �C. Theresulting moist pellets were then dried overnight in a drying

cabinet using an air blower at 38 �C until the moisture levelwas below 10%. The vitamin premix and vitamin C source

(Table 2) were then emulsified with the remaining oil and

lecithin in a KitchenAid mixer and this mixture was added to

the dry cooled pellets by top coating using a Hobart D300

food mixer with a whisk beater. The finished pellets were

then stored in plastic bins at 19–20 �C until used.

Indoor protocols

Four diets were tested with four different feeding regimes and

two pellet forms as follows:

DFF OI shrimp diet; sinking pellet; fixed ration; day

feeding with feeders.

NFF OI shrimp diet; sinking pellet; fixed ration; night

feeding with feeders.

ADF OI shrimp diet; sinking pellet; fixed 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 catfish diet; floating pellet; fed to

satiation; day feeding by hand.

CCCS Commercial catfish diet; sinking crumble; fed to

satiation; day feeding by hand.

Day feedings. four times daily (08.00, 11.00, 14.00,

17.00 hours), Night feedings: four times nightly (20.00,

23.00, 02.00, 05.00 hours), All-day feedings: eight times

during the day and night (at 08.00, 11.00, 14.00, 17.00,

20.00, 23.00, 02.00, and 05.00 hours) using battery operated

Aquarium Fish Feeders (Fish Mate F14, Pet Mate Ltd,

Hersham, Surrey, UK). The fixed feeding ration employed

was based on a shrimp daily feeding guide developed at the

Oceanic Institute (Table 4).

In the case of satiation feeding animals were fed to satiation

four times daily (08.00, 11.00, 14.00, 17.00 hours); latex

gloves were used for all feedings and handling of feed. All

experimental animals were weighed individually at bi-weekly

intervals for the duration of the experiment, and feeding rates

adjusted weekly; animals blotted with an absorbent towel and

weighed on a Mettler Toledo PB 3002 (Mettler-Toledo Inc.,

Hightstown, NJ, USA) electronic balance.

Outdoor protocols

Four diets were tested with four different feeding regimes and

two pellet forms as follows:

Table 2 Formulation of the Oceanic Institute (OI) experimental

sinking pelleted shrimp diet used in the 8-week feeding trial

Ingredient [crude protein (%)/crude lipid (%); OI shrimp dietingredient cost – US$ kg^1] (g kg^1dry weight)

Fishmeal ^ LT 94 (71.83/11.14; 1.28)1 245.0Squid meal (58.94/4.19; 2.85)2 25.0Soya bean meal, dehulled, solventextracted (43.84/1.69; 0.16)3

95.0

Wheat, whole hard red winter(13.88/1.76; 0.12)4

469.4

Wheat gluten meal (72.97/1.06; 1.00)4 40.0Brewers yeast (40.30/0.29; 0.62)5 30.0Krill hydrolysate (59.38/10.45; 8.30)6 20.0Soya lecithin, liquid (0.88)7 20.0Marine fish oil, Menhaden (0.96)8 30.0Cholesterol-FG (60%; potency 64%) (22.00)9 2.34OI mineral premix LV99.1 (64.75)10 0.6Potassium phosphate, dibasic(17.78% P, 44.9% K; 2.40)11

5.6

Calcium phosphate, monobasic(26.46% P,17.12% Ca; 2.40)11

5.6

Sodium phosphate, dibasic (21.82%P, 32.39% Na; 2.40)11

5.6

OI vitamin premix ^ LV99.1 (47.74)12 4.0Choline chloride (60%;52% potency) (1.21)13 1.154Vitamin C (35% ascorbic acid potency) (15.00)14 0.714

1SSF Sildolje-og Sildemelindustriens Forskningsinstitut, Norway.2 Agribrands Purina Mexico, S.A. de C.V., Mexico (by courtesy of).3 Land-o-Lakes, Seattle,WA, USA.4 Hawaii Flour Mills, Honolulu, HI, USA.5 Williams Bio-Products, Decatur, IL, USA (by courtesy of).6 Specialty Marine Products,WestVancouver, BC, Canada (by courtesy of).7 Central Soya Company Inc, FortWayne, IN, USA (by courtesy of).8 Omega Protein Inc., Reedville,VA, USA.9 Solvay Pharmaceuticals B.V.,Veenendaal,The Netherlands.10 OI mineral premix LV99.1 ^ to supply the following elements (mg kg)1

diet): zinc (Zn, as sulphate) 72 mg, iron (Fe, as sulphate) 36 mg,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(Cr, trivalent, as chloride) 0.8 mg, selenium (Se, as selenate) 0.2 mg, andmolybdenum (Mo, as molybdate) 0.2 mg.11 ICN Biomedicals, Inc., Aurora, OH, USA.12 OI vitamin premix LV99.1 ^ to supply the following vitamins (mg orIU kg^1 diet): thiamine 40 mg, ribo£avin 60 mg, pyridoxine 60 mg,pantothenic acid 180 mg, niacin 80 mg, biotin 0.6 mg, inositol 400 mg,folic acid 6 mg, cyanocobalamine 0.10 mg, vitamin A 6000 IU, vitamin D32000 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, bycourtesy of).13 Choline 60% ^ to supply 600 mg of active choline kg^1 diet (RocheV|tamins Inc., Parsippany NJ; by courtesy of).14 Stay-C 35% ^ to supply 250 mg of active vitamin C kg^1 diet (RocheV|tamins Inc, Parsippany NJ; by courtesy of).

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

Composition (g kg^1as feed basisDiets1

except as noted) DFF^DFS CSS CCFS CCCS

Proximate composition

Moisture 61.1 64.6 47.2 46.8Crude protein (N · 6.25) 351.7 349.5 374.7 376.0Crude lipid 82.4 91.1 54.3 49.6Cholesterol 2.7 1.46 0.97 0.97Ash 62.8 102.8 103.4 104.9Gross energy (MJ kg^1) 19.00 18.28 18.45 18.29Amino acid composition

Aspartic acid 29.74 34.96 37.07 37.07Serine 15.44 17.62 17.65 17.65Glutamic acid 69.94 52.49 59.97 59.97Glycine 17.40 20.32 29.36 29.36Alanine 18.53 20.83 24.01 24.01Taurine 1.96 1.12 0.98 0.98Cystine 4.73 5.79 4.73 4.73Tyrosine 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 6.121Potassium (K, g kg^1) 8.09 4.986 7.186 7.186Calcium (Ca, g kg^1) 5.733 19.84 25.23 25.23Magnesium (Mg, g kg^1) 1.635 2.385 2.182 2.182Sodium (Na, g kg^1) 4.103 2.292 2.544 2.544Manganese (Mn, mg kg^1) 32.88 117.1 116.8 116.8Iron (Fe, mg kg^1) 135.9 397.3 430.7 430.7Copper (Cu, mg kg^1) 27.35 38.65 8.789 8.789Zinc (Zn, mg kg^1) 104 138.5 122.8 122.8Boron (B, mg kg^1) 1.164 9.252 9.409 9.409Feed cost5, US$ kg^1 1.04 0.61 0.43 0.43Fatty acids (% total fatty acids)C12:0 0.1 0.2 0.1 0.1C14:0 5.1 8.0 4.6 4.6C14:1 0.2 0.1 0.2 0.2C15:0 0.4 0.5 0.4 0.4C16:0 19.2 20.5 20.3 20.3C16:1n-7 5.3 9.3 6.1 6.1C16:2n-4 1 1.4 1 1C16:3n-4 0.9 1.4 0.9 0.9C16:4n-1 0.1 nd6 nd ndC17:0 0.3 0.4 0.4 0.4C18:0 2.4 3 4.3 4.3C18:1n-9 15.0 16.4 24 24C18:1n-7 nd 0.1 nd ndC18:1n-5 0.2 nd nd ndC18:2n-6 21.3 16.1 14.2 14.2C18:2n-4 0.1 0.2 nd ndC18:3c 0.1 0.2 0.2 0.2C18:3n-4 0.3 0.4 0.2 0.2

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DFF OI shrimp diet; sinking pellet; fixed ration; day

feeding by hand.

NFF OI shrimp diet; sinking pellet; fixed ration; night

feeding by hand.

ADF OI shrimp diet; sinking pellet; fixed ration; day and

night feeding by hand.

DFFP OI shrimp diet; sinking pellet; fixed ration; day

feeding by hand; plastic tank cover.

CCFF Commercial catfish diet; floating pellet; fixed ration;

day feeding by hand.

CCCF Commercial catfish diet; sinking crumble; fixed

ration; day feeding by hand.

CSF Commercial shrimp diet; sinking pellet; fixed ration;

day feeding by hand.

Day feedings. four times daily (08.00, 11.00, 14.00,

17.00 hours); Night feedings: four times nightly (20.00,

23.00, 02.00, 05.00 hours); All-day feedings: eight times

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

the shrimp daily feeding guide described above (Table 4);

allotted daily feed allocations were equally divided into four

or eight portions per day as required. Sinking feed was

applied using a feeding tube directly onto a 0.12-m–2

submersible feeding tray placed 0.6 m below the water

surface on one side of the tank. During the last 2 weeks of

Table 3 (continued)

Composition (g kg^1as feed basisDiets1

except as noted) DFF^DFS CSS CCFS CCCS

C18:3 n-3 2.5 1.7 1.6 1.6C18:4 n-3 1.6 1.1 1.1 1.1C18:4 n-1 0.1 0.2 nd ndC20:0 0.1 0.1 nd ndC20:1n-9 2.7 0.7 1.6 1.6C20:1n-7 0.1 0.1 nd ndC20:2 n-6 0.1 0.1 0.2 0.2C20:3 n-6 0.2 0.2 0.3 0.3C20:3 n-3 0.1 0.1 nd ndC20:4 n-6 0.5 0.9 0.6 0.6C20:4 n-3 0.6 0.6 0.9 0.9C20:5 n-3 6.5 8.0 6.8 6.8C21:5 n-3 0.1 0.1 0.5 0.5C22:1n-11 2.2 0.1 1.1 1.1C22:4 n-6 0.2 0.2 0.6 0.6C22:5 n-3 1.1 1.4 1.3 1.3C22:6 n-3 7.6 3.8 5.3 5.3C23:0 0.3 0.5 0.1 0.1C24:1n-9 0.2 nd nd ndTotal n-6 22.3 17.5 15.9 15.9Total n-3 20.1 16.8 17.5 17.5Unknown peaks 1.3 2.1 1.0 1.0

1DFF^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^1diet ingredient costs only, excludesmanufacturing costs; all other costs are from Rangenprice list, F.O.B. Buhl, Idaho;FTL: full truck load quantities (5 July1999).6 nd = Not detected or value lower than 0.05%.

Table 4 Feeding rates for shrimp in 8-week feeding trials

Percentage of estimated shrimp biomass

Shrimpbody weight (g) 21^24 �C 24^28 �C 28^32 �C

1^3 8 6 73^5 7 5 65^7 6.5 4.5 5.57^9 6 4 59^11 5.5 3.5 4.511^13 5 3 413^15 4.5 2.5 3.515^17 4 2.5 317^30 3 2 2.5

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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 dissolvingthe 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

A.G.J. Tacon et al.

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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.

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

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.

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 0.02Mean final body weight (g) 5.92ab 5.98ab 6.71a 5.78b 5.17b 3.66c 3.67c 0.18Shrimp feed intake

Mean feed intake (g shrimp^1 day^1) 0.21 0.24 0.22 0.17 0.14 0.12 0.12Shrimp growth response

Total weight gain (%) 268.4ab 272.6ab 323.8a 264.5ab 223.7b 133.4c 130.2c 13.0Meanweekly weight gain (g week^1) 0.54ab 0.55ab 0.64a 0.52ab 0.45b 0.26c 0.26c 0.02Specific growth rate (% day^1)3 2.33ab 2.34ab 2.57a 2.31ab 2.10b 1.51c 1.48c 0.07Shrimp feed utilization

Feed efficiency (%)4 35.8ab 28.9bc 40.4a 42.4a 45.6a 26.1bc 21.8c 0.13Total 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 withfeeders; 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; £oatingpellet; 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).

Diet, culture and shrimp growth

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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 wereclearly 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 concentrationsin 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

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 0.03Mean final body weight (g) 17.20a 17.34a 18.89a 16.78a 11.35b 10.46b 12.94b 0.68Shrimp feed intake

Mean feed offered (g shrimp)1 day)1) 0.56 0.44 0.53 0.54 1.76 0.54 0.55Shrimp growth response

Total weight gain (%) 977.4ab 1015.0a 1098.2a 956.6ab 609.5c 554.7c 728.0bc 52.1Mean weekly weight gain (g week)1) 1.95a 1.97a 2.16a 1.90a 1.22b 1.11b 1.42b 0.09Specific growth rate (% day)1) 4.24a 4.30a 4.43a 4.20ab 3.50c 3.35c 3.76bc 0.10Shrimp feed utilization

Apparent feed efficiency (%)3 49.7ab 63.1a 57.7a 47.1ab 3.5c 25.6bc 34.1ab 6.17Apparent feed nitrogen efficiency (%)4 28.45ab 35.05a 32.01a 26.61ab 1.79c 13.01bc 18.99ab 3.46Apparent feed phosphorus efficiency (%)5 33.44ab 46.56a 39.42ab 31.19abc 2.06d 12.05cd 23.93bc 4.14Total shrimp production

Total initial shrimp biomass (kg m)3) 0.11a 0.10a 0.11a 0.11a 0.11a 0.11a 0.10a 0.01Total final shrimp biomass (kg m)3) 0.75abc 0.92a 0.89ab 0.68abc 0.14 0.39c 0.50bc 0.08Total biomass increase (kg m)3) 0.64abc 0.82a 0.78ab 0.57abc 0.04 0.29c 0.40bc 0.08Total feed offered (kg) (as fed basis) 1.93 1.94 2.03 1.82 1.58 1.67 1.76Shrimp survival (%) 64.7a 79.3a 69.7a 61.0a 19.0b 56.7a 58.3ab 6.2

Feed cost kg)1shrimp 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 handfeeding; plastic tank cover; CCFF = commercial cat¢sh diet; £oating pellet; ¢xed ration; day feeding by hand; CCCF = commercial cat¢sh diet; sinkingcrumble; ¢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 theconsumption 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.

A.G.J. Tacon et al.

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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 �Cfeeding 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

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).

Within all the microcosm tanks there was a rapid

development of a microbial food chain, initially in the visible

form of a green algal-based autotrophic microbial food web,

with a bacterial-based heterotrophic microbial food web

developing later; the latter was visible in the water column as

suspended particulate matter or ‘microbial floc’ (‘floc’).

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.

Week0 2 4 6 8

Shr

imp

wei

ght (

g)

0

2

4

6

8

10

12

14

16

18

20

22

24

DFFNFFADFDFFPCCFFCCCFCSF

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.

Diet, culture and shrimp growth

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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

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.

A.G.J. Tacon et al.

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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 lowermean 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

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 2.22DFF 74.54ab 19.82a 1.63a 2.62a 1.39NFF 73.89b 19.19a 1.49a 2.88a 2.61ADF 74.73ab 19.41a 1.41a 2.69a 1.76DFFP 74.58ab 19.48a 1.67a 2.61a 1.66CCFF 76.06a 18.49a 1.51a 2.50a 1.44CCCF 75.29ab 19.00a 1.53a 2.64a 1.54CSF 76.65a 17.86a 1.47a 2.51a 1.51SEM3 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 handfeeding; plastic tank cover; CCFF = commercial cat¢sh diet; £oating pellet; ¢xed ration; day feeding by hand; CCCF = commercial cat¢sh diet; sinkingcrumble; ¢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 BTreatment1 (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 2.116DFF 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 handfeeding; plastic tank cover; CCFF = commercial cat¢sh diet; £oating pellet; ¢xed ration; day feeding by hand; CCCF = commercial cat¢sh diet; sinkingcrumble; ¢xed ration; day feeding by hand; CSF = commercial shrimp diet; sinking pellet; ¢xed ration; day feeding by hand.2 Standard error of the means.

Diet, culture and shrimp growth

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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.

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.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

DFF NFF ADF DFFP CCFF CCCF CSF

Treatment

Wee

kly

gro

wth

(g)

Week 0 2 Week 2 4

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

(g week–1) 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; 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 Com-

Commercial catfish diet; sinking crum-

ble; fixed ration; day feeding by hand;

CSF ¼ Commercial shrimp diet; sinking

pellet; fixed ration; day feeding by hand.

A.G.J. Tacon et al.

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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

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).

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.

Diet, culture and shrimp growth

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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 157Moisture (g kg^1) 58.6^73.1 66Crude protein (N · 6.25) (g kg^1) 292.0^343.3 312Crude lipid (g kg^1) 25.7^26.3 26.0Cholesterol (g kg^1) 0.47^0.49 0.48Ash (g kg^1) 255.5^318.1 282Gross energy (MJ kg^1) 10.3^12.8 12Phosphorus (P) (g kg^1) 3.6^21.2 13.5Potassium (K) (g kg^1) 1.3^8.9 6.4Calcium (Ca) (g kg^1) 5.6^28.6 17Magnesium (Mg), (g kg^1) 1.2^4.5 2.6Sodium (Na) (g kg^1) 4.1^43.1 27.5Manganese (Mn) (mg kg^1) 8.9^46.8 28.5Iron (Fe) (mg kg^1) 170.8^521.0 320Copper (Cu) (mg kg^1) 3.8^88.6 22.8Zinc (Zn) (mg kg^1) 78.3^577.9 338Boron (B) (mg kg^1) 8.8^45.7 27.3Amino acid (g kg^1)Aspartic acid 30.3^31.1 31.1 12.25 9.20 9.85Serine 12.7^13.8 13.2 5.21 4.78 4.13Glutamic acid 31.7^34.3 33.0 13.01 21.66 14.67Proline 12.1^12.8 12.5 4.91 Not analysed 6.76Glycine 16.8^17.6 17.2 6.78 5.39 8.04Alanine 17.6^19.4 18.5 7.31 5.74 5.60Taurine 0.34^0.36 0.35 0.14 0.61 0.75Cystine 3.9^4.1 4.0 1.57 1.46 1.03Tyrosine 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 100E/NE ratio6 50.4:49.6 52.6:47.47 50.2:49.8

1OI control shrimp diet (Table 2).2 Estimated dietary amino acid requirement pro¢le of shrimp (Litopenaeus vannamei) calculated according to themethod 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 OIshrimp diet and all day feeding regime, with animals growing froman initial body weight of1.58 g (whole body tissue containing alanine 0.97%, arginine1.42%, 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%, methionine0.36%, 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) toa ¢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%, proline1.25%, 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 byArai (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).

A.G.J. Tacon et al.

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As a result of the work carried out in this and other trials,

further studies examining the relationship of dietary nutrient

levels, development of ‘floc’ throughout the growth cycle and

growth of shrimp in high-density culture conditions are being

undertaken.

In view of the fact that zero-water exchange culture systems

are usually operated as closed farming systems (with no solids

removal or water exchange in the present case), it is not

surprising that many essential nutrients will be progressively

depleted from the water column (including those additional

mineral elements required by the ‘floc’ and resident phyto-

plankton) and that other digestive/excretory metabolites or

feed contaminants (including possible heavy metal contam-

inants) could progressively accumulate to toxic levels within

the culture system with time (McNeil 2000). In the present

instance, the last 2 weeks of the outdoor feeding trial saw a

progressive deterioration in water quality, as evidenced by a

decrease in pH, a marked increase in nitrite (following a peak

in ammonia at week 6), and consequent reduced shrimp

growth (Figs 4 & 5). Clearly, closed zero-water exchange

culture systems can only biologically support a certain level of

nutrient input and shrimp biomass without the system

‘crashing’ and compromising shrimp growth and survival.

For example, McIntosh (2000) reports that organic loadings

could reach as high as 500 kg ha –1 day–1 within zero-water-

exchange shrimp ponds operated in Belize. Interestingly, this

is equivalent to a daily loading rate of 50 g feed tank–1 day–1,

which is similar to that reached during the final weeks of the

present outdoor feeding trial (Fig. 3). However, considerably

higher loading rates and shrimp yields (as high as 8 kg m–2)

have been reported within experimental indoor zero-water-

exchange culture systems in Montana (USA) operated with

continuous illumination, buffer input, cation addition, and

solids management (R. McNeil, personal communication,

August 2000; McNeil 2000).

Despite the encouraging results obtained with zero-water-

exchange culture systems, it is clear from the two feeding

trials that the nutrition and feeding of shrimp reared under

closed culture conditions will be different from that of

animals reared under open running water culture conditions.

Apart from the obvious differences in terms of natural food

availability, it is almost impossible to view shrimp feed

consumption or feeding behaviour in zero-water-exchange

culture systems because of ‘floc’ production in the culture

tanks. Clearly, the nutrition and feeding of the target species

must be studied under conditions which mimic as closely as

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

increased biosecurity (Bullis & Pruder 1999) and reduced feed

costs and water use (Chamberlain & Hopkins 1994; Boyd

2000), and by doing so increase the possibility of moving the

Table 10 Fatty acid composition of lipids within ‘microbial floc’

(‘floc’) collected from the outdoor shrimp rearing tanks. Values are

expressed as percentage total recovered fatty acids

Fatty acid Range Mean

C6:0 0.0^0.2 0.1C8:0 0.1^0.2 0.15C10:0 0.2 0.2C12:0 0.6 0.6C13:0 nd ndC14:0 5.2^7.2 6.2C14:1 4.0^4.7 4.35C15:0 1.1^1.2 1.15C15:1 nd ndC16:0 22.7^23.3 23C16:1n-7 11.3^13.7 12.5C16:2n-4 0.5^1.0 0.75C16:3n-4 1.6^2.9 2.25C16:4n-1 0.5 0.5C17:0 0.5^0.7 0.6C17:1 nd ndC18:0 2.1^2.5 2.3C18:1n-9 5.5^6.2 5.85C18:1n-7 4.2^5.5 4.85C18:1n-5 0.2^0.3 0.25C18:2n-6 3.9^4.6 4.25C18:2n-4 nd ndC18:3c 0.2^0.3 0.25C18:3n-4 nd ndC18:3n-3 3.4^4.5 3.95C18:4n-3 0.2 0.2C18:4n-1 nd ndC20:0 0.3 0.3C20:1n-9 0.6^1.3 0.95C20:1n-7 0.0^0.3 0.15C20:2n-6 0.1^0.2 0.15C20:3n-6 0.1^0.2 0.15C20:3n-3 nd ndC20:4n-6 1.2^2.1 1.65C20:4n-3 0.0^0.1 0.05C20:5n-3 2.1^3.9 3C21:5n-3 0.1^0.2 0.15C22:0 nd ndC22:1n-11 0.1^0.7 0.4C22:4n-6 0.1^0.2 0.15C22:5n-3 0.6^0.8 0.7C22:6n-3 1.0^1.7 1.35C23:0 0.1^0.2 0.15C24:0 nd ndC24:1n-9 nd ndTotal saturated 34.5^35.0 34.75Total monounsaturated 28.4^30.2 29.3Totaln-6 6.3^6.4 6.35Totaln-3 9.3^9.4 9.25Unknown peaks 15.9^16.7 16.3

nd = Not detected (value lower than 0.05%).

Diet, culture and shrimp growth

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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.

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