Ammonia toxicity as a criterion for the evaluation of larval quality in the prawn macrobrachium rosenbergii

Thuy, Mathieu Wille, Patrick Sorgeloos Laboratory of Aquaculture and Artemia Reference Center, Uni6ersity of Gent, Rozier44 , 9000Ghent, Belgium Received 17 August 1999; received in revi

Ammonia toxicity as a criterion for the evaluation of larval

quality in the prawn Macrobrachium rosenbergii

Ronaldo O Cavalli *, Els Vanden Berghe, Patrick Lavens, Nguyen T.T Thuy,

Mathieu Wille, Patrick Sorgeloos

Laboratory of Aquaculture and Artemia Reference Center, Uni6ersity of Gent, Rozier44 , 9000Ghent, Belgium

Received 17 August 1999; received in revised form 12 November 1999; accepted 18 November 1999

Abstract

The feasibility of a short-term ammonia toxicity test as an evaluation criterion for larval quality was assessed in three

trials In each one, Macrobrachium rosenbergii larvae originating from the same spawn were nutritionally differentiated

in two groups by feeding them either a nutrient-rich (Artemia nauplii enriched for 24 h with n-3 highly unsaturated fatty acids (HUFA) and ascorbic acid (AA)) or a nutrient-poor diet (Artemia nauplii starved for 24 h) Throughout their

development, larvae from both treatments were exposed during 24 h to six concentrations of total ammonia (NH4++

NH3) and a control (no ammonia added) Based on mortality rates, the median lethal concentration for 50% of the population (LC50) was estimated As expected from earlier work, larvae fed the optimal diet presented higher n-3 HUFA and AA contents as well as higher growth and metamorphosis rates From the moment the effect of diet quality was analytically detectable in the tissues of the larvae, the ammonia test was able to distinguish both groups of larvae Differences in ammonia tolerance were observed as early as larval stage 4 and remained evident throughout larval development The short-term ammonia toxicity test proved to be a valuable, sensitive and reproducible criterion for the establishment of larval quality © 2000 Elsevier Science Inc All rights reserved

Keywords:Ammonia toxicity; Larval quality; Macrobrachium rosenbergii; Prawn; Stress test; Aquaculture; Highly unsaturated fatty

acids; Ascorbic acid

1 Introduction

World aquaculture production has increased

tremendously over the last two decades It is

estimated that only from 1987 to 1996 the volume

of production rose by 250% (FAO, 1998) To

support this expansion, the technology for the

mass production of fry was established for several

species of economic importance Nowadays,

com-mercial aquaculture operations rely mainly on the

production of hatchery-reared fry For the most successful species current hatchery practices en-able the supply of sufficient numbers of fry and, hence, larval quality is becoming of major con-cern Unfortunately, up to the present limited information is available on standardised methods for the detection of larval and postlarval quality Probably one of the first developments of a specific test for the evaluation of fry quality took place in Japan when Watanabe et al (1983)

mea-sured the survival of red sea bream (Pagrus

ma-jor) larvae 24 h after exposure to the air for 5 s.

Since then, several methods have been proposed These usually involve the exposure of the animals

* Corresponding author Tel.: + 9-264-3754; fax: +

32-9-264-4193.

E-mail address: ronaldo.cavalli@rug.ac.be (R.O Cavalli)

0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc All rights reserved.

PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 1 1 3 - 9

R.O Ca6alli et al./Comparati6e Biochemistry and Physiology, Part C125 (2000) 333 – 343 334

to a short but extreme environmental stress

(salin-ity, temperature, pH, or formalin) at which their

physiological condition will determine their ability

to survive (Tackaert et al., 1989; Briggs, 1992;

Dhert et al., 1992; Fegan, 1992; Ako et al., 1994;

Samocha et al., 1998) Other methods use more

subjective observations, e.g body pigmentation,

colour and activity, morphological (muscle to gut

ratio) indicators (Bauman and Jamandre, 1990),

biochemical (fatty acid) composition (Arellano,

1990) and even a score comprising several

parameters like chromatophore development,

body deformity, fouling, and muscle opaqueness

(Fegan, 1992) Most often these criteria are

con-sidered complementary and therefore used

to-gether to evaluate larval quality However, most

of them were conceived to evaluate the quality of

fry in later developmental stages, i.e at the end of

the hatchery cycle and prior to their release in

grow-out facilities For the early larval stages,

other than the usual estimates of spawn size, egg

size and hatching rate, time to hatching and larval

survival, very few evaluation methods have been

devised For penaeid shrimps, Bray et al (1990)

suggested the use of protozoea I length, while

Browdy (1992) indicated that larval quality could

be evaluated according to the phototactic

re-sponse of the nauplius stage A similar principle

was used to separate ‘healthy’ and ‘weak’ larvae

of the freshwater prawn Macrobrachium

rosenber-gii (Singh and Philip, 1995) Although all these

criteria are useful, none of them is considered

sufficiently sensitive or standardised to provide a

conclusive and reliable assessment of the

physio-logical condition of larvae

Ammonia is the principal excretory product in

aquatic animals (Kinne, 1976) and its mechanisms

of toxicity and lethal concentrations to various

fishes and crustaceans of commercial importance

are relatively well documented (Tomasso, 1994)

In water, ammonia is found primarily as the

ammonium ion (NH4+) and the non-ionised

molecule (NH3), which co-exist in an equilibrium

reaction governed mainly by pH (Emerson et al.,

1975) Higher pH levels increase the concentration

of NH3 in relation to NH4+ Non-ionised

ammo-nia freely diffuses across cell membranes in the

direction favoured by its pressure gradient

(Fromm and Gillette, 1968) Therefore, if

ammo-nia levels increase in water, ammoammo-nia excretion

diminishes, and levels of ammonia in blood and

other tissues increase This might result in an

elevation of blood pH and adverse effects on membrane stability and enzyme-catalysed reac-tions (Tomasso, 1994), which may eventually lead

to death

Since acute static bioassays are a standard prac-tice in aquatic toxicology studies, a similar proce-dure could also prove valuable to ascertain the physiological condition of larval stages of aquatic animals Accordingly, this study aimed to assess the feasibility of a short-term ammonia toxicity test as a larval quality evaluation criterion The

freshwater prawn M rosenbergii was chosen as a

model species since it easily reproduces under captive conditions and its larvae can be cultured following standardised hatchery practices

2 Materials and methods

2.1 Origin of animals

Prawn larvae were obtained from broodstock imported from Thailand and reared indoors in a standardised maturation system (Cavalli et al., 1999a,b) The broodstock animals were fed to satiation twice a day with a shrimp broodstock feed (INVE Technologies N.V., Baasrode, Belgium)

2.2 Experimental procedures

Three independent trials were repeated in time

In each trial, newly hatched larvae originating from the same spawn were divided in two groups and reared under standard conditions in an exper-imental hatchery system In order to create two different quality groups, the larvae were fed either

a nutrient-rich or a nutrient-poor diet

Dietary supplementation of ascorbic acid (AA) and n-3 highly unsaturated fatty acids (HUFA) are known to increase the survival and stress

tolerance of M rosenbergii postlarvae (Devresse

et al., 1990; Merchie et al., 1995; Romdhane et al., 1995) Accordingly, the nutrient-rich diet

con-sisted of Artemia franciscana (Great Salt Lake,

USA) enriched with a n-3 HUFA rich emulsion (ICES enrichment emulsion 50/0.6/C, containing 50% n-3 HUFA) to which 20% ascorbyl palmitate

was added The enrichment of Artemia was

per-formed according to Merchie et al (1995) Freshly hatched nauplii were enriched for 24 h Two doses

of 0.3 g emulsion l− 1 were added at 12-h

inter-vals The density of nauplii for enrichment was

around 200 ml− 1 After 24 h, the enriched

meta-nauplii were harvested and rinsed with tap water

over a 120-mm sieve to remove any remaining

emulsion

The nutrient-poor diet consisted of freshly

hatched Artemia nauplii starved for 24 h under

the same conditions as the enriched Artemia

group (stocking density of 200 nauplii ml− 1,

tem-perature 28 9 1°C, and natural seawater)

2.3 Lar6iculture system and feeding

For each trial, the two groups of larvae were

reared to postlarvae in separate recirculation units

at an initial density of 100 l− 1 Each unit

con-sisted of a 100-l black cylindro-conical PVC tank

connected to an 80-l submerged biological filter

and a 20-l overhead tank Water was continuously

pumped from the filter to the overhead tank at a

rate of 2.5 l min−1and then forced back through

the bottom of the rearing tank by gravity An

outlet screen (150 mm) at the top of the rearing

tank led the water back to the biological filter and

at the same time retained the larvae and Artemia

within the rearing tank Two shortcuts, at the

pump outlet and the rearing tank inlet, enabled

the adjustment of the water flow rate The filter

screen was cleaned twice a day to prevent

overflow

A 300-W thermostatic heater placed in the filter

maintained the water temperature at 28 9 1°C

Water salinity at 12 9 1 g l− 1 was obtained

through the mixture of deionised water and

natu-ral seawater Deionised water was added to the

system to compensate for losses due to

evapora-tion The photoperiod was automatically

con-trolled at 12 h light and 12 h dark Aeration in the

rearing tanks and filters assured oxygen levels

above 5 mg O2 l−1

Ammonium, nitrite and ni-trate were below 0.2 mg NH4

+

l−1

, 0.03 mg NO2

l−1 and 50 mg NO3 l−1, respectively, while pH

varied from 7.8 to 8.2

Prawn larvae were fed solely on Artemia

meta-nauplii, which were maintained in the rearing

tanks at a density of 10 – 15 nauplii ml− 1 Feeding

started on the second day after hatching and was

carried out twice a day (10:00 and 18:00 h) The

enriched and starved groups were prepared daily

and stored at 4°C (Merchie, 1996) for the evening

feeding Every morning, Artemia remaining from

the previous day were filtered out of the

larvicul-ture tanks to maintain a constant quality of the prey organisms This was accomplished by in-creasing the water flow rate (to around 19 l min−1) and the filter screen mesh sizes (from 150

mm for normal functioning to 350 mm, for the first

7 days, and 500 mm from day 8 onwards)

At least 30 larvae from both treatments were periodically sampled and conserved in 4% forma-lin for the determination of total length (from the tip of rostrum to the tip of the telson; TL) and larval staging (Uno and Kwon, 1969) The larval stage index (LSI) was then estimated according to Maddox and Manzi (1976):

LSI = SS i/N

where S i is the stage of the larvae (i = 1 – 12) and

N is number of larvae examined.

2.4 Ammonia toxicity test set-up

The general procedures for the ammonia toxic-ity tests were based on the methodology presented

by the Standard Methods for the Examination of Water and Wastewater (Greenberg et al., 1992) The tests were performed in duplicate in a series

of 1-l glass cones immersed in a water bath at

28 9 1°C Starting from newly hatched larvae (day 0) up to complete metamorphosis to postlar-vae, groups of 30 animals from both treatments were exposed during 24 h to six increasing con-centrations of total ammonia (TAN; NH4+

+

NH3) and a control (no ammonia added) TAN concentrations were based on preliminary tests to identify suitable ranges for each LSI, and varied according to the age of the larvae The ammonia stock solution was prepared with reagent grade

NH4Cl and was added to the glass cones immedi-ately before stocking the test animals

Measurements of pH were carried out at the beginning (after adding the NH4Cl solution) and

at the end of the tests Initial values ranged from 7.8 to 8.3 Ammonium (NH4+) concentrations were also measured at the beginning and at the end of trial 1 with a selective ammonium electrode (model 6833; Consort, Turnhout, Belgium) As in the larval rearing tanks, salinity and temperature were kept at 12 9 1 g l−1 and 28 9 1°C, respec-tively The animals were not fed and water was not renewed during the exposure

The concentrations of non-ionised ammonia (NH3) were estimated according to the general formula for bases (Albert, 1973) for the mean

R.O Ca6alli et al./Comparati6e Biochemistry and Physiology, Part C125 (2000) 333 – 343 336

values of pH, salinity and temperature as

pre-sented by Armstrong et al (1978):

[NH3] = [TAN]/1 + 10[pK − pH]

where pK = 9.31 at a temperature of 28°C and

salinity of 12 g l−1; pH is the mean value

mea-sured at the beginning and the end of test

After 24 h of exposure, larvae presenting no

movement of appendages and not responding to

mechanical stimuli were considered dead Based

on the mortality rates, the mean lethal

concentra-tions for 50% of the population (24-h LC50) were

estimated with the Trimmed Spearman Karber

Method (Hamilton et al., 1977)

2.5 Salinity stress test

Postlarvae from trial 2 and larvae (LSI = 8.8 –

9.0) from trial 3 were also subjected to salinity

stress tests (Tackaert et al., 1989) The tests were

run in five replicates, with groups of 10 larvae or

postlarvae being transferred to 1-l plastic beakers

containing water at 28 9 1°C and a salinity of 65

or 45 g l−1, respectively The test medium was a

mixture of natural seawater (33 g l− 1

) and artifi-cial salts (Instant Ocean, Aquarium System,

Sar-rebourg, France)

The mortality was monitored at 3-min intervals

during 1 h The animals presenting no movement

of pleopods and giving no reaction to prodding

with a pipette were considered dead The

sensitiv-ity to the salinsensitiv-ity stress was expressed by the

cumulative stress index (CSI), which was

calcu-lated as the sum of cumulative mortality observed

over the test period Higher sensitivity to osmotic stress resulted in earlier and/or higher mortalities and thus a higher value of CSI (Dhert et al., 1992)

2.6 Biochemical analyses Enriched and starved Artemia meta-nauplii,

prawn larvae and postlarvae were periodically sampled for the determination of total lipids (TL), fatty acid methyl esters (FAME) and AA levels Larvae and postlarvae were food deprived for at least 2 h prior to sampling All samples were rinsed with tap water over a sieve and frozen until analysis Samples for TL and FAME were main-tained at − 20°C, while those for AA were kept

at − 80°C TL were determined by Folch et al (1957), modified by Ways and Hanahan (1964), while FAME analysis followed the methodology

of Coutteau and Sorgeloos (1995) AA levels were analytically determined according to Nelis et al (1997)

2.7 Statistical analysis

Data were subjected to one-way analysis of

variance-ANOVA (P B 0.05) and, where

appro-priate, Tukey’s Honest Significant Difference

(HSD) Student’s t-test was used to establish

dif-ferences between nominal and measured ammonia concentrations Differences on the LC50 values were graphically determined through the compari-son of polynomial regressions

3 Results

In all ammonia tests performed in trial 1, the nominal concentrations were significantly related

to the mean ammonium values measured (P B

0.05) Therefore, no measurements were per-formed in trials 2 and 3 Only low pH variations within single tests and among subsequent tests were observed, hence these were considered negligible

The effects of enrichment and starvation on the

biochemical composition of Artemia are

sum-marised in Table 1 The content of TL, n-3 HUFA, 20:5n-3, 22:6n-3 and AA were

signifi-cantly higher in the enriched Artemia than in the starved ones (P B 0.05) The differences in the

biochemical composition of Artemia were

Table 1

Biochemical composition (mean 9 S.D.) of enriched and

starved Artemia meta-nauplii fed to M rosenbergii larvaea

Enriched Starved Total lipids (% DW) 22.5 9 6.3 b 14.1 9 3.2 c

Selected fatty acids

(mg g −1 DW)

20:5n-3 40.3 9 7.5 b 3.8 9 0.5 c

0.3 9 0.1 c

22:6n-3 15.8 9 2.9 b

61.7 9 10.9 b

Sn-3 ( ± 20:3n-3) 5.3 9 0.8 c

7.5 9 2.8 c

15.9 9 2.8 b

Sn-6 ( ± 18:2n-6)

230.7 9 31.7 b

600.7 9 35.8 c

Ascorbic acid (mg g −1 3911.8 9 36.7 b

DW)

a Within rows, superscripts express significant differences

between treatments (PB0.05).

Fig 1 Total length (mm) as a function of age (days after

hatching) of M rosenbergii larvae fed enriched or starved

Artemia meta-nauplii in trials 1 and 2 *Significant differences

between means; PL indicates postlarvae.

(P B 0.05) in morphological development (LSI)

between larvae of the same age but from different treatments started to be detected 9 and 6 days after hatching in trials 1 and 2, respectively (Fig 2)

The evolution of the LC50 values according to the morphological development of the larvae (LSI) from both treatments is shown in Figs 3 and 4 LC50values for both groups were differen-tiated after a certain larval stage, usually around LSI 4 This trend of differentiation was even more apparent as larval development proceeded For both total and non-ionised ammonia, differences

in LC50 values between treatments were clearer in trial 2 than in trial 1 A significantly higher toler-ance to the salinity stress was observed for the

postlarvae fed enriched Artemia compared to those fed starved Artemia (Fig 5) On the other

hand, no difference to the response to the salinity

Fig 2 Larval stage index (LSI) as a function of age (days after

hatching) of M rosenbergii larvae fed enriched or starved

Artemia meta-nauplii in trials 1 and 2 *Start of significant

differences between treatments.

reflected in the tissue composition of larvae fed

the different diets (Table 2) Animals fed enriched

Artemia presented comparatively higher levels of

TL, n-3 HUFA and AA than those fed starved

Artemia The biochemical composition of day 0

larvae differed between trials, i.e larvae from trial

1 had an average AA content of 149 mg g−1DW

while those from trial 2 had mean AA levels of

265 mg g− 1 DW

Higher growth (Fig 1) and metamorphosis

rates (Fig 2) were observed for larvae fed

en-riched Artemia, as expected (Merchie et al., 1995;

Romdhane et al., 1995) Significant differences

Table 2

Biochemical composition of M rosenbergii larvae (Lv) and postlarvae (PL) fed enriched (rich) or starved (stv) Artemia in trials 1 and 2a

rich

n.a.

Fatty acids (mg g −1 DW)

12.6

9.6

Sn-3 ( ± 20:3n-3)

105.9 83.8 184.4 39.1 62.3 279.0 388.8 66.0 113.4

579.6 148.8 910.7 451.6 652.0 487.2 n.a n.a n.a 193.6 265.1 844.4 384.9 835.7 447.7 484.9 442.5 191.9 Ascorbic acid (mg g −1 DW)

a

n.a., not available.

Fig 3 Polynomial regressions of the estimated 24-h LC50values for total ammonia (mg NH4+ +NH3l −1 ) according to the larval stage

index of M rosenbergii larvae fed enriched or starved Artemia meta-nauplii in trials 1 and 2.

shock was detected between larvae fed enriched or

starved Artemia (Fig 5).

4 Discussion

As expected, the biochemical composition of

Artemia nauplii was influenced by the enrichment

and starvation procedures, which in turn affected

the composition of the larvae that fed upon them

Higher TL, n-3 HUFA and AA contents were

found in the tissues of larvae fed enriched Artemia versus those fed starved Artemia Larvae fed en-riched Artemia also presented higher growth and

metamorphosis rates than those fed starved

Artemia These results confirm the positive effects

of these nutrients in the enhancement of larval development and correspond well with previous findings (Devresse et al., 1990; Merchie et al., 1995; Romdhane et al., 1995) Hence, our goal to

R.O Ca6alli et al./Comparati6e Biochemistry and Physiology, Part C125 (2000) 333 – 343 340

produce two groups of larvae with distinct quality

properties as study material for the ammonia

testing was considered achieved

Comparison of the ammonia tolerance (LC50)

of larvae at the same stage of morphological

development (LSI) indicated pronounced

differ-ences between treatments The estimated LC50

values for both total and non-ionised ammonia

were consistently higher for larvae fed enriched

Artemia. Similarly, postlarvae fed enriched

Artemia presented a higher tolerance to the

salin-ity stress All this indicates the superior abilsalin-ity of

the animals fed enriched Artemia to cope with

changing environmental conditions, i.e a better physiological condition Furthermore, these ob-servations confirm the possibility of differentiat-ing larval quality in terms of ammonia tolerance and, more importantly, demonstrate the potential

of the short-term ammonia toxicity test as a crite-rion for the establishment of larval quality

It is also worth noting that the ammonia test was sensitive enough to detect differences in the

Fig 4 Polynomial regressions of the estimated 24-h LC50values for non-ionised ammonia (mg NH3l −1 ) according to the larval stage

index of M rosenbergii larvae fed enriched or starved Artemia meta-nauplii in trials 1 and 2.

Fig 5 Mean ( 9 S.D.) cumulative stress index (CSI) of M.

rosenbergii postlarvae and larvae (LSI of 8.8 – 9.0) fed enriched

or starved Artemia meta-nauplii in trials 2 and 3, respectively.

broodstock, the inherent variation in brood qual-ity, and minor differences in management and/or environmental conditions during the distinct lar-val rearing periods

The salinity stress test is probably the most used method to estimate fry quality It is based on the principle that a short-term exposure to a salinity shock will indicate the physiological con-dition of the animals However, as the osmoregu-latory capability of crustaceans changes with ontogenetic development (Sandifer et al., 1975; Charmantier et al., 1988), only animals with a similar morphological development should be used for comparison For instance, differences in gill development at early developmental stages may affect the capability of the animals to toler-ate changes in salinity (Ribeiro, 1998) and hence invalidate the results of the salinity stress test In this regard, our results suggest that the salinity stress test was not sensitive enough to distinguish both groups of larvae and therefore should have its use restricted to later developmental stages In contrast, the ammonia stress test was able to differentiate the larval groups, proving to be an appropriate tool to evaluate larval quality One possible application of the ammonia test would be in broodstock nutrition studies Previ-ously it was shown that 8-day-old larvae originat-ing from females with different nutritional backgrounds could be distinguished by an ammo-nia stress (Cavalli et al., 1999a) This result was in line with the superior broodstock performance observed in some of the production parameters considered (fecundity and hatching rate) In a

similar study, the response of M rosenbergii

fe-males to the dietary supplementation of phospho-lipids was assessed and no significant differences were detected in any of the performance parame-ters measured (Cavalli et al., 1999b) In this case, the ammonia test also found no differences in the 24-h LC50 values of larvae from the different treatments

Also, one should not rule out possibilities for more practical applications, such as the measure-ment of fry quality before leaving the commercial hatchery and thus before submitting them to the stress of transportation and/or transfer to nursery

or grow-out ponds In this case, however, as an alternative to the methodology presented here, the procedures for the ammonia test could be sim-plified to minimise the needs for labour, equip-ment and time For instance, reliable results may

early stages of larval development From the

mo-ment the effect of diet quality was analytically

detectable in the larval tissues, the ammonia test

was able to distinguish both groups of larvae, i.e

as early as larval stage 4 Furthermore, this

pat-tern of differentiation remained evident

through-out the experimental period

Another fundamental aspect in the

determina-tion of the feasibility of the ammonia test as a

quality criterion is its reproducibility over time

All trials had overall results with similar patterns,

demonstrating that the test is indeed reproducible

However, a within-trial comparison also revealed

differences in the LC50 values of larvae with

simi-lar LSI This indicates that some variation may be

found when working with different batches of

larvae Although the reasons for these differences

are not clear, this may be due to differences in the

nutritional background of the broodstock, as

sug-gested by the differences in AA content in the

batches of newly hatched larvae Other sources of

variation could be the genetic differences among

R.O Ca6alli et al./Comparati6e Biochemistry and Physiology, Part C125 (2000) 333 – 343 342

be obtained with fewer ammonia concentrations

The use of a single concentration might also be

feasible (Cavalli et al., 1999a), but this would

require a previous knowledge of the LC50 values

for each larval or postlarval stage (as ammonia

tolerance varies with development) and the

maintenance of similar pH levels for the different

treatments (to ensure that the larvae from

differ-ent groups are exposed to similar NH3

concentrations)

Finally, it is concluded that the ammonia test is

a sensitive and reproducible criterion for the

es-tablishment of larval quality, even for the early

stages of development One can assume that this

test could also be used as a general larval quality

method for other aquatic species, perhaps even as

a predictive indicator of larval viability

Regard-less of the species under consideration, further

work is warranted to examine the precise

relation-ship between larval quality, as evaluated by the

ammonia test, and the future performance under

grow-out conditions

Acknowledgements

This work was partially supported by a grant

from the Brazilian Council for Science and

Tech-nology (CNPq), which is gratefully acknowledged

The authors express their gratitude to Nopadol

Phuwapanish, Department of Fisheries, Thailand,

for providing the broodstock prawns, and to

Geert Vandewiele and Petra Rigole for their

tech-nical assistance

References

Ako, H., Tamaru, C.S., Bass, P., Lee, C.S., 1994

Enhancing the resistance to physical stress in larvae

of Mugil cephalus by the feeding of enriched Artemia

nauplii Aquaculture 122, 81 – 90

Albert, A., 1973 Selective Toxicity Chapman and Hall,

London

Arellano, E., 1990 Fatty acid composition of wild and

cultured Penaeus 6annamei as a method to evaluate

postlarval quality In: Abstracts of the World

Aqua-culture Society Meeting National Research Council

Canada, Ottawa, p 50

Armstrong, D.A., Chippendale, D., Knight, A.W.,

Colt, J.E., 1978 Interaction of ionized and

un-ion-ized ammonia on short-term survival and growth of

prawn larvae, Macrobrachium rosenbergii Biol Bull.

154, 15 – 31

Bauman, R.H., Jamandre, D.R., 1990 A practical

method for determining quality of Penaeus monodon

(Fabricius) fry for stocking in grow-out ponds In: New, M., Saram, H., Singh, T (Eds.), Technical and Economic Aspects of Shrimp Farming Proceedings

of the Aquatech 90 Conference Infofish, Kuala Lumpur, pp 124 – 131

Bray, W.A., Lawrence, A.L., Lester, L.J., 1990

Repro-duction of eyestalk-ablated Penaeus stylirostris fed

various levels of total dietary lipids J World Aquacult Soc 21, 41 – 52

Briggs, M.R.P., 1992 A stress test for determining

vigour of post-larval Penaeus monodon Fabricius.

Aquacult Fish Manage 23, 633 – 637

Browdy, C., 1992 A review of the reproductive biology

of Penaeus species: perspectives on controlled

shrimp maturation systems for high quality nauplii production In: Wyban, J (Ed.), Proceedings of the Special Session on Shrimp Farming, World Aqua-culture Society Meeting World AquaAqua-culture Soci-ety, Baton Rouge, LA, pp 22 – 51

Cavalli, R.O., Lavens, P., Sorgeloos, P., 1999a

Perfor-mance of Macrobrachium rosenbergii broodstock fed

diets with different fatty acid composition Aquacul-ture 179, 387 – 402

Cavalli, R.O., Menschaert, G., Lavens, P., Sorgeloos, P., 1999b Maturation performance, offspring

qual-ity and lipid composition of Macrobrachium

rosen-bergii females fed increasing levels of dietary

phospholipids Aquacult Int., submitted

Charmantier, G., Charmantier-Daures, M., Bouraricha, N., Thuet, P., Aiken, D.E., Trilles, J.P., 1988 On-togeny of osmoregulation and salinity tolerance in

two decapod crustaceans: Homarus americanus and

Penaeus japonicus Biol Bull 175, 102 – 110.

Coutteau, P., Sorgeloos, P., 1995 Intercalibration exer-cise on the qualitative and quantitative analysis of

fatty acids in Artemia and marine samples ICES

Coop Res Rep 211, 30 pp

Devresse, B., Romdhane, M., Buzzi, M., Rasowo, J., Le´ger, P., Brown, J., Sorgeloos, P., 1990 Improved larviculture outputs in the giant freshwater prawn

Macrobrachium rosenbergii fed a diet of Artemia

nauplii enriched with w3-HUFA and phospholipids World Aquacult 21 (2), 123 – 125

Dhert, P., Lavens, P., Sorgeloos, P., 1992 Stress evalu-ation: a tool for quality control of hatchery-pro-duced shrimp and fish fry Aquacult Eur 17 (2),

6 – 10

Emerson, K., Russo, R.C., Lund, E.R., Thurston, R.V., 1975 Aqueous ammonia equilibrium calcula-tions: effect of pH and temperature J Fish Res

Bd Can 32, 2379 – 2383

FAO, 1998 Aquaculture Production Statistics 1987 –

1996 FAO Fisheries Circular No 815, Revision 10 FAO, Rome, 197 pp