Aquaculture Research, 2010, 1^10 doi:10.1111/j.1365-2109.2009.02457.

Protein lipid ratio for the growth and survival of juvenile

crayfish Procambarus acanthophorus (Hobbs 1972)
reared under controlled conditions

Enrique Cervantes-Santiago1, Martha P. HernaŁndez-Vergara1, Carlos I. Pe¤rez-Rostro1 &

Miguel A. Olvera-Novoa2
1
Laboratorio de Nutricio¤n Acu|¤ cola, Divisio¤n de Estudios de Posgrado e Investigacio¤n, Instituto Tecnolo¤gico de Boca del R|¤ o,
Veracruz, Me¤xico
2
Centro de Investigacio¤n y de Estudios Avanzados del I.P.N., Unidad Me¤rida,YucataŁn, Me¤xico

Correspondence: M P HernaŁndez-Vergara, Laboratorio de Nutricio¤n Acu|¤ cola, Divisio¤n de Estudios de Posgrado e Investigacio¤n.
Instituto Tecnolo¤gico de Boca del R|¤ o, Km. 12 Carr, Veracruz-Cordoba, C.P. 94290, Veracruz, Me¤xico. E-mail: mphv1@yahoo.
com.mx

Abstract
The in£uence of dietary protein/lipid levels on
growth and survival in juvenile cray¢sh (Procam-
barus acanthophorus) was evaluated during a12-week
nutritional trial. Twenty experimental diets were for-
mulated containing di¡erent protein (200, 250, 300,
350 and 400 g kg  1) and lipid (60, 80, 100 and
120 g kg  1) levels. A bifactorial model (5  4) was
used with three replicates and 15 cray¢sh
(0.014  0.006 g) per replicate, in a recirculation sys-
tem with 60^15 L rectangular tanks and a biological
¢lter, the culture conditions were a photoperiod of
12:12 h L:D and temperature 24.7  1.0 1C. Total sur-
vival ranged from 66.6% to 86.6%, with no di¡er-
ences attributed to treatments. Dietary protein
levels had a signi¢cant e¡ect on growth gain and nu-
tritional e⁄ciency; in contrast, lipid levels had no ef-
fect on juvenile performance. The 250/120 (protein/
lipid) treatment promoted the most e⁄cient growth
(¢nal weight: 3.09 g, weight gain: 21995%, speci¢c
growth rate: 3.35% day  1); the less e⁄cient growth
was observed in juveniles fed with 400 g kg  1 pro-
tein diets. The quadratic regression model indicated
279 g kg  1 as the optimal protein level for cray¢sh
growth, while levels between 211 and 232 g kg  1
could minimize the feeding costs without a¡ecting
growth.
Keywords: cray¢sh, Procambarus acanthophorus,
protein requirements, growth, lipid, survival
Introduction
Commercial aquaculture with crustaceans in Mexi-
co, as in other places around the world, has begun
with the culture of exotic species. Native organisms
are rarely used, largely due to the market demand
for large and traditional crustaceans (prawns,
shrimp, crabs and lobsters), the short supply informa-
tion on these species and the limited aquaculture fa-
cilities using them (DOF 2006). These factors may
have delayed the development of culture biotechnol-
ogies for native organisms, but aquaculture requires
diversi¢cation and Procambarus sp. is a promising op-
tion. Some authors promote the diversi¢cation and
research to develop technologies that allow culture
of biological and commercial important native spe-
cies instead of introduced ones, which will have irre-
versible environmental impacts (AŁlvarez-Torres,
Soto, Avile¤s-Quevedo, D|¤ az-Luna & Trevino-Carrillo
1998; Galvao & Bueno 2000; Rodr|¤ guez-Serna Car-
mona-Osalde, Olvera-Novoa & Arredondo-Figueroa
2000).
A good option for crustacean aquaculture is cray-
¢sh, which are cosmopolitan organisms found in all
the continents, except Africa, and include about 500
species (Fowler, Lorio & Lutz 1994). The cray¢sh in-
dustry in the Unites States is an important activity
with good economic yields, but in most countries,
including Mexico, it is only used in small ¢sheries,
for biological and conservation research. Actually,
some cray¢sh species have a huge potential for

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Journal Compilation r 2010 Blackwell Publishing Ltd 1
Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al. Aquaculture Research, 2010, 1^10
aquaculture facilies including Procambarus acan-
thophorus, because of their simple cycle life, and
adaptation to di¡erent environments and cavtivity
conditions (Cervantes-Santiago 2008). The culture
of any aquatic species requires knowledge of its biolo-
gic aspects, environment preferences and feed re-
quirements to develop diets that cover its metabolic
and growth needs. The nutritional requirements in
crustaceans vary widely in relation to the species,
age and individual physiological condition, feeding
habits, dietary protein components and culture tech-
nique (D’Abramo & Sheen 1994; HernaŁndez-Vergara,
Rouse & Olvera-Novoa 2001).Vital roles in crustacean
nutrition are recognized in the protein and lipid le-
vels because they provide the energy required for
maintenance, muscle generation, growth and repro-
duction (Guillaume 1997; Thompson, Muzinic, Eng-
ler, Morton, & Webster 2004); for this reason, it is
necessary to know the requirements in crustaceans
with aquaculture potential.
In some cray¢sh, the dietary protein requirement
is between 200 and 300 g kg  1 and lipid from 40 to
60 g kg  1 for optimum growth (Jover, FernaŁndez-
Carmona, Del R|¤ o & Soler 1999; HernaŁndez-Vergara
et al. 2001; HernaŁndez-Vergara, Rouse, Olvera-Novoa
& Davis 2003). The noble cray¢sh Astacus astacus
need around 200^300 g kg  1 dietary protein with
at least150 and 200 g kg  1 animal protein for proper
growth (Lovell 1989), while red swamp cray¢sh Pro-
cambarus clarkii require between 250 and 350 g kg  1
(Guillaume 1997). In terms of lipid requirements,
white river craw¢sh Procambarus acutus acutus need
60 g kg  1 dietary lipid for good growth; in contrast
with the ¢rst one, cray¢sh Procambarus llamasi re-
quire at least a 200/120 g kg  1 [protein/lipid (P/L)]
ratio for acceptable growth under controlled culture
conditions, whereas a 100/60 g kg  1 ratio produces
low growth (Carmona-Osalde, Olvera-Novoa, &
Rodr|¤ guez-Serna 2005). HernaŁndez-Vergara et al.
(2001) indicated that redclaw cray¢sh Cherax quadri-
carinatus larvae and juveniles require between 250
and 350, and 40 and 80 g kg  1 lipid. A similar pro-
tein requirement was reported by Corte¤s-Jacinto,
Villarreal-Colmenares, Civera-Cerecedo and Mart|¤ -
nez-Cordova (2003) for optimum growth in C. quadri-
carinatus (342 g kg  1), but they recommend a
310 g kg  1 dietary protein level for juveniles (1g).
The variations in the protein and lipid requirement
data are associated with the material source, quality
and inclusion levels, as well as the presence of other
energy sources in diet formulations (D’Abramo 1989);
for this reason, it is important to conduct some trials
2 Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10
seeking the best diet for the organism in culture. The
objective of the present study was to determine the
most e⁄cient P/L ratio in diets for juvenile P. acantho-
phorus, to increase growth and survival and promote
sustainable cray¢sh culture using diets that inte-
grated their nutritional requirements at the lowest
cost.
Materials and methods
Juvenile cray¢sh P. acanthophorus (initial weight
0.014  0.006 g) were selected from a stock main-
tained in the reproduction area of the Native Crusta-
ceans Laboratory, of the InstitutoTecnologico of Boca
del R|¤ o, Veracruz, Mexico, for a 12-week nutritition
trial (December 2007 to February 2008) to evaluate
growth and survival under controlled conditions.
Experimental design and system
A bifactorial (5  4) design was used to evaluate 20
experimental diets formulated containing di¡erent
protein (200, 250, 300, 350 and 400 g kg  1) and lipid
(60, 80, 100 and 120 g kg  1) ratios, with three repli-
cates and 15 organisms per replicate in a completely
randomized design. The experiment was conduced
under controlled environmental conditions (12 h
L:12 h D photoperiod, and 24.7  1.0 1C temperature)
in a recirculation system with tap water and 60^15 L
rectangular plastic tanks with lateral drainage, and a
constant water £ow (1L min  1), mechanical and
biological ¢lter and 15 sections of a PVC tube of 4 cm
length  1.99 cm diameter for each experimental
unit, used for juvenile refuge in the ecdysis period
(HernaŁndez-Vergara 2001).
Experimental diets
The cray¢sh P. acanthophorus has the ability to digest
vegetal source protein in arti¢cial diets (Cervantes-
Santiago, HernaŁndez-Vergara & Pe¤rez Rostro 2007);
for this reason, in the experimental diets, a 75/25
soy meal/ ¢sh meal ratio as was used the protein
source, as well as ¢sh and corn oil at a 1:1ratio, wheat
bran, corn starch, vitamins and minerals, soy le-
cithin, pigment and carboxymethylcellulose as a bin-
der (Table 1).
Diets were formulated using a spreadsheet (EXCEL,
Microsoft) based on the protein, lipid, vitamin, miner-
al and energy requierements of cray¢sh (HernaŁndez-
Vergara 2001). The dry ingredients were ground to
0.250 mm, and then mixed for 15^20 min in a mixer
r 2010 The Authors
 Table 1 Formulation and proximate composition of experimental diets containing di¡erent protein/lipid ratios fed Procambarus acanthophorus

r 2010 The Authors

Diets
1
Ingredients (g kg ) 200/60 200/80 200/100 200/120 250/60 250/80 250/100 200/120 300/60 300/80 300/100 300/120 350/60 350/80 350/100 350/120 400/60 400/80 400/100 400/120

Fish meal 77.1 77.1 77.1 77.1 96.3 96.3 96.3 96.3 115.6 115.6 115.6 115.6 134.8 134.8 134.8 134.8 154.1 154.1 154.1 154.1
Soya mealw 327.9 327.9 327.9 327.9 409.8 409.8 409.8 409.8 491.8 491.8 491.8 491.8 573.8 573.8 573.8 573.8 667 681 695.1 702.6
Wheat branz 58.1 58.1 58.1 58.1 72.6 72.6 72.6 72.6 87.2 87.2 87.2 87.2 101.7 101.7 101.7 101.7 88.3 53.5 18.6 0.0
Fish oil‰ 22.3 32.3 42.3 52.3 20.4 30.4 40.4 50.4 18.4 28.4 38.4 48.4 16.5 26.5 36.5 46.5 14.6 24.6 34.6 44.6
Corn oilz 20.8 30.8 40.8 50.8 18.6 28.6 38.6 48.6 16.3 26.3 36.3 46.3 14 24 34 44 12.4 23.3 34.2 44.7
Corn starch 431.3 411.3 391.3 371.3 319.8 299.8 279.8 259.8 208.3 188.3 168.3 148.3 96.7 76.7 56.7 36.7 1.1 1.0 0.9 0.0
Vitamin and mineral 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0
premixk
Carboxymethylcellulose 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0
Soya lecithin 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
Pigment 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
Vitamin C7 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Proximate composition (g kg  1 dry base)
Moisture 63.1 54.9 63.6 59.4 66.2 61.3 59.4 52.3 92.5 82.4 82.9 79.1 62.7 57.4 75.5 53.7 66.5 75.2 67.4 56.1
Crude protein 217.6 219.6 220.0 227.7 276.9 271.9 271.2 268.2 317.9 316.9 315.4 319.4 377.2 373.0 376.5 381.5 434.4 424.5 425.9 420.9

Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10

Ether extract 68.3 95 116.9 134.8 67.5 91.1 114.7 136 62.6 81.4 98.4 112.8 63.4 84.1 110.7 135.1 62.3 86.4 104.9 126.1
Ash 42.3 46.0 44.8 45.9 56.4 55.3 54.8 54.6 65.5 63.9 64.5 64.6 74.1 77.3 74.5 77.3 83.5 83.5 82.1 99.4
Crude fiber 10.7 11.9 11.0 12.3 10.8 16.1 20.4 20.9 25.8 22.9 21.0 20.1 29.3 31.6 34.1 40.8 34.8 36.5 30.4 28.2
NFE 598.0 572.6 543.7 519.9 522.2 504.3 479.5 468.0 435.7 432.5 417.8 404.0 393.3 376.6 328.7 311.6 318.5 293.9 289.3 269.3
Energy (MJ 100 g  1) 18.92 18.83 20.01 17.24 18.42 18.67 18.75 17.24 18.29 18.25 18.46 19.00 18.54 18.92 19.17 18.83 18.08 18.04 17.58 18.46

Fish meal: protein 65.08%; lipids 8.85%, ash 12.5%; moisture 7.6%; and ¢ber 0.2%. Source: El Pedregal Silver Cup balanced feeds.
wSoya meal: protein 42.70%; lipids 2.18%; ash 7.58%; moisture 7.57%; ¢ber 38.32%. Source: (SERCA) Servicios Cient|¤ ¢cos Agropecuarios.
zWheat bran: protein 17.2%; lipid 3.46%; ash 5.40%; moisture 10.81%; ¢bre 41.87%.
‰Shark liver oil.
zCorn oil, Mazola.
kVitamin and mineral premix (Laboratorios Brovel, Me¤xico): Vitamin A 200 000 UI; Vitamin B1 220 mg; Vitamin B2 30 mg; nicotinamide 400 mg; pantothenic acid 100 mg; Vitamin B6 0.5 mg; Vitamin B12;
Vitamin C 120 mg; Vitamin D3 15000 UI; Vitamin E 100 UI; Folic acid 3 mg; calcium 1500 mg; cobalt 3 mg; phosphorous 1150 mg; iron 150 mg; magnesium 200 mg; potassium 25 mg; manganese 25 mg;
selenium 0.1mg; sodium 3000 mg; zinc 400 mg. Vit C (Sigma, Me¤xico).

3
Aquaculture Research, 2010, 1^10 Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al.
Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al. Aquaculture Research, 2010, 1^10
(Crypto JC32 Peerles Rota bowl mixer, Crypto, Lon-
don, UK); the oils and soy lecithin were also added
and mixed for a further 15 min. The carboxymethyl-
cellulose was pre-gelatinized separately in warm
(60 1C) water and mixed into the mixture until a
homogeneous mass was formed (HernaŁndez-Vergara
et al. 2003). The mixture was passed through a 2-mm-
diameter meat grinder (Moulinex HV8, DKA1,
France) to form pellets, which were dried in a forced-
air oven (RIOS ROCHA S.A., Mexico) at 60 1C for 12 h
until the moisture content was o100 g kg  1. Dried
pellets were stored at  4 1C in zipper plastic bags
until use. Diets were analysed for proximate compo-
sition in the beginning of the experiment following
standard methods (AOAC 1990). Crude protein
(g Kg  1) and energy content were determined using
an elemental analyzer (CN Flash EA Thermo Quest,
Basingstoke Haunt, UK).
Experimental procedure
Cray¢sh were fed once daily (15:00 hours). At the be-
ginning of the trial, feeding rates were 20% of the to-
tal biomass and were gradually adjusted downward
during the ¢rst 2 weeks to 5% of the biomass at the
end of the study (Cervantes-Santiago 2008). At the
beginning and the end of the experiment, the cray-
¢sh were individually measured from the tip of the
rostrum to the telson using a vernier, and weighed
in an electronic scale balance (OHAUS, Scout Pro,
CS200, NJ, USA; 0.01g) by placing them beforehand
on an absorbent towel to remove excess water, and
then counted. Additionally, every 2 weeks, the juve-
niles in each experimental unit were counted and
their individual wet body weights were determined
for growth and survival calculations, feeding adjust-
ment and e⁄ciency estimations. On the second day of
the experiment, all organisms died after the initial
measurements were replaced with an equal-size ju-
venile, with no further replacements made there-
after.
Diet quality and nutritional e⁄ciency were evalu-
ated in terms of survival, feed e⁄ciency and weight
gain by applying standard models (HernaŁndez-Ver-
gara, Olvera-Novoa & Rouse 2004). Percent survival
was expressed as
Sð%Þ ¼ 100ðNf =Ni Þ
where Nf is the ¢nal number of organisms per treat-
ment and Ni is the initial number of organisms.
4 Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10
Percent weight gain
WGð%Þ ¼ 100½ðWf =Ni Þ=Wi 
whereWf is the individual ¢nal weight and Wi is the
initial individual weight. Daily weight gain
(mg day  1), DWG 5 sIWG week days  1. The speci¢c
growth rate was estimated as
WGð%Þ ¼ 100½ðWf  Wi Þ=Wi 
per days, and the feed conversion ratio as FCR 5 IFI/
WG based on the individual feed intake (IFI) (dry
matter basis) and total weight gain in all juveniles
(WG) (wet weight).
Water quality
Water quality in the experimental system was moni-
tored twice a day (9:00 and 18:00 hours) during the
trial period. Temperature ( 1C) was measured using a
s
digital thermometer (Lifegard Temp AlertTM, Life-
gard Aquatic Digital Temp Alert, WI, USA), dissolved
oxygen (DO) using a YSI556 MPS (Yellow Spring In-
strument, OH, USA) and water hardness (CaCO3
mg L  1), pH, total ammonium (NH4), N-nitrite
(NO2) and N-nitrate (NO3) using a rapid colorimeter
kit (Hagen, Nutra¢n, Mans¢eld, USA).
Statistical analysis
Results were analysed using a multifactor explora-
tory analysis to identify the factor with the maxi-
mum e¡ect on the response parameters. A mixed
multivariate ANOVA (95% con¢dence interval) was ap-
plied to identify the di¡erences between treatments
(protein, lipid) and a possible interaction (pro-
tein  lipid). Because there were no di¡erences in
the lipid content and an interaction was not ob-
served, factors were combined in a variable P/L ratio
as shown in Table 2. A surface response model
and generalized linear models were applied from
quadratic regression models: yi 5 a1b1X11b2X221ei.
The survivance data were arcsine transformed be-
fore statistical analyses. All statistical analyses were
performed using the STATISTICA version 7.0 software.
Results
Water quality parameters (average  SD) were within
the ranges recommended for cray¢sh growth and survi-
val: temperature: 24.7  1.0 1C; DO: 5.80  0.29
r 2010 The Authors
Aquaculture Research, 2010, 1^10 Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al.

Table 2 Results of the mixed multivariate ANOVA for nutritional trial in Procambarusacanthophorus fed experimental diets

Survival Final Weight SGR IWG IFI

Factor (%) weight (g) gain (%) (%.day  1) (g day  1) (g day  1) FCR PER

Protein P 5 0.961735 P 5 0.000000 P 5 0.000000 P 5 0.000000 P 5 0.000000 P 5 0.000001 P 5 0.360972 P 5 0.000000

Lipid P 5 0.444496 P 5 0.666190 P 5 0.666190 P 5 0.648131 P 5 0.666190 P 5 0.102146 P 5 0.728465 P 5 0.428748
Proteinlipids P 5 0.333173 P 5 0.101207 P 5 0.101207 P 5 0.090823 P 5 0.101207 P 5 0.608254 P 5 0.916980 P 5 0.927956

Protein and lipids levels and the interaction protein  lipids (P  0.05).
SGR, speci¢c growth rate; IFI, indicidual feed intake; IWG, individual weigh gain; FCR, food convertion rate; PER, protein e⁄ciency rate.

Table 3 Means of proximate composition (dry matter) of the female and male Procambarus acanthophorus fed with experi-
mental diets containing di¡erent protein/lipid ratios

Females Males

Moisture Protein Lipid Calories Moisture Protein Lipid Calories

P/L (g kg  1) (%) (%) (%) (MJ100 g  1) (%) (%) (%) (MJ100 g  1)

200/60 72.2 11.0 3.5 17.99 75.6 9.8 2.5 16.07

200/80 70.7 16.9 4.2 19.29 73.1 10.9 3.1 17.24
200/100 72.4 10.9 4.3 18.49 73.9 10.5 3.1 17.33
200/120 72.5 14.4 3.9 19.66 74.0 11.5 3.2 18.17
250/60 74.1 11.6 3.1 18.20 74.1 11.9 2.1 17.12
250/80 71.9 12.7 3.5 18.33 72.7 13.8 2.3 18.54
250/100 71.6 13.3 3.2 19.37 73.7 12.8 2.4 18.42
250/120 74.2 12.1 3.5 19.37 72.8 11.9 2.6 15.15
300/60 73.1 13.5 2.5 18.33 74.3 12.2 1.7 18.29
300/80 72.1 12.8 3.0 18.33 74.5 12.7 1.8 17.08
300/100 74.1 11.5 3.0 17.91 76.4 12.1 2.1 17.66
300/120 74.9 13.0 2.9 17.74 75.3 11.8 2.2 17.29
350/60 73.2 12.7 1.9 18.16 77.4 13.2 1.6 19.55
350/80 73.6 13.7 2.9 15.69 74.8 13.0 2.0 17.75
350/100 73.9 15.1 3.3 19.71 76.6 12.6 2.0 16.32
350/120 73.8 12.6 3.6 20.21 75.8 12.2 2.3 17.33
400/60 73.9 11.4 2.1 16.32 76.7 14.2 1.4 16.99
400/80 74.3 10.7 2.3 16.48 75.3 11.7 1.7 16.83
400/100 74.2 11.7 2.7 16.78 77.2 11.4 1.6 16.53
400/120 75.3 12.0 2.3 16.86 78.5 11.4 1.5 16.90

mg L  1; ammonium: 0.06  0.05 mg L  1; N-nitrite:
0.10  0.0 mg L  1; N-nitrate: 5  0.0 mg L  1; pH:
8.67  0.13; and total hardness: 358  103.05 mg L  1
CaCO3.
The nutritional trial was stopped after 84 experi-
mental days because reproductive behaviour was ob-
served in di¡erent units. The mixed multivariate
analysis of variance to determine whether there were
di¡erences between treatments, and a possible inter-
action between factors (protein and lipids), only
showed the e¡ect of the protein (Po0.05) in all vari-
ables, except for survival and FCR, with no e¡ect of
lipids, or an interaction between the two factors
(Table 2).
In the treatments 200/120, 250/60 and 400/120
ovigerous females were found. To determine whether
the reproductive behaviour has some e¡ect on the
growth, and carcass body composition by sex, a prox-
imate composition analysis was performed. The car-
cass body analysis showed no di¡erences by sex or
treatment, although lipid (%) and body caloric en-
ergy (MJ 100 g  1) contents in females were slightly
higher than the males for all treatments, without sig-
ni¢cant di¡erences; for this reason, the results were
analysed as a group (Table 3).
The cray¢sh survival ranged from 66% to 86%,
without di¡erences between treatments. Because of
the lowest survival recorded in the juvenile fed with
the 250/12 g kg  1 diet, although this treatment
yielded the most e⁄cient parameters (WG, SGR and
DWG), a correlation analysis was performed between
weight gain and survival to determine a possible

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Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10 5
Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al. Aquaculture Research, 2010, 1^10

Table 4 Comparation of means between treatments for the di¡erent traits evaluated in Procambarus. acanthophorus fed ex-
perimental diets containing di¡erent protein/lipid ratios

Ratio
Weight SGR IWG Feed Intake
P L Survival (%) Gain (%) (% day  1) (mg day  1) (mg day  1) FCR PER

200 60 82.22 16 448abc 2.64abc 27.41abc 33.45ab 1.23 3.52ab

200 80 68.89 17 686abc 2.68ab 29.48abc 33.55ab 1.15 3.79a
200 100 73.33 15 924abc 2.62abc 26.54abc 31.25ab 1.20 3.61ac
200 120 75.56 17 233abc 2.66abc 28.72abc 33.85ab 1.17 3.54ad
250 60 73.33 19 186ab 2.72ab 31.98ab 36.28ab 1.14 2.97abe
250 80 77.78 20 543ab 2.75ab 34.24ab 33.91ab 1.00 3.47ab
250 100 86.67 19 995ab 2.74ab 33.33ab 33.65ab 1.01 3.48ab
250 120 66.67 21 995a 2.79a 36.66a 39.95a 1.10 3.32ab
300 60 71.11 18 829ab 2.71ab 31.38ab 39.58a 1.28 2.27bde
300 80 77.78 16 067abc 2.62abc 26.78abc 31.25ab 1.21 2.50abe
300 100 71.11 21 376a 2.77a 35.63a 35.35ab 0.99 2.93abe
300 120 73.33 19 186ab 2.72ab 31.98ab 36.43ab 1.16 2.71abe
350 60 77.78 18 495ab 2.70ab 30.83ab 34.70ab 1.13 2.22be
350 80 82.22 16 471abc 2.64abc 27.45abc 29.03ab 1.06 2.41bcde
350 100 71.11 15 567abc 2.61abc 25.94abc 28.97ab 1.12 2.21be
350 120 71.11 18 710ab 2.71ab 31.18ab 33.23ab 1.07 2.33bcde
400 60 68.89 14 186bc 2.56bc 23.64bc 25.61b 1.10 1.98e
400 80 80.00 14 233bc 2.57bc 23.72bc 27.66ab 1.16 1.87e
400 100 80.00 11 662c 2.46c 19.44c 23.08b 1.22 1.84e
400 120 73.33 11 733c 2.47c 19.56c 22.95b 1.17 1.94e

Values with the same letter superscript in the same column are not statistically di¡erent (P  0.05).
SGR, speci¢c growth rate; IWG, individual weigh gain; FCR, food convertion rate; PER, protein e⁄ciency rate.

in£uence of mortality on growth. The weight gain^

survival ratio was not signi¢cant (r2 50.037;
P 5 0.1365), indicating that cray¢sh growth was only
a¡ected in the experimental diets.
The cray¢sh fed with 250,300 and 350 g kg  1 pro-
tein levels showed signi¢cantly better growth than
the organisms fed with the 200 and 400 g kg  1 pro-
tein treatments without any relation to the lipid con-
tent. The lipid levels for all diets had no signi¢cant
e¡ect on cray¢sh growth (Table 4).
Better WG (%), SGR (% day  1) and DWG
(mg day  1) were observed in cray¢sh fed with the
250/120 and 300/100 diets, with no statistical
di¡erence between them, with the being signi¢-
cantly higher (Po0.05) than those with 400/60,
400/80, 400/100 and 400/120 levels. Feed intake of
the experimental cray¢sh in the di¡erent treat-
ments was proportional to biomass increase and Figure 1 Response surface model of the e¡ects of dietary
nutrient assimilation e⁄ciency. Cray¢sh fed with protein and lipid levels on weight gain in Procambarus
the diets 250/120 and 300/60 had signi¢cantly acanthophorus.
higher (Po0.05) IFI values than the 400/60, 400/
100 and 400/120 treatments. No di¡erences were The e¡ect of P/L levels in the cray¢sh weight gain
observed between treatments for cray¢sh FCR, (%) was evaluated using the response surface model.
with the highest e⁄ciency (0.99) in the organisms Data showed a protein level between 200 and
of 300/100 and the lowest (1.28) with the 300/60 300 g kg  1 for the best cray¢sh growth, without an
treatment. e¡ect of the lipid ratio (Fig. 1).

r 2010 The Authors

6 Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10
Aquaculture Research, 2010, 1^10 Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al.
26000
WG (%) = –14864.28+251.14x –0.45x2; 0.95 Conf. Int.
24000
22000
20000
Weight gain (%)
18000
16000
14000
12000
10000
8000
180 200 220
Figure 2 Relationship between weight gain and dietary protein level for optimum growth (Xmax) and a lower protein
level (X0) for Procambarus acanthophorus.
The equation for the response surface model was
WGð%Þ ¼ 17013:33 þ 277:41x  53:48y
 0:45x2  0:29xy þ 0:81y2
when
x ¼ Protein level in the diet ðg kg1 Þ and
y ¼ Lipid level in the diet ðg kg1 Þ
The optimal dietary protein level for the maximum
growth of P. acanthophorus was 279 g kg  1; these
data were determined graphically based on the WG
(%) results, using a second-order quadratic regres-
sion analysis. The 95% con¢dence limits help to de-
termine the dietary protein level (between 211 and
232 g kg  1) that is able to minimize feed costs with-
out a¡ecting growth (Fig. 2).
Discussion
The present reseach combined di¡erent levels of pro-
tein and lipid to determine the e¡ects on growth, sur-
vival and carcass composition. The carcas body
composition in males and females cray¢sh fed the ex-
perimental diets in the present study was similar,
without any e¡ect related to the experimental diets
(P/L ratio). In contrast, HernaŁndez-Vergara et al.
(2003) found a di¡erence between sexes in the lipid
metabolic routes in pre-adults C. quadricarinatus,
where females had a higher carcas lipid level than
r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10
r2 = 0.5428; r2 adjusted = 0.5267
240 260 280 300 320 340 360 380 400 420
Dietary protein levels (g Kg–1)
males with the same age. Results were associated
with storage of lipid reserves for egg development or
vitellogenesis for all treatments (4%, 8% and 12%
lipid ratio). In the present study, a similar carcass
composition and growth could be associated with
the age of the cray¢sh. Our observations in the la-
boratory with the species suggest a reproductive be-
haviour 2 or 3 weeks before the beginning of gonadic
development, a period when females begin to store
more energy for vitellogenensis. In the 200/120,
250/60 and 400/120 treatments, ovigerous females
were found, but the eggs did not complete their devel-
opment, probably because the females did not have
enough energy store in the eggs.
The survival average in the present trial (66^86%)
was similar to previous results under laboratory con-
ditions for P. acanthophorus (Cervantes-Santiago et al.
2007), which could be because the species has a sig-
ni¢cantly less aggressive and territorial behaviour
than other cray¢sh. In agreement with these data,
Cruz-Ordonez (2009) indicated no aggressive beha-
viour in P. acanthophorus did not increase at a density
as high as 100 org m  2, without signi¢cantly di¡er-
ences in growth and survival rates. Related data were
observed by Carmona-Osalde et al. (2005) in P. llamasi
mantained under controlled conditions (87^100%
survival). In contrast, Webster, Goodgame-Tiu, Tid-
well and Rouse (1994) obtained 50^71% survival
rates for juvenile C. quadricarinatus, when the mortal-
ity rate was associated with cannibalism. Jones, De
7
Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al. Aquaculture Research, 2010, 1^10
Silva and Mitchell (1996) obtained rates of 95^100%
for ‘Common yabby Cherax destructor Austin, 1996’
and 100% for ‘White yabby C. albidus Austin, 1996’
when individual contained were used in the experi-
mental period.
The lipid ratio tested in the present research (60,
80, 100 and 120 g kg  1) had no signi¢cant e¡ect on
the survival, growth or feed e⁄ciency in P. acantho-
phorus, as apparently all energy and essential fatty
acid requirements were being met through the lipid
source (1:1 animal/vegetal source). The result sug-
gested a speci¢c essential fatty acid requirement,
more than the dietary lipid level. Similar data were re-
ported by Davis and Robinson (1986), who estimated
the dietary lipid requirement level of the White cray-
¢sh P. acutus acutus with no di¡erences in growth
with a dietary lipid level between 0 and 120 g kg  1;
for this, 40 g kg  1 was suggested for diet formula-
tion. Other authors recommended a lipid level be-
tween 40 and 120 g kg  1, and no more than
120 g kg  1 in crustacean diets (D’Abramo, Wright,
Wright, Bordner & Conklin 1985; New 1987; Corte¤s-
Jacinto, Villarreal-Colmenares, Cruz-SuaŁrez, Civera-
Cerecedo, Nolasco-Soria & HernaŁndez-Llamas 2005).
Diets must also be supplemented with 0.5%
cholesterol to promote e⁄cient lipid metabolism
(HernaŁndez-Vergara, Olvera-Novoa & Rouse 2004).
The best growth and survival in A. astacus are ob-
tained with dietary lipid levels between 55 and
100 g kg  1, with a lower survival rate with a high le-
vel (160 g kg  1) (Ackefors, Castell, Boston, Rty &
Svensson 1992). In intensive and semi-intensive cul-
tures of C. quadricarinatus, practical diets can contain
42 g kg  1 lipids without adverse e¡ects on the
growth, when the natural production of a nutrient
supplement occurs in the culture tanks (HernaŁndez-
Vergara et al. 2003).
The results of the present research suggested a
dietary protein requirement of P. (A.) acanthophorus
between 232 and 279 g kg  1 (minimum 211), with a
60 g kg  1 lipid ratio. These results suggested good
nutritional e⁄ciency capacity of the species for use
as energy in the diets from di¡erent sources (P/L).
The 250/120 (P/L) treatment provided the best
growth and nutritional e⁄ciency, but the data sug-
gest that it is possible to use di¡erent P/L ratios
without a¡ecting the growth, to improve the feed
costs. Protein levels below 250 g kg  1 and above
400 g kg  1 yielded lower growth rates, probably due
to the cray¢sh-speci¢c protein requirement, in which
low levels cover basal metabolic requirements but do
not allow for growth, while a level above the require-
8 Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 1^10
ment produces metabolic stress in the organism be-
cause it expends extra energy in eliminating excess
nutrients, thus reducing the feed e⁄ciency and
growth rate. The dietary protein requirement
found in this research for P. acanthophorus is similar
to previous reports for other cray¢sh. Jover et al.
(1999) reported a minimum protein requirement
of 220 g kg  1 for P. clarkii, and recommended a range
of 220^260 g kg  1; lower levels decreased the
growth rates. Ackefors et al. (1992) suggested that
commercial diets for A. astacus must contain 300^
350 g kg  1 protein, 200^250 g kg  1 carbohydrates
and no more than 100 g kg  1 lipid. For ‘Signal cray-
¢sh Pacifastacus leniusculus, Dana, 1852’, Celada, Car-
ral, Gaudioso,Temino and FernaŁndez (1989) reported
a protein requirement between 270 and 330 g kg  1
for good growth, which could be improved by supple-
menting with fresh aquatic plants. Webster et al.
(1994) indicated that a 330 g kg  1 protein level is
adequate for recently enclosed C. quadricarinatus lar-
vae. This agrees with the 330 g kg  1 protein reported
as producing the most e⁄cient growth in C. quadri-
carinatus juveniles (weight 41g) when compared
with lower levels (Keefe & Rouse 1999). HernaŁndez-
Vergara et al. (2001) reported protein requirements
ranging from 300 to 350 g kg  1 and a lipid require-
ment of 40^80 g kg  1 for good growth rates in larvae
and juveniles of C. quadricarinatus. In most cases, the
protein requirement for some cray¢sh species is
higher than those found for P. acanthophorus; this
could be related to the trophic place of cray¢sh in
the environment, when detritivore species are at the
bottom of the trophic chain with a very low metabolic
requirement, and consequently the low protein re-
quirement observed here is not unexpected.
The present results indicate that under controlled
conditions, the cray¢sh P. acanthophorus requires a
diet containing 279 g kg  1 protein and 60 g kg  1
lipid for optimum growth, with a protein proportion
of 75/25% vegetal/animal source. The low protein
requirement and e⁄cient feed conversion rate of
P. acanthophorus make it a very promising species for
aquaculture. Feed costs can be minimized without
a¡ecting the growth performance with a protein le-
vel as low as 211^232 g kg  1.
Acknowledgements
This research was supported by ITBOCA, and the
Mexican Science and Thechnology Council (CONA-
CYT) with a Master’s in Science sholarship 201573.
r 2010 The Authors
Aquaculture Research, 2010, 1^10 Protein lipid ratio for growth and survival of juvenile cray¢sh E Cervantes-Santiago et al.
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