Aquaculture Reports 20 (2021) 100709

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Effect of nursery system and stocking density to produce juveniles of

whiteleg shrimp Litopenaeus vannamei
Delia Rodríguez-Olague a, Jesús T. Ponce-Palafox a, b, *, 1,
Sergio G. Castillo-Vargasmachuca a, b, *, 1, Eulalio Arámbul-Muñoz a, Raúl C. de los Santos a,
Héctor M. Esparza-Leal c
a
Maestría en Ciencias Biológicas y Agropecuarias (CBAP), Universidad Autónoma de Nayarit, Mexico
b
Laboratorio de Bioingeniería Costera. Escuela Nacional de Ingeniería Pesquera. Universidad Autónoma de Nayarit, Mexico
c
Departamento de Acuacultura, Instituto Politécnico Nacional-CIIDIR Unidad Sinaloa, Guasave, Sinaloa, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: Indoor nursery systems are becoming increasingly popular for high-quality whiteleg shrimp production. These
Clean-water systems allow for a significant degree of control and management in a biosecure manner. However, it is still
Photo-heterotrophic unclear as to what type of production system is best suited for indoor whiteleg shrimp nurseries. This study
Biofloc technology
evaluated and compared clean-water recirculating aquaculture system (CW-RAS), photo-heterotrophic (PT), and
Carrying capacity
Indoor tanks nursery
biofloc technology (BFT) systems in terms of water quality, growth performance, and production costs of
whiteleg shrimp Litopenaeus vannamei at three stocking densities (500, 1000 and 1500 org/m3) for 35 d in the
nursery phase. In this study, twenty-seven, 100-L circular plastic tanks (microcosm) were randomly assigned to
one of the treatments and stored with postlarvae shrimps (0.016 g). Throughout the experiment, water quality
parameters remained within acceptable ranges for whiteleg shrimp growth. High concentrations of total
ammonia-nitrogen (TAN) were determined in the CW-RAS system and nitrates and nitrites in the BFT system.
Furthermore, the growth parameters showed significant differences (P < 0.05) between the treatments; higher
values in PT and BFT systems at 500 org/m3 and lower survival in the CW-RAS system. These results suggest that
a dense-dependent effect was observed from 1000 org/m3 onwards. In general, postlarvae, feed, labor and energy
accounted for higher costs. The study findings further indicate that CW-RAS had the highest production costs for
1000 juveniles.

1. Introduction
The culture of whiteleg shrimp, Litopenaeus vannamei continues to
expand worldwide, reaching a production of 4,966,200 MT (FAO,
2020). Over the past three years, the production increased at a com­
pound annual growth rate (CAGR) of 5.7 percent (Anderson et al.,
2019). However, in recent times, environmental degradation, climate
change, and the proliferation of pathogens have emerged as key chal­
lenges that are preventing the sustainable development of the world’s
whiteleg shrimp farming industry (Jory, 2019). Additionally, interna­
tional market prices, feed, fuel costs, and whiteleg shrimp diseases
continue to have a negative influence on Latin America’s whiteleg
shrimp producers (Anderson et al., 2019).
Over the last five decades, whiteleg shrimp farming has changed
from the original model Generation 1 (large ponds and low production)
to the current Generation 5 (small ponds, total water recirculation, and
waste capture with more environmental controls for high production),
which signifies a model of high biosecurity (McIntosh, 2019). The latter
model generally includes controlled systems with smaller ponds and
tanks, shaded, and covered to reduce environmental fluctuations.
Additionally, it requires healthy postlarvae free from diseases and
modern hatcheries with sanitation, quality control, automation, recy­
cling of water, and reduction in the use of fresh food (Jory, 2019).
As with other industries, productivity remains a key factor affecting
the bottom line of whiteleg shrimp producers. Consequently, the two-
phase system that includes a nursery stage followed by the grow-out
stage has been implemented for more than two decades in order to
improve the quality of whiteleg shrimp production at a super-intensive

* Corresponding authors at: Laboratorio de Bioingeniería Costera. Escuela Nacional de Ingeniería Pesquera. Universidad Autónoma de Nayarit, Mexico.
E-mail addresses: jesus.ponce@usa.net (J.T. Ponce-Palafox), sergio_machuca_@hotmail.com (S.G. Castillo-Vargasmachuca).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.aqrep.2021.100709
Received 11 August 2020; Received in revised form 20 April 2021; Accepted 20 April 2021
Available online 30 April 2021
2352-5134/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
D. Rodríguez-Olague et al.
level (Samocha and Lawrence, 1998). In whiteleg shrimp production
intensification, the density of the shrimp population remains the most
important parameter. Several physico-chemical variables and popula­
tion parameters, such as specific growth rate (SGR) and survival rate,
tend to be critical at extreme limits, in high densities. On the basis of
these parameters, the most appropriate density of the whiteleg shrimp
L. vannamei population in the grow-out phase in the biofloc technology
(BFT) system is 500–625 org/m2 (Da Silveira et al., 2020). However, it is
imperative to note that the quality of postlarvae management in the
nursery phase is a key factor for their growth and survival as well as the
basis for juveniles’ maximum growth performance in the grow-out phase
(Zelaya et al., 2007).
In the whiteleg shrimp farming industry, the nursery systems provide
numerous benefits, including improved inventory, increase in size uni­
formity, less predation, reduced pond grow-out duration, an increase in
the number of harvests per year, better feed conversion efficiencies, and
greater biosecurity (Persyn and Aungst, 2001; Arnold et al., 2006). Over
the past two decades, a series of strategies have been used to intensify
the nursery phase of whiteleg shrimp production. This includes the use
of high-protein feeds, fertilizers, indoor recirculating raceways, and
artificial substrates (Moss and Moss, 2004). Consequently, in the middle
of the last decade, recirculating aquaculture systems (RAS) technology
for nursery whiteleg shrimp production has emerged as the most
advanced and successful system with high survival (52.0%–97.5%) and
high densities of 3,000–8,300 org/m3 (Davis and Arnold, 1998; Cohen
et al., 2005; Mishra et al., 2008; Tierney et al., 2020).
Currently, new farming techniques and systems are required to
maximize production in the nursery and grow-out phases, mitigate the
negative effects of heightened stocking density, and generate high
quality (premium price) whiteleg shrimp at the lowest cost. This has
resulted in the development of improved whiteleg shrimp farming sys­
tems in intensive and hyper-intensive controlled systems including CW-
RAS, PT, and BFT systems. Today, these technologies have been suc­
cessful on a commercial level and can be applied depending on the re­
sources, conditions, and market strategies. Although the CW-RAS
maintains high water quality and density, the system can have higher
start -up and operating costs (Table1). PT system allows for high growth
at low production costs. Furthermore, whiteleg shrimps show good
growth and survival in the BFT system, which, entails higher costs than
the PT system.
The PT system is based on the balance between photoautotrophic
(phytoplankton) and heterotrophic (bacteria) organisms (McIntosh,
2001; Huda et al., 2013; Moreno-Figueroa et al., 2018). In PT and BFT
systems, the density is determined in the nursery and grow-out phases.
In the nursery phase, low densities of 1500 org/m3 have been used to
high densities of 9000 org/m3, finding the best specific growth rate
(SGR) at 1500 org/m3 and demonstrating that growth in the nursery
phase is density-dependent (Esparza-Leal et al., 2015). In addition, when
a substrate is integrated into the BFT, the lateral area of the tank in­
creases, which, in turn, causes a higher growth of biofilms and natural
feeds. Consequently, it improves water quality, growth, feed conversion
ratio (FCR), and immune parameters of whiteleg shrimp culture (Fer­
reira et al., 2015; Kumar et al., 2019).
Table 1
Factor and level of the different types of systems studied in the culture of white
shrimp L. vannamei. Clear-water recirculating aquaculture systems (CW-RAS),
photo-heterotrophic (PT), and biofloc technology (BFT).
Factors RAS PT
Water quality
+++ ++
Productivity natural – +
Density +++ ++
Specific growth rate + +++
Survival ++ +++
Costs +++ + ++
Importance level factor: +, low; ++, medium; +++, high; -, nil.
Aquaculture Reports 20 (2021) 100709
An analysis of the effect of stocking density of L. vannamei in the BFT
system has been undertaken comparing with CW-RAS system as a
controlled environment. Most studies suggest that no differences exist
between the two systems with a non-significant trend for better growth
(Esparza-Leal et al., 2015; Ray et al., 2017; Martinez-Porchas et al.,
2020). The implementation and selection of these RAS for the produc­
tion of whiteleg shrimp L. vannamei in the nursery phase remain reliant
on the location. However, it is also essential to consider the availability
of inputs and production strategies as well as the understanding of
physical-chemical, biological, and economic factors for
decision-making. (Juarez and Martinez-Cordero, 2004). Thus, the study
of production costs from the nursery phase, along with other factors,
helps deepen the understanding of the whiteleg shrimp industry’s
overall economy. The operation costs particularly depend on services,
supplies, size of the operation, design of the nursery system (Naegel,
2010), and energy expenditure. Accordingly, a bioeconomic analysis
and stochastic model have been performed to elucidate the single-phase
production dynamics of a photo-heterotrophic system (Moreno-Figueroa
et al., 2019), where feed and postlarvae account for the main costs. Thus
far, no studies have been carried out in the nursery phase. The com­
parison of different RAS is required to include the elements of decision
for their best application. This study aimed to compare three nursery
systems for whiteleg shrimp production – CW-RAS, PT, and BFT in terms
of water quality, growth performance, survival, and production costs at
different stocking densities.
2. Materials and methods
2.1. Location and animals
The experiments were performed in the Coastal Bioengineering
Laboratory of the National School of Fishery Engineering, University of
Nayarit, San Blas Nayarit, Mexico (21◦ 29′ N, 105◦ 12′ W). The postlarvae
of L. vannamei were supplied by a commercial laboratory (Acopio de
Larvas y Asesoría en Proyectos S.A. de C.V.®) located in Matanchen Bay,
San Blas, Nayarit. Mexico.
2.2. System and experimental design
A completely randomized design method with two independent
variables was used in this study. These variables include treatment
systems (CW-RAS, PT, and BFT) and stocking densities (500 (D500),
1000 (D1000) and 1500 (D1500) org/m3). Nine treatments were tested
in triplicate for 35 d. The study was conducted in twenty-seven circular
indoor polyethylene nursery tanks (0.1 m3) stocked with 28-d postlarvae
L. vannamei (initial mean weight of 0.016 g ± 0.002 g) that were hand-
counted into the tanks.
Water oxygenation in each tank was provided with 1 air diffuser
stone and generated by an integrated system of air of 4.5 HP regenera­
tive blower. Then, the aeration was conducted through a plastic hose (2′′
diameter) to the end in each tank. For all systems, the water was sup­
plied directly from the sea through PVC pipe (3′′ diameter) with a
Jacuzzi Magnum force3® type pump, which passed through two Life­
gard® Aquatic’s filters with zeolite sand and activated carbon, respec­
tively also ending in the culture units. Both aeration and water lines
were disinfected with 5% formalin and 30 % hydrochloric acid,
respectively. There was no renewal of water in all systems during the
study period; only the water lost due to evaporation was replaced. Each
BFT CW-RAS tank had an external biofilter composed of an 8-cm air diffuser;
the water passed through cotton wadding, zeolite sand, and activated
carbon, thus leaving a drain at the bottom and returning to the shrimp
+
++
+ tank through a pump "Power Head SPH-800.′′ An internal support tube
++ maintained the water level, ensuring the exchange of water between a
++ filter and the culture tank.
Cyclotella choctawhatcheeana microalgae (180,000 cel/mL) was
added to the PT system on days 1, 5, 10, and 15 of the experiment with a
2
D. Rodríguez-Olague et al.
proportion equivalent to 8% of the total volume of each tank to maintain
a C:N ratio of 2–3 (Martinez-Porchas et al., 2020). The microalgae in­
oculums were obtained from Commercial Production Laboratory “Aco­
pio de Larvas y Asesoría en Proyectos S.A. de C.V®”. In addition, a
commercial probiotic (Neutrobacter®) was added to each tank once per
week until the end of the trial (250 mL/m3) with a view to promoting a
heterotrophic microbial complex. Furthermore, 10 % of molasses per kg
of feed was added to each tank at the beginning of the experiment so as
to improve nitrification.
In the BFT system, biofloc water in the experimental tanks came from
a matured biofloc shrimp tank (with 0.935 mg/L, 0.325 mg/L, 3.392
mg/L, 0.564 mg/L, and 180.0 mg/L of total ammonia nitrogen, nitrite,
nitrate, phosphate, and total suspended solids, respectively), where
molasses, wheat flour, balanced feed, and shrimp were previously
applied. The C:N ratio for BFT was adjusted to 12:1 by adding molasses
as a carbon source weekly and putting plastic mesh in each tank as a
substrate to generate the formation and proliferation of biofilm and
biofloc (Marinho et al., 2017).
The postlarvae were fed a commercial feed containing 0.9–1.1 mm
40 % crude protein (Maltacleyton®, Mexico) at different times of day
(0800, 1400, and 2000 h). The daily feeding rates were initially at
16− 10% of estimated biomass. Subsequently, the feed was adjusted
daily according to its consumption, to determine the consumption; 10 %
of the pellet was placed in trays and the rest was distributed in the tank.
2.3. Water analysis
The experimental area’s photoperiod was 12/12 h light/dark cycle.
The water temperature was maintained by two Minisplit Mabe Cold and
Heat 12,000 BTUs placed in strategic areas that regulated the temper­
ature of the experimental room. For each tank, the dissolved oxygen (DO
mg/L) was recorded on a daily basis every 6 h (8:00, 14:00, 20:00, and
02:00 h) with YSI 550A® (YSI Incorporated, Yellow Springs, Ohio, USA).
Additionally, the salinity and pH were recorded twice a day (7:00 and
19:00) with a Vital Sine SR-6® refractometer and Hanna pHep Tester®
potentiometer, respectively. Every third day, the YSI 9500 direct-read
photometer® (YSI Incorporated, Yellow Springs, Ohio, USA) was used
to measure total ammonia nitrogen (TAN), non-ionized ammonium
(NH3-N), nitrite (NO2-N), and nitrate (NO3-N) concentrations in mg/L.
2.4. Growth and production performance
Biometrics was performed at the beginning and end of the experi­
ment by weighing, measuring, and counting all the shrimps from each
experimental tank using a digital balance (precision 0.001 g, Sartorius®)
and a stainless steel metal ruler with conversion table 30.5 ± 0.5 cm.
Initial body weight (IBW, g) and initial biomass (IB, g) were determined
at the beginning of the study. After 35 d, at the end of the study, the total
number of shrimp (TNS), final body weight (FBW, g), final length (FL,
cm), condition factor (CF), total feed consumption (TFC, g), feed con­
version factor (FCR), feed efficiency (FE), final biomass (BF, g), Pro­
duction (P, kg/m3), variation coefficient-body weight (CV-BW, %),
weight gain (WG), growth rate (GR, g/week), specific growth rate (SGR,
%/d), and survival (S, %) were determined.
2.5. Production costs
All the production costs of each treatment were recorded. Expendi­
tures were shown as costs per whiteleg shrimp produced per cubic meter
and cost per 1000 juvenile of whiteleg shrimp produced at the end. The
production costs were grouped into 8 categories: postlarvae, feed,
microalgae, probiotics, chemicals, operating supplies, labor, and energy
(Juarez and Martinez-Cordero, 2004; Naegel, 2010).
3
Aquaculture Reports 20 (2021) 100709
2.6. Data management and analysis
The normality tests (Shapiro-Wilk) and homoscedasticity (Levene)
were determined for each data group. Additionally, the variables were
analyzed using the One-way Analysis of Variance (ANOVA) and post-hoc
Tukey test (Zar, 2010). In all cases, the level of significance was found to
be 5% (P < 0.05). Before the analysis, the survival data were trans­
formed (arcsine of the square root) and expressed as mean ± standard
deviation. Using the data obtained in the study, the projections of the
density-production relationship were made up to a density of 9000
org/m3. This density was obtained under the similar conditions of pre­
vious works comparing CW-RAS with BFT in L vannamei (Esparza et al.,
2015). The extrapolation of the density-production relationship was
performed in accordance with the criteria of López-Uriostegui et al.
(2014), where the relationship produced a quadratic equation in the
form:
Production = β + β + β2d2, (1)
where β2 is a quadratic coefficient (other than 0), β1 signifies the linear
coefficient, β0 is the intercept, and d denotes the density
The maximum density (dmax) was obtained as follows:
dmax = β1/ 2β2, (2)
where dmax = maximum density.
The maximum production was given by
Productionmax = β0 + ((β1)2 / 2β2) - β1/2, (3)
3. Results
3.1. Water physical and chemical variables
The variables of temperature, salinity, pH, and OD did not show
significant differences (P > 0.05) in all treatments throughout the 35 d of
the experiment. The mean values and standard deviation (SD) for these
variables are shown in Table 2. Among all the tested densities, the
highest concentrations of TAN were observed in the CW-RAS treatment,
reaching a mean concentration of 1.23 mg/L in D1500. On the other
hand, the highest concentrations of NO2-N and NO3-N were determined
in the BFT treatment in all the tested densities, reaching a maximum
concentration from 3.45 to 3.92 mg/L and 10.65–13.48 mg/L,
respectively.
3.2. Growth and production performance
In general, differences (P < 0.05) were found in growth performance,
use of feed, and production parameters in all the treatment systems and
densities. The whiteleg shrimps grew significantly larger with greater
survival in the PT and BFT treatments than in CW-RAS (Table 3). It
caused higher biomass and production in the density of 1500 org/m3
with a better feed conversion ratio and feed efficiency in the BFT
treatment. However, the coefficient of variation of the whiteleg shrimp
was observed to be lower in the BFT treatment in all densities.
The effect of density on the whiteleg shrimp production in CW-RAS,
PT, and BFT systems was described by a quadratic (or parabolic) func­
tion – a polynomial function of the second degree described in Fig. 1.
Where the equation for CW-RAS, BF and PT were y = -2E-08x2 +
0.0003x + 0.0625, R2 = 0.9978; y = -2E-08x2 + 0.0004x + 0.3432, R2 =
0.998 and y = -2E-08x2 + 0.0004x + 0.5688, R2 = 0.9911, respectively.
The carrying capacity (CC) at 8000 org/m3 for PT and BFT and 9000
org/m3 for CW-RAS (Fig. 1a)
D. Rodríguez-Olague et al. Aquaculture Reports 20 (2021) 100709

Table 2
Physical and chemical variables of water of the L. vannamei in clear-water recirculating aquaculture systems (CW-RAS), photo-heterotrophic (PT), and biofloc
technology (BFT) treatments during the nursery phase cultured indoor at 500 (D500), 1000 (D1000) and 1500 (D1500) orgs/m3 over 35-d.

Parameters/ D500 D1000 D1500

treatment CW-RAS PT BFT CW-RAS PT BFT CW-RAS PT BFT

Temperature (ºC) 29.5 ± 0.7a 29.4 ± 0.6a 29.4 ± 0.7a 29.3 ± 0.7a 29.4 ± 0.6a 29.4 ± 0.7a 29.4 ± . 0.8a 29.4 ± . 0.1a 29.4 ± . 0.6a
Salinity 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a 34.9 ± 0.1a
pH 8.47 ± 0.07a 8.42 ± 0.11a 8.45 ± 0.11a 8.42 ± 0.13a 8.39 ± 0.12a 8.45 ± 0.11a 8.44 ± 0.06a 8.41 ± 0.11a 8.41 ± .09a
Oxygen (mg/L) 6.24 ± 0.75a 6.35 ± 0.72a 6.48 ± 0.69a 6.14 ± 0.75a 6.25 ± 0.72a 6.37 ± 0.69a 6.11 ± 0.70a 6.17 ± 0.76a 6.25 ± 0.69a
TAN (mg/L) 1.11 ± 1.17a 0.49 ± 0.29b 0.93 ± 0.72a 1.19 ± 1.28a 0.51 ± 0.34b 1.06 ± 0.85a 1.23 ± 1.17a 0.65 ± 0.46b 1.09 ± 0.93a
NO2-N (mg/L) 0.34 ± 0.26c 2.20 ± 1.15b 3.69 ± 1.18a 0.40 ± 1.26c 2.08 ± 1.16b 3.42 ± 1.11a 0.67 ± 0.31c 2.11 ± 1.18b 3.95 ± 1.12a
NO3-N (mg/L) 4.29 ± 2.12b 1.50 ± 1.02c 10.65 ± 2.30a 5.61 ± 2.98b 1.56 ± 0.89c 11.38 ± 2.67a 9.5 ± 2.16b 1.69 ± 0.85c 13.48 ± 2.35a

Within a row, values (mean ± SD) accompanied by different letters are significantly different (P < 0.05). TAN, total ammonia nitrogen; NO2-N, nitrite; and NO3-N,
nitrate.

Table 3
Growth performance, survival and distribution size of the L. vannamei in clear-water recirculating aquaculture systems (CW-RAS), photo-heterotrophic (PT), and
biofloc technology (BFT) treatments, during the nursery phase cultured indoor at 500 (D500), 1000 (D1000) and 1500 (D1500) orgs/m3 over 35-d.

Parameters/ D500 D1000 D1500

treatment CW-RAS PT BFT CW-RAS PT BFT CW-RAS PT BFT

IBW (g/org) 0.016 ± 0.016 ± 0.016 ± 0.016 ± 0.016 ± 0.016 ± 0.016 ± 0.016 ± 0.016 ±
0.007a 0.007a 0.007a 0.007a 0.007a 0.007a 0.007a 0.007a 0.007a
FBW (g/org) 0.44 ± 0.05d 1.27 ± 0.09a 1.21 ± 0.03a 0.42 ± 0.06d 0.95 ± 0.09b 0.91 ± 0.02b 0.41 ± 0.05d 0.82 ± 0.09c 0.80 ± 0.03c
FL (cm) 4.59 ± 0.11c 5.42 ± 0.50a 6.02 ± 0.36a 4.38 ± 0.51c 5.35 ± 0.56a 5.86 ± 0.16a 4.57 ± 0.33c 5.20 ± 0.57a 5.38 ± 0.18a
CF (%) 0.46 ± 0.03d 0.80 ± 0.12a 0.55 ± 0.06b 0.45 ± 0.16d 0.62 ± 0.14b 0.45 ± 0.04d 0.43 ± 0.05d 0.58 ± 0.11b 0.50 ± 0.02c
FB (g) 203.0 ± 3.2d 655.1 ± 5.5b 550.2 ± 4.9b 347.1 ± 7.2c 915.3 ± 7.7a 693.1 ± 5.8b 493.2 ± 1150.2 ± 9.6a 856.9 ± 9.8a
9.62c
P (kg/m3) 0.20 ± 0.00d 0.65 ± 0.00b 0.55 ± 0.00b 0.35 ± 0.00c 0.92 ± 0.00a 0.69 ± 0.00b 0.49 ± 0.01c 1.15 ± 0.01a 0.86 ± 0.01a
TFC (g) 21.53 ± 67.10 ± 53.87 ± 47.18 ± 92.17 ± 84.16 ± 68.18 ± 125.20 ± 118.82 ±
2.11e 11.12c 5.99c 6.23d 29.07b 10.05b 5.37c 29.83a 13.73a
FCR 1.39 ± 0.27b 1.34 ± 0.12b 1.21 ± 0.14a 1.78 ± 0.31d 1.46 ± 0.24c 1.28 ± 0.14a 1.82 ± 0.29d 1.48 ± 0.23c 1.40 ± 0.21c
FE (%) 0.72 ± 0.02b 0.72 ± 0.01b 0.83 ± 0.01a 0.56 ± 0.03c 0.68 ± 0.02b 0.78 ± 0.02a 0.55 ± 0.02c 0.68 ± 0.03b 0.71 ± 0.02b
CV-BW 9.09 ± 0.03c 7.12 ± 0.02b 2.50 ± 0.01a 13.11 ± 10.36 ± 2.64 ± 0.03a 11.44 ± 9.10 ± 0.02c 3.44 ± 0.02a
0.02c 0.01c 0.04c
WG (g/org) 0.42 ± 0.02d 1.25 ± 0.07a 1.19 ± 0.03a 0.40 ± 0.05d 0.93 ± 0.04b 0.89 ± 0.03b 0.39 ± 0.02d 0.80 ± 0.04c 0.78 ± 0.03c
GR (g/week) 0.012 ± 0.036 ± 0.034 ± 0.012 ± 0.027 ± 0.026 ± 0.011 ± 0.023 ± 0.022 ±
0.001c 0.003a 0.002a 0.001c 0.002b 0.002b 0.001c 0.003b 0.003b
SGR (%/d) 9.47 ± 0.03c 12.50 ± 12.36 ± 9.34 ± 0.33c 11.67 ± 11.55 ± 9.27 ± 0.22c 11.25 ± 0.02b 11.18 ± 0.15b
0.44a 0.36a 0.15b 0.07b
S (%) 92.1 ± 2.5b 98.9 ± 4.1a 98.5 ± 2.5a 82.5 ± 3.5b 96.3 ± 1.9a 95.2 ± 3.6a 80.1 ± 4.1b 94.6 ± 4.3a 93.3 ± 2.6a

Within a row, values (mean ± SD) accompanied by different letters are significantly different (P < 0.05). IBW, initial body weight; FBW, final body weight; FL, final
length; CF, condition factor; FB, final biomass; P, production; TFC, total feed consumed; FCR, feeding conversion ratio; FE, feeding efficiency; CV-BW, coefficient of
variation- body weight; WG: weight gain; GR: growth rate; and SGR: specific growth rate; S, survival.

3.3. Production costs

Four types of costs contributed over 85 % of the total production

costs in all treatments (Table 4). The highest costs were in the order of
importance – labor, postlarvae, energy, and feed – for CW-RAS. The
order of importance was labor, postlarvae, feed, and microalgae for PT
and labor, postlarvae, chemical, and feed for BFT.

4. Discussion

Temperature, salinity, DO, and pH remained within acceptable

Fig. 1. Relationship between production (color lines) and survival (black lines)
of whiteleg shrimp L. vannamei at different densities in nursery system. Carrying
capacity (a). The curves of the treatments of CW-RAS (circle), BFT (square) and
PT (triangle) are shown.
ranges in all treatments for the growth of L. vannamei (Ponce-Palafox
et al., 2019). The levels of TAN, nitrites, and nitrates in all treatments
were within the range recommended for whiteleg shrimp culture
(Valencia-Castañeda et al., 2019). However, the TAN was higher in the
CW-RAS treatment as compared to the PT and BFT treatments. Addi­
tionally, all the measured TAN concentrations were similar or below the
estimated safe levels for rearing L. vannamei in salinities near 35 (Lin and
Chen, 2001). In the PT and BFT treatments, the lowest concentration of
TAN showed that the three TAN elimination processes described in
aquatic systems were presented in these treatments: photoautotrophic
elimination by algae, autotrophic bacterial conversion, and heterotro­
phic bacterial conversion directly into microbial biomass (Ebeling et al.,
2006). The maximum mean nitrite concentration recorded in all

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D. Rodríguez-Olague et al. Aquaculture Reports 20 (2021) 100709

Table 4
Comparative analysis of production costs of the L. vannamei in clear-water recirculating aquaculture systems (CW-RAS), photo-heterotrophic (PT), and biofloc
technology (BFT). Treatments system during the nursery phase cultured indoor at 500 (D500), 1000 (D1000) and 1500 (D1500) orgs/m3 over 35-d.
D500 D1000 D1500
Parameters/
treatment CW- % PT % BFT % CW- % PT % BFT % CW- % PT % BFT %
RAS RAS RAS

Postlarvae 2.25 30.0 2.25 37.4 2.25 31.9 4.5 42.0 4.5 51.3 4.5 45.6 6.8 49.2 6.8 57.9 6.8 52.9
Feed 0.59 7.9 0.58 9.6 0.49 6.9 1.54 14.4 1.09 12.4 1.05 10.6 2.35 17.0 1.76 15.0 1.73 13.5
Microalgae – 0.0 0.44 7.3 – 0.0 – 0.0 0.44 5.0 – 0.0 – 0.0 0.44 3.7 – 0.0
Probiotic – 0.0 0.19 3.2 – 0.0 – 0.0 0.19 2.2 – 0.0 – 0.0 0.19 1.6 – 0.0
Chemical 0.14 1.9 0.14 2.3 1.9 26.9 0.14 1.3 0.14 1.6 1.9 19.3 0.14 1.0 0.14 1.2 1.9 14.8
Operating 0.33 4.4 – 0.0 – 0.0 0.33 3.1 – 0.0 – 0.0 0.33 2.4 – 0.0 – 0.0
supplies
Labor 2.3 30.6 2.3 38.2 2.3 32.6 2.3 21.5 2.3 26.2 2.3 23.3 2.3 16.6 2.3 19.6 2.3 17.9
Energy 1.9 25.3 0.12 2.0 0.12 1.7 1.9 17.7 0.12 1.4 0.12 1.2 1.9 13.7 0.12 1.0 0.12 0.9
Total costs ($/m3) 7.51 6.02 7.06 10.71 8.78 9.87 13.82 11.75 12.85
Cost/1000 16.3 12.2 14.3 13.0 9.1 10.4 11.5 8.3 9.2
juvenile ($)

$ = US Dollar.

treatments was lower than the maximum value calculated (25.7 mg/L)
for L. vannamei (Lin and Chen, 2003). In their study, Ray et al. (2017)
found that the nitrite concentrations in CW-RAS were lower than in BFT,
as recorded in our experiment. This could be due to significantly (P <
0.05) high rates of water renewal in CW-RAS as compared to other
systems (Silva et al., 2015). Nitrate, the final product of nitrification,
was observed to have the highest concentration (p < 0.05) in the
CW-RAS and BFT treatments (Camargo et al., 2005). The nitrate levels
recorded for all treatments meanwhile, were below the critical levels for
L. vannamei (Furtado et al., 2015; Satanwat et al., 2020). Furthermore,
the final nitrate concentration was higher in the BFT system than in
CW-RAS and the pattern was similar to that reported in nursery tanks
employed in other studies for L. vannamei (Esparza-Leal et al., 2015,
2020; Ray et al., 2017). In the PT system, microalgae have helped in
reducing the concentration of nitrates (Jiménez-Ordaz et al., 2021).
However, the heterotrophic pathway does not generate nitrate (Rajta
et al., 2020), thus justifying the lower concentrations recorded in this
system.
The growth and survival parameters in CW-RAS, PT, and BFT sys­
tems were within the range of values considered adequate for the
nursery phase in intensive whiteleg shrimp farming (Godoy et al., 2012;
Esparza-Leal et al., 2015; Tierney et al., 2020). The values recorded in
this study were higher than those recorded in most experiments of these
culture systems. Better growth and productive performance (24.2 %)
than the CW-RAS system were observed in the PT and BFT systems, as
reported in other studies (Izquierdo et al., 2006; Luis-Villaseñor et al.,
2015; Guemez-Sorhouet et al., 2019). In the nursery phase, we found an
inversely proportional relationship between the growth performance of
L. vannamei and stocking density (Wasielesky et al., 2013; Silva et al.,
2015). Additionally, in the present study, FBW, WG, GR, and SGR values
were higher in the density of 500 org/m3. Growth reduction with
increased density in the nursery phase has been attributed to decreased
water quality, natural productivity, and space (Peterson and Griffith,
1999; Wasielesky et al., 2013; Silva et al., 2015). As previously reported
in other studies, the lower FCR value of the PT and BFT systems was
attributed to the availability of microalgae and microorganisms associ­
ated with the bioflocs compared to the CW-RAS system (Marinho et al.,
2017). In the current study, the highest feed efficiency was observed in
the BFT system at 500− 1,000 org/m3. Moreover, microalgae stocking in
the PT and BFT systems in this study should have contributed to a better
performance of the whiteleg shrimp (Godoy et al., 2012; Jiménez-Ordaz
et al., 2021).
In the nursery phase, the variability in whiteleg shrimp size was
caused by size-dependent factors that modify growth and the level of
overcrowding that can further modify size heterogeneity (Peacor et al.,
2007; Araneda et al., 2013). Size uniformity remains a critical aspect in
the final harvest of whiteleg shrimp farming at a commercial level
(Zelaya et al., 2007). In the nursery phase, this parameter can have a
wide distribution of size by weight up to 24.0%–70.0% of CV-BW (Moss
and Moss, 2004; Zelaya et al., 2007). However, in our study, CV of the
weight size distribution decreased in the BFT system, which was in
agreement with the findings of other studies in the nursery phase using
the BFT system (Khanjani et al., 2020). In our study, the CV-BW was low
(2.5 a 13.11 %) and exhibited the pattern: CW-RAS > PT > BFT. This
indicates that more than 97.5 % of the whiteleg shrimps in 35 d had a
relatively uniform weight in the BFT system. Additionally, survival was
not found to be affected by stocking density, as reported in similar
studies of the three systems evaluated at different stocking densities
(Wasielesky et al., 2013; Esparza-Leal et al., 2015;Guemez-Sorhouet
et al., 2019; Tierney et al., 2020).
The relationship between production and increased density in an
intensive RAS of whiteleg shrimp L. vannamei has been established as
dense-dependent type related through a quadratic function (Williams
et al., 1996). These results suggest that a dense-dependent effect was
observed from 8000 to 9000 org/m3 onwards. This effect was critical
when reaching carrying capacity (Fig. 1, letter "a"). The production
continues to increase, albeit at a decreasing rate (Hepher, 1978). In
contrast, survival had a density-independent effect in all the systems
evaluated in this study.
In the selection of CW-RAS, PT, and FBT systems at a commercial
level, it is recommended to not only consider productive efficiency as a
deciding factor in the nursery phase, but also analyze management
strategies through economic efficiency and optimize the inputs with a
view to obtaining the best return on investment (Davis et al., 2008).
Postlarvae shrimp prices and an increase in variable costs have caused
stress in the industry. Therefore, the production costs need to be opti­
mized in principle in order to improve economic returns. In this study,
the cost structure of the nursing phase of the CW-RAS, PT, and BFT
systems was closer to that of a hatchery than the grow-out phase, in
which the proportion was higher for the costs of labor, postlarvae,
feeding, and energy (Juarez and Martinez-Cordero, 2004). In addition,
the production costs were the highest for the CW-RAS system and the
lowest for the PT system. In the CW-RAS and BFT systems, we found high
production and survival of up to 8,000–9,000 org/m3 (Davis and Arnold,
1998; Cottingham, 2015;Esparza-Leal et al., 2015).
5. Conclusion
In this study, the physico-chemical variables of water, temperature,
salinity, pH, and DO did not show significant differences in all treat­
ments. The CW-RAS system had significantly higher levels of TAN, and
nitrite-nitrate concentrations were found to be higher in BFT. A non-
significant tendency was observed to present higher concentrations of
TAN and nitrates with increasing density in all treatments. Furthermore,

5
D. Rodríguez-Olague et al.
growth performance, production, and survival were significantly
improved in the PT and BFT treatments in comparison to the CW-RAS
treatment. The dense-dependent effect of whiteleg shrimp growth in
nursery culture began to critique 7000 org/m3 and 8000 org/m3 in PT-
BFT and CW-RAS, respectively. The final results showed that the PT
system had the lowest costs compared to CW-RAS and BFT systems. The
results of this study indicate that the PT system may be a more pro­
ductive and profitable option than CW-RAR and BFT systems for nursery
whiteleg shrimp production. Finally, it was found that the stocking
density in the nursery phase, which registered the best indicators, was
500 org/m3. Moreover, the whiteleg shrimp management system and
strategy in the nursery should consider high water quality, growth rate,
and lower costs based on the production goal and conditions in the
whiteleg shrimp farm.
6. Author statement
Delia Rodríguez Olague: Bioassays, Water Quality, data analysis;
Jesus T. Ponce-Palafox: Conceptualization, Validation, Reviewing and
Editing; Sergio G. Castillo-Vargasmachuca: Conceptualization, Vali­
dation, Reviewing and Editing; Eulalio Arámbul Muñoz; Investigation,
Supervision; Raúl Claro de los Santos: Water Quality analysis; Héctor
M. Esparza-Leal: Conceptualization, Validation.
7. Ethical approval
All applicable international, national, and/or institutional guidelines
for the care and use of animals were followed by the authors.
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgements
We want to thank Universidad Autónoma de Nayarit for the financial
support to the project, from which the results of this paper were
obtained.
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