fishes
Article
Production of Marine Shrimp Integrated with Tilapia at High
Densities and in a Biofloc System: Choosing the Best
Spatial Configuration
Mariana Holanda 1 , Wilson Wasielesky, Jr. 1 , Gabriele Rodrigues de Lara 2, *
1
2
*
Citation: Holanda, M.; Wasielesky,
W., Jr.; de Lara, G.R.; Poersch, L.H.
Production of Marine Shrimp
Integrated with Tilapia at High
and Luís H. Poersch 1
Marine Aquaculture Station, Institute of Oceanography, Federal University of Rio Grande—FURG,
Rua do Hotel, n◦ 2, Cassino, Rio Grande 96210030, Brazil
Escuela de Ciencias del Mar, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330,
Valparaíso 2340000, Chile
Correspondence: gabriele.rodrigues@pucv.cl
Abstract: Integrating marine shrimp and tilapia has been shown to be a viable alternative in a system
based on bioflocs, but there is no consensus on the spatial arrangement of farmed animals. The
present study aims to (1) compare the performance of shrimp and fish in high density when subjected
to polyculture (species in the same tank) and multitrophic (species in different tanks) arrangements,
and (2) effects on water quality, especially on organic matter (biofloc). The experiment was carried
out for 30 days, and three treatments with bioflocs were evaluated in triplicate: control: shrimp
monoculture, polyculture: shrimp and tilapia in the same tank, and multitrophic: shrimp and tilapia
in separate tanks. The results show that the best product configuration is the multitrophic system,
where the biological control of bioflocs took place, and the best performance of the Pacific white
shrimp L. vannamei and Nile tilapia O. niloticus was obtained. Tilapia, as an organic consumer, was
effective in controlling bioflocs.
Keywords: organic consumer; IMTA; pacific white shrimp; tilapia; multitrophic aquaculture;
suspended solids
Densities and in a Biofloc System:
Choosing the Best Spatial
Configuration. Fishes 2022, 7, 283.
https://doi.org/10.3390/
1. Introduction
fishes7050283
Shrimp production is steadily increasing, and Pacific white shrimp Litopenaeus vannamei
continues to be one of the most produced species in the world [1]. This activity tends to
intensify using technologies that allow production in high stocking densities, such as the
biofloc system. Biofloc technology (or biofloc system or BFT system—Biofloc Technology
system) is characterized by super intensive production of aquatic organisms, minimal or
no water renewal, and cycling of the nutrients in the tank itself by the stimulation of a
microbiota that forms microbial aggregates [2]. Besides maintaining water quality in the
system, as the ammonia is removed by heterotrophic bacteria [3], these aggregates also
serve as food for the reared animals [4]. The removal of toxic nitrogen compounds in the
biofloc system is the key to the success of this technology in aquaculture.
In closed aquaculture systems, ammonia is excreted by aquatic animals and, in the
case of using biofloc technology, is assimilated by heterotrophic bacteria after the addition
of an organic carbon source [5]. For each gram of ammonia metabolized by heterotrophic
bacteria, nearly 8 g of total suspended solids (the bioflocs) are produced [3], producing an
accumulation of suspended solids in the system, which may negatively affect the reared
organisms. Samocha and Prangnell [6] recommend that for shrimp production, the total
suspended solids (TSS) concentrations should not exceed 350 mg L−1 . For this purpose,
mechanical clarifiers, which function as particle decanters for the removal of particulate
organic matter by gravitational action in a slow radial water flow, are generally used [7].
Academic Editor: Andrew G. Jeffs
Received: 31 August 2022
Accepted: 2 October 2022
Published: 12 October 2022
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Fishes 2022, 7, 283
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This equipment is relatively easy to operate but requires extra space in the facility and
depends directly on the type of biofloc [8], as the size and nature of the biofloc will directly
influence the speed and efficiency of sedimentation [9], and clarifiers may not be as efficient
in some specific cases. Furthermore, recent studies have shown that clarifiers can modify
the structure of the microbial community present in bioflocs [10] and may open doors for
opportunistic microorganisms in the culture system [11].
Another way to remove the excess of TSS is by integrating other species of interest
in aquaculture, which consumes these particles, reducing the TSS accumulation. This
technique, designated Integrated Multitrophic Aquaculture (IMTA), has been gaining importance in recent decades due to the need to boost environmentally friendly technologies
while maintaining productivity in aquaculture systems. IMTA is based on the concept
that residues, such as unconsumed feed, feces, and metabolic excretion of one species, are
useful to feed species of different trophic levels in the same culture environment [12,13].
Therefore, the coupling of biofloc and IMTA systems could be an effective way to deal
with the accumulation of TSS generated by the rapid formation of biofloc and diversify the
production, taking advantage of the nutritional benefits of the aggregates.
The Nile tilapia Oreochromis niloticus (hereafter referred to as tilapia) is a potential
candidate to compose the multitrophic system with shrimps. Tilapia has been considered a
species of great interest [14,15] for its rusticity, tolerance to a wide range of salinity, and
because it presents better growth rates when cultivated in environments rich in natural
productivity. Furthermore, as an omnivorous species, tilapia can consume the bioflocs of
the system, as already observed in the integrated system with Pacific white shrimp [16–18].
The major ecological advantage of the IMTA system is the conversion of waste into
protein, such as excess TSS from shrimp production in the biofloc system. This ecological
advantage can improve the production system. Some studies have already successfully
reported the integration of tilapia to L. vannamei culture in the biofloc system, even highlighting its role as an organic extractivist in the system, consuming the biofloc present
in the system and transforming this excess into fish biomass [18]. However, there is no
study that identifies which is the best spatial arrangement to integrate tilapia into the
system, which can combine the biological advantage and the economic advantage of this
integration. Therefore, the present work investigates the use of two different spatial conformations: tilapia in the same tank as the shrimp and tilapia in a separate tank, to optimize
the integrated culture of white shrimp and tilapia in order to obtain a better zootechnical
performance of the shrimp and higher consumption of TSS by the tilapia.
2. Materials & Methods
2.1. Study Location and Origin of the Animals
The study was conducted at the Marine Aquaculture Station (EMA), Institute of
Oceanography of the Federal University of Rio Grande (FURG), located in Cassino beach,
Rio Grande, RS, southern Brazil. The study lasted 30 days. Juvenile tilapias (O. niloticus)
were provided from a commercial fish farm (Piscicultura Amorim, Rio Grande do Sul,
Brazil). Before the experiment, the tilapia juveniles were acclimatized in a biofloc system
for two weeks. Shrimp juveniles of L. vannamei were obtained from a commercial hatchery,
and they were acclimated in a biofloc system for 2 weeks. The experiment was approved by
the Ethics and Animal Welfare Committee of FURG (Case number 23116.005895/2016-42).
2.2. Experimental Design
The experiment consisted of three treatments in triplicate, randomly distributed:
MONO—monoculture of shrimp; IMTA ST—integrated tilapia and shrimp in the same
tank; IMTA DT—integrated culture tilapia and shrimp in different tanks. In all treatments,
the shrimp density was 204 shrimp m−3 (45 shrimp tank−1 ), and the fish density was
100 fish m−3 (18 fish tank−1 ). At the beginning of the experiment, shrimp and fish had
initial weights of 2.67 ± 0.17 g and 7.44 ± 1.18 g, respectively. Shrimps and fish were
individually weighed before being placed in the experimental units. A biofloc inoculum
Fishes 2022, 7, 283
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was used in the experimental units stocked with mature bioflocs, corresponding to 20% of
the volume of the experimental tank (44 L of inoculum + 176 L of seawater), according to
Krummenauer et al. [19]. The inoculum was taken from a 60-day culture of L. vannamei
with a density of 400 m−3 in 35 m3 tanks from L. vannamei cultivation in a greenhouse.
Molasses was used as a carbon source in the initial phases of cultivation for ammonia
control. The inoculum’s initial concentration was TSS ± 350 mg L−1 and ±70 mg L−1 of
nitrate, indicating that the nitrification process was taking place in this matrix tank.
2.3. Recirculation System
A blend of seawater (previously chlorinated and neutralized with ascorbic acid, according to Roselet et al. [20]) and fresh water from the local supply company was prepared
to achieve salinity 15. In IMTA DT treatment, water from each fish tank was constantly
circulated to tanks containing shrimp with a submerged pump (Sarlo Better® , SB 1000C,
São Caetano do Sul, São Paulo, Brazil, 13 W and 720 L h−1 flow rate) and returned to the
fish tanks by gravity. Twelve tanks with 220 L useful volume (bottom area of 0.36 m2 ) were
used. Water was constantly aired through air stones (two air stones per tank with 3.4 cm2
and 13 cm in length) to maintain the suspension of the biofloc and adequate dissolved
oxygen concentration. The stability of water temperature was obtained using heaters with
thermostats (Stealth, ETP250, Ohio, USA, 250 W).
2.4. Feed Management
The animals were fed twice a day (9:00 a.m. and 5:00 p.m.). Shrimp feeding was
carried out following the methodology described by [21] with commercial feed containing
38% crude protein (Poty Active 38, 1.6 mm, Guabi Aqua, Guabi Nutrition and Animal
Health S.A., Campinas, São Paulo, Brazil). Tilapias were fed with a 36% crude protein
commercial feed (Guabitech Mirim QS 2–3 mm, Guabi Aqua, Guabi Nutrition and Animal
Health S.A.). The amount of food provided to tilapias was restricted to 1% of the tank’s
biomass in order to stimulate the consumption of biofloc by the fish. The total feed used
(shrimp + fish) during the experiment was calculated using the average of the sum of
feed that went into each replicate of each treatment. The nitrogen input was calculated
according to [3], taking into account 38% crude protein in the shrimp feed and 36% in the
fish feed.
2.5. Shrimp and Fish Performance
The growth of the shrimp (15 shrimp/tank) and fish (10 fish/tank) was assessed by
performing weekly and biweekly biometrics, respectively, using a digital scale with 0.01 g
precision (Marte® científica AS2000; Santa Rita do Sapucaí, MG, Brazil). In these instances,
the amount of food provided to the animals was adjusted according to the increase in
biomass in the tanks. To perform the biometrics, the tilapias were anesthetized with
50 mg L−1 benzocaine hydrochloride in an aqueous solution, and after complete recovery,
they were returned to the experimental units. At the end of the experimental period, the
remaining animals were counted for the determination of survival and growth rates. The
parameters analyzed were: Survival (%) = (final shrimp or fish number/initial shrimp or
fish number) ×100; Final mean weight (g): ∑ final weight of live animals (g)/total number
of animals; total biomass (g): ∑ final weight of all live animals (g); feed conversion ratio
(FCR) = offered feed (g)/(final biomass (g)—initial biomass (g)); productivity (kg m−3 ):
[(final biomass (kg) − initial biomass (kg)) × 1000]/useful tank volume (L); specific growth
rate (g week−1 ): weight gain (g)/number of weeks.
For IMTA DT and IMTA ST treatments, some parameters were calculated considering
the system integrated by shrimp and fish. The parameters analyzed were:
1.
System productivity (Kg m−3 ): (Fsb + Ffb) − (Isb + Ifb)/Total useful volume (m3 ),
where: Fsb is final shrimp biomass; Ffb is final fish biomass; Isb is initial shrimp
biomass; Ifb is initial fish biomass; The total volume considered in the IMTA DT
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2.
3.
treatment was 0.44 m3 and the total volume considered in the IMTA ST treatment
was 0.22 m3 ;
Feed conversion rate (FCR) = (Fos+Fof)/(Fsb + Ffb) − (Isb − Ifb), where: Fos = feed
offered to the shrimp; Fof = Feed offered to the fish
Total final biomass (g): Σ final biomass of shrimps and biomass of the fish.
2.6. Water Quality
Dissolved oxygen, temperature, and pH were checked twice a day after feeding the
organisms with a multiparameter probe (YSI, model Pro-20, Ohio, USA) and a benchtop
pH meter (Mettler Toledo, FEP20, Brazil), respectively. The salinity was verified once a
week with an optical refractometer (ATC, RTP-20ATC, São Paulo, Brazil).
Turbidity and total suspended solids (TSS) were analyzed twice a week, turbidity
with a portable turbidimeter (Hach 2100P, Hach Company; Loveland, CO, United States)
and total suspended solids (TSS) were measured according to the methodology described
by [22]. Alkalinity was verified twice a week following the methodology proposed by
APHA (2017). Total ammoniacal nitrogen (TAN) and nitrite (N–NO2 ) were measured daily
according to the methodology described by [23] and [22], respectively. Organic fertilization
with molasses occurred whenever concentrations of total ammoniacal nitrogen (TAN)
exceeded 1mg L−1 , according to [3,5]. Nitrate (N–NO3 ) and orthophosphate (P–PO4 3 )
were measured once a week, following the methodology proposed by [22]. When pH and
alkalinity values dropped below 7.2 and 120 mg CaCO3 L−1 , respectively, hydrated lime
[Ca(OH)2 ] was used according to [24] for correction. Fresh water was added weekly to
compensate for the evaporation in the tanks. The water quality parameters and addition of
hydrated lime in the IMTA DT treatment tanks were measured in both tanks and the data
presented are an average of these measurements.
2.7. Statistical Analysis
All data were analyzed by one-way analysis of variance (ANOVA), except the zootechnical performance data of the tilapia that were analyzed by the t-test. Homoscedasticity and
normality were analyzed by the Levene test and the Kolmogorov–Smirnov test, respectively.
The Tukey test was applied when significant differences were detected (p < 0.05). Survival
results were arcsine transformed prior to the analysis [25].
3. Results
3.1. Water Quality
The mean temperature (>28 ◦ C), dissolved oxygen (>6 mg L−1 ), and pH (>7.9) values
were similar in all treatments. In addition, the mean values of ammonia, nitrite, nitrate,
and orthophosphate did not present significant differences among the treatments (Table 1).
The mean alkalinity, on the other hand, was significantly higher in the IMTA DT than in
the IMTA ST and MONO treatments (Table 1). The average TSS and turbidity values were
higher in the MONO treatment and lowered in the IMTA DT. The IMTA ST treatment did
not differ statistically from the last two (Table 1).
subsectionShrimp and Tilapia Performances
Survival, final average weight, weekly weight gain, final biomass, and shrimp productivity were higher in MONO and IMTA DT treatments, with no significant difference
between them, and lower in IMTA ST treatment. Shrimp FCR was lower in MONO
and IMTA DT treatments and did not present significant differences between these two
treatments. The FCR was significantly higher in IMTA ST treatment.
The opposite was observed for tilapia. Final average weight, weekly weight gain, final
biomass, and productivity were higher in IMTA ST treatment and lower in IMTA DT, while
FCR was lower in IMTA ST and higher in IMTA DT treatment.
For the system as a whole (shrimp and tilapia together), the total final biomass was
alike in IMTA DT and IMTA ST, with significantly lower values in MONO treatment. The
system productivity was significantly higher in IMTA ST than in Mono and IMTA DT
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treatments. The system FCR, on the other hand, was higher in MONO than in IMTA DT
and IMTA ST treatments, which did not differ significantly between them (Table 2).
Table 1. Water quality parameters (mean±standard deviation) of L. vannamei and O. niloticus during
the experiment in the different treatments: MONO, IMTA DT, and IMTA ST.
Temperature ◦ C
DO (mg L−1 )
pH
Alkalinity (mg CaCO3 L−1 )
TSS (mg L −1 )
Turbity (NTU)
TAN (mg L −1 )
NO2 -N (mg L−1 )
NO3 -N (mg L−1 )
PO4 3 (mg L−1 )
MONO
IMTA DT
IMTA ST
29 ± 2
(25.7–31.2)
6±0
(5.2–7.3)
8±0
(7.5–8.0)
125 ± 45 ab
(70–255)
294 ± 146 a
(115–540)
127 ± 70
(44–244)
0.2 ± 0.3
(0–1.0)
0.9 ± 0.5
(0.21–1.89)
31.3 ± 27.6
(6–66)
2.2 ± 2.1
(0.2–4.2)
29 ± 2
(25.7–31.3)
7±0
(5.5–7.3)
8±0
(7.7–8.2)
150 ± 49 a
(60–270)
211 ± 79 b
(95–340)
110 ± 57
(29–188)
0.1 ± 0.2
(0–0.84)
1.2 ± 0.7
(0.24–2.73)
32.3 ± 33.9
(6–66)
2 ± 1.9
(0.2–4.2)
28 ± 2
(25–31.1)
6±0
(5.2–7.3)
8±0
(7.5–8.1)
115 ± 43 b
(50–250)
247 ± 106 ab
(140–455)
101 ± 50
(34–217)
0.3 ± 0.8
(0–3.57)
1.5 ± 1.6
(0.15–5.25)
31.5 ± 29.7
(6–75)
2.1 ± 2.1
(0.2–4.6)
Different letters in the same row represent significant differences (p < 0.05) between treatments after one-way
ANOVA followed by Tukey’s test. OD (dissolved oxygen), TSS (total suspended solids), TAN (total ammonia). In
parentheses, the minimum and maximum values are represented.
Table 2. Zootechnical performance (mean ± standard deviation) of L. vannamei and O. niloticus during
the experiment in the different treatments: MONO (shrimp monoculture), IMTA DT (integrated tilapia
and shrimp in the same tank), and IMTA DT (integrated culture tilapia and shrimp in different tanks).
MONO
IMTA DT
IMTA ST
Shrimp
Survival (%)
Final mean weight (g)
FCR
WWG (g week −1 )
Final biomass (g)
Yield (kg m−3 )
98 ± 2 a
8.1 ± 0.5 a
1.4 ± 0.1 a
1.3 ± 0.1 a
356 ± 23 a
1.1 ± 0.2 a
96 ± 7 a
8.0 ± 0.3 a
1.4 ± 0.1 a
1.3 ± 0.1 a
353 ± 15 a
1.1 ± 0.0 a
79 ± 16 b
5.1 ± 0.8 b
3.1 ± 1.3 b
0.6 ± 0.2 b
226 ± 38 b
0.5 ± 0.2 b
Tilapia
Survival (%)
Final mean weight (g)
FCR
WWG (g week −1 )
Final biomass (g)
Yield (kg m−3 )
-
92 ± 7
15.2 ± 1.2 b
0.6 ± 0.0 b
2.1 ± 0.3 b
274 ± 22 b
0.7 ± 0.1 b
88 ± 0
27.2 ± 2.4 a
0.3 ± 0.0 a
4.7 ± 0.5 a
490 ± 44 a
1.6 ± 0.2 a
Shrimp + Tilapia
Total feed use (g)
Total nitrogen input (g)
Total nitrogen input (g m −3 )
Total final biomass (g)
Total Yield (kg m−3 )
FCR
174.4 ± 12.6 b
10.6 ± 0.7 b
48.2 ± 3.5 b
356.4 ± 22.6 b
1.1 ± 0.2 b
1.4 ± 0.1 a
214.3 ± 7.7 a
12.9 ± 0.4 a
29.27 ± 1.0 a
627.1 ± 37.9 a
1.0 ± 0.0 b
1.0 ± 0.1 b
200.6 ± 11.5 ab
12 ± 0.7 ab
54.7 ± 3.1 b
716.1 ± 46.6 a
2.0 ± 0.0 a
0.8 ± 0.01 b
Different letters after mean values ± standard deviation in the same row represent significant differences
(p < 0.05) among treatments after one-way ANOVA followed by Tukey’s test. FCR = Feed conversion rate,
WWG: weekly weight gain. For system calculations, a useful volume of 0.22 m3 was considered for the Mono and
IMTA ST treatments and 0.44 m3 for the IMTA DT treatment.
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In terms of feed use, there was a higher input of feed in the treatments where tilapia
was present and a lower input in the Mono treatment. The same pattern was observed for
the total nitrogen (N) input in the ponds. When the N-input m−3 was calculated, IMTA
DT showed the lowest nitrogen input m−3 and Mono and IMTA ST showed the highest Ninput m−3 , with no significant difference between the latter two (Table 2).
4. Discussion
4.1. Water Quality
Integrating tilapia with L. vannamei culture either in the same pond or in separate
ponds did not affect water quality parameters for either cultured species [18,26–28].
Nitrogen compounds also did not undergo any significant variation during the experiment as a result of the use of biofloc inoculum [19]. These values did not reach toxic levels
for shrimp or tilapia [29–33]. Despite the high nitrate values at the end of the experimental
period, the pH values were above 7.9, without acidification of the medium, because of the
successive pH and alkalinity corrections during the experiment, as described by Furtado
et al. [24]. Despite the decrease in the mean value of TSS, the presence of tilapia did not affect the microbial community in the biofloc system since there was no significant difference
in the mean value of nitrogen compounds with the presence of fish.
Total suspended solids (TSS) and turbidity values were kept at acceptable levels for
both species [34,35]. Despite the higher input of feed and, consequently, nitrogen in the
treatments where tilapia was present, the average TSS values were significantly higher
where tilapia was not present, even though the biomass produced with shrimp + fish
integration was higher in these treatments. That is, the higher nitrogen input did not
reflect in bacterial biomass accumulation as expected in a shrimp monoculture biofloc
system [3,36], suggesting the consumption of solids by tilapia, which demonstrates an
ecological benefit.
Despite higher feed input in IMTA DT and IMTA ST treatments when compared to
the MONO treatment, orthophosphate levels were not affected by the presence of the fish.
According to Poli et al. [37], tilapia can retain up to 223% more phosphorus in an integrated
system with shrimp than in a shrimp monoculture. Therefore, it can be shown that the
presence of tilapia in integration with shrimp is a viable alternative for greater recycling of
nutrients in the culture system.
4.2. Shrimp and Tilapia Performances
The presence of tilapia in the integrated system with shrimp in the same pond negatively affected the husbandry performance of the shrimp. There are some theories for the
favored husbandry performance of tilapia: (1) due to the high biomass (shrimp + fish) in
the same pond. Shrimp growth is density dependent [27,36], i.e., the higher the stocking
density, the lower the weight of the individual. The density of shrimp combined with the
density of fish in a small area (bottom area of 0.36 m2 ), together with a total initial biomass
2.2 times higher than the shrimp that were grown in the Mono and IMTA DT treatments,
contributed to the worse zootechnical performance of the shrimp in the tilapia in treatment;
(2) the tilapia may have ingested the shrimp feed or ingested the shrimp themselves: This
explains the better performance of the tilapia when grown in the same pond as the shrimp.
The total suspended solids were a possibly significant source of energy for the tilapia,
as the FCR was 0.6 in IMTA DT and 0.3 in IMTA ST treatments. Poli et al. [37] observed
low feed conversion rates for tilapia (from 0.21 to 0.24) when fed with 1% of the biomass
and shrimp FCR values close to those found in this study.
In IMTA ST treatment, half of the FCR was found, with double the growth and
productivity of tilapia when compared to IMTA DT. This result shows that if we increase
the feeding rate of the tilapia when growing in separate tanks from the shrimp, tilapia will
possibly still exert its ecological role in the integrated system as a consumer of bioflocs and
show higher growth rates, as seen in the IMTA ST treatment. Even though undernourished,
the tilapia, consuming the flake, showed weekly growth expected for culture in the biofloc
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system. Similar results of biofloc consumption by tilapia when growing in integrated
shrimp culture have been observed in other studies [15,38–40].
According to Troell et al. [13], IMTA is the only practical remediation approach with
the prospect of generating revenue by diversifying production, whereas all other biomonitoring approaches usually only represent additional costs to the producer. The goal is to
increase long-term sustainability and profitability per culture unit (and not by species, as
practiced in monocultures) by recovering some of the nutrients and energy lost in monocultures, turning them into additional products with commercial value. The integrated culture
of white shrimp and tilapia is a way to diversify production and improve the profitability
of aquaculture. The final biomass of IMTA DT and IMTA ST treatments doubles the final
biomass of shrimp monoculture, justifying the use of IMTA and proving it to be an ecologically and economically viable alternative. It is worth noting that it was possible to increase
the total biomass produced in the IMTA DT treatment by 175% without compromising the
specific growth rate and feed conversion of the shrimp.
5. Conclusions
Tilapia proved to be efficient in consuming and maintaining TSS levels in the integrated
system with shrimps and did not affect water quality. The zootechnical performance of
shrimp L. vannamei was impaired when growing in the same tank as the tilapia. Therefore,
the best integrated super-intensive system for L. vannamei and O. niloticus in the tilapiato-shrimp ratio of 0.49 is in separate tanks, as observed in the IMTA DT treatment. It is
recommended that under the experimental conditions tested, shrimp and fish be grown in
separate tanks so that the producer can have two final products for marketing at the end of
the production cycle without negatively affecting shrimp production.
Author Contributions: Conceptualization, M.H., W.W.J. and L.H.P.; methodology, M.H., W.W.J. and
L.H.P.; software, M.H. and G.R.d.L.; validation, M.H.; W.W.J.; G.R.d.L. and L.H.P.; formal analysis,
M.H. and G.R.d.L.; investigation, M.H.; W.W.J.; G.R.d.L. and L.H.P.; resources, W.W.J.; G.R.d.L.
and L.H.P.; data curation, M.H. and G.R.d.L.; writing—original draft preparation, M.H. and L.H.P.;
writing—review and editing, M.H.; W.W.J.; G.R.d.L. and L.H.P.; visualization, M.H.; W.W.J.; G.R.d.L.
and L.H.P.; supervision, W.W.J. and L.H.P.; project administration, W.W.J., G.R.d.L. and L.H.P.;
funding acquisition, W.W.J. and L.H.P. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by ASTRAL Project—H2020 grant Agreement 863034.
Institutional Review Board Statement: All applicable international, national, and/or institutional
guidelines for the care and use of animals were followed by the authors. The experiment was approved
by the Ethics and Animal Welfare Committee of FURG (Case number 23116.005895/2016-42).
Acknowledgments: The authors are grateful for the financial support provided by the European
Union (ASTRAL Project—H2020—Grant Agreement 863034), National Council for Scientific and
Technological Development (CNPq), and Coordination for the Improvement of Higher-Level Personnel (CAPES). W.W.J., and L.H.P., are research fellows of CNPq. Special thanks to GUABI Nutrition
and Animal Health S.A. for donating the commercial diets.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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