Biofloc technology

1. Aquaculture 547 (2022) 737470
Available online 10 September 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
Short communication
Masculinization protocol for Nile tilapia (O. niloticus) in Biofloc technology
using 17-α-methyltestosterone in the diet
Rodrigo Zhouri Costa e Silva a
, Érika Ramos Alvarenga a
, Sylvia Velloso Matta a
,
Gabriel Francisco de Oliveira Alves a
, Ludson Guimarães Manduca a
, Marcos Antônio Silva a
,
Thomás Toshio Yoshinaga a
, Arthur Francisco Araújo Fernandes b
, Eduardo Maldonado Turra a,*
a
Escola de Veterinária da Universidade Federal de Minas Gerais, Av. Antônio Carlos, n◦
6627, Caixa Postal 567, Campus da UFMG, CEP 30123-970 Belo Horizonte,
MG, Brazil
b
Department of Animal and Dairy Sciences, University of Wisconsin – Madison, 472 Animal Science Building 1675, Observatory Dr., Madison, WI 53706, USA
A R T I C L E I N F O
Key-words:
BFT
Masculinization
Sex control
Uniformity of fingerlings
Fish
A B S T R A C T
Sexual control through hormone treatments is widely used in Nile tilapia due to its simplicity and high efficiency.
However, possible impacts of hormone residues introduction on the environment are still a topic of concern. One
way to minimize the environmental impact of hormonal treatments is to use close aquaculture systems, such as
the Biofloc Technology (BFT). However, BFT system can provide constant additional feed and there is the
possibility that the feed with masculinization hormone offered would not be fully ingested, resulting in a lower
ratio of males in relation to the traditional hormonal inversion protocols. Hence, different feeding frequencies
and hormonal concentrations, superior to conventional protocols of masculinization, should be tested. Thus, our
goal was to elaborate a masculinization protocol that would allow better growth and higher masculinization
percentages for Nile tilapia (O. niloticus) in BFT. Nile tilapia fry were raised in BFT and submitted to different
concentrations of 17α-methyltestosterone (60, 90, 120, 150 and 180 mg ⋅ kg− 1
) which was offered five or eight
times a day. The control group consisted of fry raised in clear water tanks fed with 60 mg ⋅ kg− 1
hormone diet
provided five times a day. The experimental design was a factorial arrangement (5 × 2), plus control, on two
random blocks, each block having two replicates per treatment. Stocking density applied was of 2 fry⋅ L− 1
(300
fry⋅ tank− 1
). Water quality variables did not diverge between BFT systems and control group, except for settable
solids, a result already expected. Animals submitted to BFT systems presented higher survival (%) and uniformity
when compared to the clear water group. Higher hormone concentration treatments presented less proportion of
males in contrast to 60 mg ⋅ kg− 1
groups and no differences were found between feed frequencies tested. In
conclusion, it was possible to achieve ≥94% masculinization rates in tilapia using 17α-methyltestosterone at 60
mg ⋅ kg− 1
on BFT systems, under a five times per day feeding regime.
1. Introduction
In Aquaculture, many strategies have been used for sex control, such
as production of monosex populations through the induction of sex
reversal by using hormonal treatment (Wassermann and Afonso, 2003;
El-Sayed, 2006; Baroiller and Cotta, 2018), chromosomal manipulation
for the production of triploid individuals (Arai and Fujimoto, 2018;
Alvarenga et al., 2020), hybridization, and selection, or a combination of
these (El-Sayed, 2006; Baroiller and Cotta, 2018; Wang and Shen, 2018).
From the sex control strategies, the production of male monosex popu­
lation has many other advantages in Nile tilapia (O. niloticus)
production, such as: faster growth reaching a larger harvest size, and
greater uniformity; better flesh quality and appearance, and reducing
the energy cost of gonad development (Hines and Watts, 1995; Beard­
more et al., 2001; El-Sayed, 2006; Singh, 2013; Wang and Shen, 2018).
Two different approaches for all-male offspring deserve particular
attention as they are environmentally friendly an follow the legislation
of several countries, mainly European: the production of all-male
offspring (a) from YY-male × XX-female mating, and (b) from the
treatment of fries by high temperatures.
YY males (“supermales”) can be produced from the mating of XY
males and XY phenotypic females (feminized larvae). The production of
* Corresponding author.
E-mail address: eduturra@ufmg.br (E.M. Turra).
Contents lists available at ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture
https://doi.org/10.1016/j.aquaculture.2021.737470
Received 17 May 2021; Received in revised form 16 August 2021; Accepted 8 September 2021

2. Aquaculture 547 (2022) 737470
2
“supermales” requires a set of laborious steps and several progeny tests,
which can take almost 5 years to complete the entire process (Baroiller
and Cotta, 2018). Furthermore, the effects of genes with minor effects on
sex determination can significantly interfere in the proportion of 100%
male progeny (Mair et al., 1995). To guarantee a high percentage of
males in progeny of YY parents, Beardmore et al. (2001) suggested the
need for a selection program that increases the allelic frequency in the
population of genes with minor effects that contribute to the determi­
nation of male sex. Fortunately, recent techniques developed for the
identification of sex by genotypic markers (Baroiller and Cotta, 2018)
can ensure a reduction of the time spent in steps that require progeny
testing. However, the effort and time spent to form a YY strain can lead
to an increase in costs and a genetic gap, in addition to a great difficulty
to avoid high inbreeding rate.
The production of lots of fingerlings with high proportions of males is
also achieved with heat treatment (around 36 ◦
C) for a period of 10 to
30 days of 10 dpf age larvae (days after fertilization). Since thermo­
sensitivity has genetic components, it is possible to increase the pro­
portion of males with this type of treatment by selecting more
thermosensitive families. However, the difficulties of this type of
method lies mainly in its realization on a large scale, and technical so­
lutions for that are still in its beginning (Baroiller and DttaCotta, 2018).
Most farmers use hormonal treatments, especially 17α-methyl­
testosterone (MT), for obtaining male monosex populations of Nile
tilapia. MT is preferentially chosen because it is a simple, highly effi­
cient, reliable, and cheap approach (El-Sayed, 2006; Baroiller and Cotta,
2018), and this will probably continue to be the most important method
to obtain all male offspring for a long time in the most important tilapia
producing countries. The 17 α-methyltestosterone androgen has been
tested in over 25 species within the Salmonidae, Cichlidae, Cyprinidiae,
Anabantida, Poecilidae and Cyprinodontidae. Generally, the Cichlidae
require lower doses of androgen compared to other families (Beardmore
et al., 2001). Immersion protocols for this hormone administration have
been applied but have not been successful in sex reversal of tilapias as in
temperate species (Beardmore et al., 2001). Besides that, the mascu­
linization rate of immersion protocols is lower than those via dietary
supplementation (Phelps and Popma, 2000). Moreover, the use of this
technique in a large scale, mainly in pond systems, seems to be pro­
hibitive. The administration by feed has been the most important and
used method in many countries (Popma and Green, 1990; Phelps and
Popma, 2000; Beardmore et al., 2001; Baroiller and Cotta, 2018). The
androgen is spread in the feed using ethanol as vehicle and during 28
days the most conservative and common length period, this mixture is
offered (around 20% of feeding rate) to fries under 11 mm length or with
10 dpf of age (Baroiller and Cotta, 2018). It is important to note that MT
dosages will depend upon the farming conditions. If the fry are treated
directly in systems where plankton and/or microorganisms develop, and
with possible loss of part of the feed, high dosages are often suggested
(60 mg ⋅ Kg− 1
of feed), whereas, in indoor systems, lower dosages
(30–45 mg ⋅ Kg− 1
of feed) can have the same efficiency (Baroiller and
Cotta, 2018).
Due to its rapid degradation, it is likely that MT does not accumulate
in treated fish or the environment. It is important, however, that there is
a regular management of sediments from production systems such as
ponds, ensuring aerobic conditions to increase the speed of its degra­
dation. Meanwhile, it is still possible to consider a risk in this type of
system, especially with the accumulation of MT in the adipose tissue of
tilapias around hapas that can escape, or even wild fish in water bodies
that receive effluent from tilapia farms, where they frequently consume
leftover feed with MT, serving as biotransporters of the hormone and
promoting effects on the physiology of local predators (Baroiller and
Cotta, 2018). Thus, the production of masculinized fingerlings by MT in
systems such as ponds, which have a regular discharge of effluent to the
environment and have less control over fish escapes, is potentially less
environmentally friendly. Recirculating aquaculture systems and Biofloc
technology (BFT), as (like) closed systems, can be an alternative for
tilapia hatcheries that use MT treatments.
The BFT emerged as an alternative, sustainable method to produce
aquatic animals (Avnimelech, 2009; Crab et al., 2012; Ahmad et al.,
2017). This technology enables the reduction of water and land use for
aquaculture due to its features, such as minimal water changes, higher
stocking densities (in comparison to ponds), and recycling of the nu­
trients present in the water resulting in less emission of pollutants
(Naylor et al., 2000; Avnimelech, 2009; Crab et al., 2012; FAO, 2016;
Ahmad et al., 2017). The best growth out indexes for the production of
tilapia in BFT have been under study. It is possible to produce tilapia
with more than 500 g body weight with 15 to 75 fish ⋅ m− 3
(Rakocy
et al., 2004; Green et al., 2019; Manduca et al., 2021) and to harvest Nile
tilapia from a breeding program in BFT (Turra et al., 2012a; Turra et al.,
2012b; Fernandes et al., 2015; Turra et al., 2016; Turra et al., 2018) with
an average body weight of 750 g in less than 200 days of culture (initial
body weight of 6 g) and with feed conversion ratio below 1.3 (Cavatti
Neto et al., data yet to be published). Tilapia juveniles (about 20 g body
weight) can be produced at stocking densities of 400 individuals ⋅ m− 3
,
in moderately saline environments (4 to 8 mg ⋅ L− 1
) by controlling the
toxicity of nitrite peaks (Alvarenga et al., 2018), although this indication
is not suitable for the masculinization phase (Valle et al., data yet to be
published).
Despite the recent increase in the interest on the use of BFT for Nile
tilapia culture, to the best of the authors’ knowledge, an efficient
masculinization protocol for tilapia using this system has not yet been
developed. The use of BFT for the production of all male Nile tilapia
juvenile could enhance the fingerling production, since tilapia can use
the biofloc as an additional nutritional source, and with minimal envi­
ronmental impact, due to its capacity to recycle nutrients (Azim and
Little, 2008; Avnimelech, 2009). Nevertheless, based on the initial hy­
pothesis that the BFT system can provide constant additional feed
(Ekasari et al., 2014; Silva et al., 2018), there is the possibility that the
feed with masculinization hormone offered would not be fully ingested.
Therefore, less feed consumed by the fish could mean less hormone
ingested, resulting in a lower ratio of males in relation to the traditional
hormonal inversion protocols. Hence, different feeding frequencies and
hormonal concentrations, superior to conventional protocols of mascu­
linization should be tested. Corroborating this hypothesis, David-Ruales
et al. (2019) compared the sexual inversion of red tilapia induced by
17-α-methyltestosterone (60 mg ⋅kg− 1
of feed) in Recirculating Aqua­
culture System (RAS) and BFT and found a proportion of males of 91%
and 64%, respectively. Therefore, we aimed to investigate the concen­
tration of MT in the diet and daily feeding frequency to determinate a
protocol for masculinization of Nile tilapia in BFT systems.
2. Materials and methods
2.1. Animals and experimental design
The experiment was conducted in a greenhouse under natural light at
the Aquaculture Laboratory of Veterinary School of the Federal Uni­
versity of Minas Gerais (Laboratório de Aquacultura - LAQUA, Escola de
Veterinária/Universidade Federal de Minas Gerais - UFMG), Brazil.
Experimental procedures were carried out in compliance with animal
welfare laws, policies, and guidelines. All procedures were previously
reviewed and approved by the Counsel of ethical practices in animals of
the Federal University of Minas Gerais (CEUA) under protocol number
75/2018.
For fry production, 24 females and 16 males of Nile tilapias (average
body weight of 600 g) from Chitralada line were selected for repro­
duction in clear water per week (different breeders per week), during
two non-consecutive weeks (two blocks). In each block, 6600 post yolk
sac absorption larvae were randomly collected (from at least 10
spawnings) and distributed equally on 22 tanks with a storage volume of
150 L, resulting in 300 fry ⋅ tank− 1
(stocking density = 2 fry ⋅ L − 1
), as
described by Lara-Flores and Olvera-Novoa (2013).
R.Z. Costa e Silva et al.

3. Aquaculture 547 (2022) 737470
3
The experiment was designed in a factorial arrangement (5 × 2) with
five hormonal concentration and two feeding frequencies (described
below), plus control, in random blocks. Therefore, the experiment was
composed by 11 treatments, and each treatment (control and combi­
nation of dose and feeding frequency) had four replicates with a total of
44 experimental units. Because of the limited number of tanks, the
experiment was divided into two blocks of time, each containing 22
tanks, therefore, 11 treatments and two replicates per block.
During the first 28 days of post yolk sac absorption, the Nile tilapia
fry were fed with a commercial ration (Propescado-Nutriave Foods)
containing 55% crude protein enriched with MT. Five different con­
centrations of hormone were tested, 60, 90, 120, 150 and 180 mg ⋅ kg− 1
on the fish feed. The lower dose of 60 mg ⋅ kg− 1
was chosen considering
that it is often used in outdoor systems (Baroiller and Cotta, 2018). Also,
since David-Ruales et al. (2019) used MT in a concentration of 60 mg ⋅
kg− 1
of feed in BFT and found a proportion of males of only 64% in red
tilapia, we hypothesized the higher doses could be necessary. Therefore,
we evaluated if 60 mg ⋅ kg− 1
of feed and four doses above it could be
effective for the masculinization of Nile tilapia in BFT. We also tested
two feeding frequencies - five or eight times a day - with the same total
amount of feed per day. As positive control, we used MT at 60 mg ⋅ kg− 1
of feed in clear water with a frequency of five times a day, which is the
protocol often used in outdoor systems.
Results of different masculinization rates between Nile tilapia fam­
ilies have already been obtained and presented in the literature
(Beardmore et al., 2001; Baroiller and Cotta, 2018). To avoid a family ×
treatment interaction effect, more than 20 females that spawned for
larvae harvest came from a large genetic basis broodstock, a large
number of founders being of four different origins of Chitralada line in
Brazil (>400 males and females of each line), with an inbreeding rate of
less than 1.3%. Since the evaluation of seven generations of this
breeding line presented an average female/male proportion of 45/55%
(in a breeding program in BFT), a negative control was not necessary
(feed without hormone).
The specific quantity of hormone for each treatment was measured in
a precision balance (©Marte Científica, Brazil) and dissolved in 200 mL
of absolute alcohol, then sprinkled to the fish feed during a mixing
process. The feed was then stocked in a dark room for alcohol evapo­
ration and hormone fixation, and after 24 h the fish feeds were stored in
dim white buckets and properly tagged for each treatment. The buckets
were stored in a freezer under − 20 ◦
C, to keep hormone levels
throughout the trial according to Barry et al. (2007).
The feeding rate applied was 30% of the fish weight for the first
week, 25% for the second, 20% for the third, and 15% for the fourth
week. The percentage was adjusted to lower levels each passing week, as
described by Luthada and Jerling (2013), Daudpota et al. (2016), and
recommendations of Phelps and Popma (2000) and El-Sayed (2006).
The amount of feed offered was based on the same average body weight
of fry for each experimental unit at the beginning of each week (week 1
= 0.01 g, week 2 = 0.15 g, week 3 = 0.4 g and week 4 = 0.8 g), according
to results of previous experiments. An expected mortality of 2% from
one week to another was also considered. Thus, in the first week, 3 mg of
feed / day, containing 180 ng of MT (1260 ng for the first week) were
offered for each fry; in the second week 37 mg of feed / day, containing
2220 ng of MT (15,540 ng for the second week), were offered; in the
third week 60 mg of feed / day, containing 3600 ng of MT (25,200 ng for
the third week) were offered, and in the fourth week 120 mg of feed /
day, containing 7200 ng of MT (50,400 ng for the fourth week) were
offered for each fry.
2.2. Water quality
The 20 fish tanks were filled to 150 L in the beginning of the
experimental essay with biofloc previously developed in our laboratory.
The initial biofloc total ammonia nitrogen (TAN) and nitrite (NO2
−
)
concentrations were of 0 mg ⋅ L− 1
. Temperature, salinity, pH, and
dissolved oxygen (DO) were measured three times per week using the
YSI 6920 V2 (Yellow Springs Incorporated - YSI, OH, USA) multipa­
rameter probe. TAN and N-NO2
−
were also analyzed three times per
week using spectrophotometry (Biochrom Libra S22), according to
UNESCO (1983) and Bendschneider and Robinson (1952), respectively.
Settable solids (SS) were collected and measured once a week through
the ImHoff cone (Avnimelech, 2009). Alkalinity and total suspended
solids (TSS) were monitored once a week through protocols adapted
from APHA, 1998 and Strickland and Parsons (1972), respectively.
During the experiment, when ammonia exceeded 0.5 mg ⋅ L− 1
, molasses
(estimated with 50% carbon) was added to the system in order to
maintain the proportion of 6:1 (C:N), according to Ebeling et al. (2006).
Also, sodium chloride was added until a 1.5 g ⋅ L− 1
concentration was
reached to reduce nitrite toxicity in all the tanks (adapted from Yanbo
et al., 2006).
The control treatment (clear water system) of water quality was
maintained with a water renewal rate of 50% per day. To avoid differ­
ence in temperatures between treatments, the water renewal was made
with water that was previously heated (27–28 ◦
C).
2.3. Larval biometry and sexing
At the beginning of each block of the experiment, a batch of more
than 100 randomly selected post yolk sac absorption larvae was dried in
paper towel and weighted together for the initial body weight deter­
mination. In two moments of the experiment (15 and 21 days),
approximately 50 fish were randomly selected from each tank and
weighed together. These fish were then euthanized and submitted to
Hematoxylin-Eosin (HE) histology analyses for early sex determination.
At the end of the 28 days of experiment, 75 fingerlings were indi­
vidually weighed and 60 were randomly chosen for microscopic ana­
lyses of gonads and sex determination. These fish were stored in Bouin
liquid for 24 h and then transferred to an alcohol solution at 70%. We
used the classification method to define males, females and undiffer­
entiated fish using aceto-carmin squash technic as described by Guerrero
and Shelton (1974), validated by Wassermann and Afonso (2003) and
reviewed by Makino et al. (2008). Fish were classified as intersex when
they presented both spermatogonia and oocytes.
We used a precision balance (©Marte Científica, Brazil) to weigh the
batch of post yolk sac absorption larvae and the fries on day 15, 21, and
at the end of the experiment.
2.4. Growth performance and survival
Growth performance was evaluated based on the final body weight
(BWf), final biomass (BMf), final stocking density (SDf), daily weight
gain (DWG), specific growth rate (SGR) and survival. BWf was the
average measure of weight acquired on the 28th day of experiment from
75 fingerlings per tank.
BMf was acquired by BMf = (BW15 × N15) + (BW21 × N21) + (BWf ×
Nf). BW15 is the body weight and N15 is the number of fish weighed on
the 15th day, BW21 and N21 on the 21st day, and Nf represented the final
number of remaining fish.
SDf was acquired by SDf = BWf / useful tank volume (i.e., 0.15 m3
).
DWG calculation was DWG = (BWf - BWi)/t, where BWi represents the
initial weight and BWf is the final weight and t is the duration of the trial
(days). Survival (S) was calculated as S = 100 × [(Nf + N15 + N21)/Ni],
where Ni represents the initial number of fish per tank. SGR was ob­
tained as described by Ekasari et al. (2015) by [(BWf /BWi)1/t
− 1] ×
100.
2.5. Statistical analyses
Water variables were submitted to the analysis of variance (ANOVA).
Then, the residuals were tested by Shapiro-Wilk and Bartlett test. When
residuals were normally distributed and the variances were
R.Z. Costa e Silva et al.

4. Aquaculture 547 (2022) 737470
4
homogeneous, the results were analyzed by ANOVA and post-tested
depending on CV, < 10% by Tukey, > 10% and < 20% by SNK and
above 20% by Duncan test. The values that presented p < .05 in Shapiro-
Wilk or Bartlett test were submitted to Kruskal-Wallis test.
The performance variables were analyzed using two statistical stra­
tegies. First, all treatments were compared using the mean, since the
control group is a qualitative treatment. In this comparison, if the re­
siduals of the response variable presented normal distribution with zero
mean, they would be evaluated by ANOVA and post test, as described
above. In a second statistical strategy, we considered only BFT treat­
ments and analyzed the data by factorial ANOVA, including the effect of
block, concentration, frequency, and its interactions. When the effect of
concentration or frequency or its interactions was not significant it was
removed from the final model. Regression models were also fitted for all
growth performance variables.
In order to compare body size uniformity between control and BFT,
an individual measurement of weight was made in 75 individuals per
tank (150 individuals per treatment in each block) and the homogeneity
of variance was tested by Bartlett and F-test to compare the two vari­
ances. Since the Bartlett and F tests do not consider the effect from
blocks, the entire procedure was performed using results from each
block.
Masculinization data was transformed in arcsine of the square root to
properly normalize the values and then submitted to ANOVA and Tukey
tests. A linear regression model was fitted. For a better evaluation of the
treatment effects over masculinization, an analysis of different scenarios
was established: we assumed a first scenario where 50% of undifferen­
tiated fish obtained would turn into females and 50% into males, and a
second scenario where the same proportion of masculinization obtained
for differentiated fish would repeat itself with the remaining undiffer­
entiated individuals. The results considering the scenarios were then
transformed in arcsine square root and then submitted to linear
regression analyses. Infostat (Di Rienzo et al., 2015) and R (R Core
Team, 2016) software were used for the analysis.
3. Results and discussion
Sexual control by hormone treatments is used in Nile tilapia because
this species can reproduce before reaching market size, which generates
many problems to tilapia farmers, such as overpopulation, reduced
growth, and die-off (Baroiller and Cotta, 2018; Brämick et al., 1995;
Farahmand et al., 2007; Hussain et al., 1995). Despite the environmental
issues related to hormone use, it remains widely used due to its
simplicity and high efficiency (Baroiller et al., 2009; Baroiller and Cotta,
2018; Joshi et al., 2019). Since BFT is an aquaculture system with
minimal to zero water discharge to the environment, it could minimize
the possible environmental problems generated by hormones. However,
before assessing whether BFT would be a solution for reducing hormone
residues, it is necessary to establish an efficient masculinization protocol
in this system, once its high concentration and availability of live food in
the water, above a traditional larvae culture system in ponds (and well
above a clear water system), could interfere with the ingestion of food
containing the daily amount of androgen necessary for the masculini­
zation process. Since the percentage of male tilapia obtained after a
masculinization protocol using MT in BFT was insufficient in a previous
study (David-Ruales et al., 2019), the present study investigated more
treatment possibilities, based on higher levels of MT on feed than used
pond and clear water systems, and established a protocol for mascu­
linization of Nile tilapia in BFT.
Water quality parameters were within the recommended range for
the development of tilapia (Table 1). As expected, TSS and SS were
different between treatments, with higher values found in BFT in com­
parison to the control group. However, these differences did not result in
negative effects on fingerlings growth and survival in BFT. The control
group presented an inferior survival rate in comparison to most BFT
treatments, similar results were described by Ekasari et al. (2015) and
Table
1
Water
quality
variables
(mean
or
median)
and
their
coefficients
of
variation
(CV)
or
minimum
and
maximum
data
(between
brackets)
for
Nile
tilapia
larvae
reared
in
clear
water
(control)
or
biofloc
technology
(BFT),
during
28
days
of
masculinization
under
different
17
α
-methyltestosterone
(MT)
concentration
in
the
diet
and
two
different
feeding
strategies
(5
×
⋅
day
−
1
and
8
×
⋅
day
−
1
).
Variables
Control
(Clear
water)
BFT
CV
5×
⋅
day
−
1
5×
⋅
day
−
1
MT
concentration
(mg
⋅
Kg
−
1
feed)
8×
⋅
day
−
1
MT
concentration
(mg
⋅
Kg
−
1
feed)
60
60
90
120
150
180
60
90
120
150
180
Temperature
(
◦
C)*
26.58
26.54
26.59
26.83
26.76
26.71
26.44
26.78
26.73
26.82
26.45
1.79
DO
(mg
⋅
L
−
1
)*
6.57
6.48
6.48
6.50
6.47
6.37
6.49
6.45
6.46
6.41
6.47
2.13
pH**
8.08
(8.01–8.18)
7.53
(7.17–7.83)
7.42
(7.10–7.91)
7.56
(7.07–8.03)
7.52
(7.06–7.99)
7.52
(7.06–7.91)
7.45
(7.03–7.84)
7.52
(7.10–7.93)
7.56
(7.05–8.06)
7.51
(7.18–7.95)
7.39
(7.02–8.06)
–
TAN
(mg
⋅
L
−
1
)**
0.26
(0.18–0.32)
0.22
(0.12–0.23)
0.22
(0.14–0.38)
0.19
(0.15–0.22)
0.17
(0.15–0.18)
0.21
(0.14–0.22)
0.23
(0.11–0.25)
0.18
(0.12–0.23)
0.19
(0.14–0.25)
0.18
(0.14–0.27)
0.17
(0.15–0.37)
–
Nitrite
(mg
⋅
L
−
1
)**
0.08
(0.05–0.10)
0.07
(0.02–0.15)
0.05
(0.03–0.14)
0.06
(0.03–0.10)
0.07
(0.03–0.10)
0.09
(0.04–0.16)
0.07
(0.03–0.18)
0.06
(0.04–0.12)
0.07
(0.05–0.10)
0.07
(0.04–0.16)
0.07
(0.03–0.11)
–
SS
(ml
⋅
L
−
1
)***
0.26
a
10.83
bc
12.83
b
9.75
bc
9.03
bc
8.09
c
10.01
bc
9.98
bc
9.61
bc
10.03
bc
11.55
bc
26.4
TSS
(mg
⋅L
−
1
)***
109.75
a
366.56
b
351.79
b
361.50
b
303.94
b
285.88
b
322.63
b
430.00
b
383.63
b
322.13
b
337.88
b
31.0
Alkalinity(mg
of
CaCO
3
⋅
L
−
1
)**
101.42
(65.7–127.6)
56.57
(49.5–99.2)
71.44
(52.5–90.4)
76.99
(54.2–87.8)
62.40
(52.7–82.2)
72.27
(57.5–83.6)
70.40
(57.3–88.8)
56.09
(53.2–80.2)
78.07
(57.5–83.6)
78.60
(45.0–95.7)
70.65
(66.2–89.8)
–
Salinity
(g
⋅
L
−
1
)**
1.49
(1.29–1.92)
1.86
(1.59–2.00)
1.88
(1.67–1.94)
1.70
(1.62–1.77)
1.61
(1.57–1.65)
1.81
(1.64–1.96)
1.79
(1.57–1.95)
1.90
(1.65–1.96)
1.65
(1.59–1.82)
1.72
(1.65–1.91)
1.92
(1.53–1.96)
–
Reference
values
for
tilapia
culture:
Temperature
=
27–32
◦
C
(El-Sayed,
2006);
nitrite
<8
mg
⋅
L
−
1
(El-Sayed,
2006);
DO
(dissolved
oxygen)
>
4
mg
⋅
L
−
1
(Wedemeyer,
1996);
pH
=
5.5–9
(Rebouças
et
al.,
2016);
TAN
(total
ammonia
nitrogen)
<
1
mg
⋅
L
−
1
(El-Sayed,
2006);
alkalinity
>50
mg
of
CaCO
3
⋅
L
−
1
(Boyd
et
al.,
2016);
SS
(Settleable
solids)
<
100
ml
⋅
L
−
1
(Avnimelech,
2009);
TSS
(Total
suspended
solid)
<
1000
mg
⋅
L
−
1
(Avnimelech,
2009);
salinity
≤
8
g
⋅
L
−
1
(Alvarenga
et
al.,
2018)
*Means
in
the
same
line
did
not
differ
according
to
ANOVA
and
Tukey
test
(p
>
.05).
**Median
in
the
same
line
did
not
differ
according
to
Kruskal-Wallis
test
(p
>
.05).
***Means
with
different
letters
in
the
same
line
differ
according
to
ANOVA
and
Duncan
test
(p
<
.05).
R.Z. Costa e Silva et al.

5. Aquaculture 547 (2022) 737470
5
Pérez-Fuentes et al. (2016).
We did not find differences in the growth performance variables
evaluated between animals from BFT and control group. However, an
interesting higher uniformity of animals reared in BFT was observed in
comparison to those of fish in control. The weight distributions of all
BFT treatments differed from control according to Bartlett and F tests, p
< .05. For example, we compared the body weight of animals from the
same dose (60 mg ⋅ kg− 1
of feed) in different systems and feed fre­
quencies (Fig. 1). For control, five and eight times of daily feed fre­
quencies in BFT, we did not found differences between the body weight
averages. On the other hand, the variances of body weight in control
system were higher than those in BFT (p < .001), being their values 0.09
g2
, 0.03 g2
, 0.03 g2
in block 1, and 0.33 g2
, 0.18 g2
, 0.19 g2
in block 2,
respectively. This result can be attributed to the availability of natural
food in BFT, accessible 24 h (Avnimelech, 1999; Hargreaves, 2013). This
could imply reduced cost and time to select equally sized fingerlings for
sales in BFT.
In the second statistical strategy, results from the control group were
not included (qualitative treatment), and linear regression was used to
evaluate the effects of treatments in BFT over several performance
variables. The effects of interactions and feeding frequency were not
significant for growth performance variables and a higher feeding fre­
quency tested did not improve the growth nor survival of the animals.
Therefore, the linear models presented were composed only by the ef­
fects of block and hormone concentration. All the performance vari­
ables, except survival, were negatively affected by the increase of
hormone concentration, that is, when submitted to higher hormone
concentration the fingerling growth was lower (Table 2).
A histological analysis of fish sections stained by Hematoxylin-Eosin
(HE) applied on the individuals harvested at 15 and 21 days after yolk
sac absorption was tested as an attempt on earlier quantification on the
success of the masculinization protocol. However, it proved to be inef­
ficient since there was a high proportion of undifferentiated individuals.
This analysis is also more laborious than the aceto-carmin squash with
higher processing time, thus these results indicate that this procedure
should not be applied for commercial purposes. Therefore, this is the
reason the masculinization analysis in this work was only made in the
individuals of 28 days post yolk sac absorption through the aceto-carmin
squash technic.
When we compared the results between masculinization obtained
from treatments under BFT with the result from the clear water group
(Fig. 2), we did not find differences in the masculinization rate between
Fig. 1. The weight distributions of all BFT treatments differed from control (clear water) according to Bartlett and F tests. We plotted, as an example, the weight
distributions of treatments where 60 mg of 17-α-methyltestosterone were used as hormone concentration per kg of feed: five times of daily feed frequency in BFT
(green), eight times of daily feed frequency in BFT (red), and five times of daily feed frequency in control (blue). Data were analyzed and plotted per block due its
significant effect. Observe the higher variation of weigh in clear water. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
R.Z. Costa e Silva et al.

6. Aquaculture 547 (2022) 737470
6
the groups. Several masculinization protocols, including the use of MT in
diet, have been tested in the past decades. Literature extensively men­
tions that, in order to achieve successful masculinization, the feeding
frequency must be at least four times a day (Meurer et al., 2012; Luthada
and Jerling, 2013; Baroiller and Cotta, 2018). Based on the initial hy­
pothesis that the BFT systems provide constant additional feed (the
biofloc) to the fish, there was the possibility that the diet containing MT
would not be fully ingested, resulting in less consumption of hormone.
During the oral masculinization treatment, it is possible that by
increasing the feeding frequency higher concentrations of hormone in
the blood are obtained due to the short half-life of MT. This could
potentially assure higher masculinization, therefore resulting in less
quantity of females and intersex fish (Meurer et al., 2012). However, this
was not the case for our study, where both frequencies tested, five and
eight times a day, presented similar results, indicating that a feeding
frequency of five times a day is enough for the masculinization of Nile
tilapia in BFT.
In fact, we obtained no difference between masculinization rate from
fish reared in clear water and from BFT with the same feed frequency
and concentration, thus the initial assumption based on less hormone
consumption was not proved. David-Ruales et al. (2019) found lower
masculinization induced by MT (60 mg ⋅ kg− 1
of feed) in red tilapia
reared in BFT (male proportion of 64%) as compared to animals reared
in RAS (male proportion of 91%). However, it is worth to note these
authors did not describe the feed frequency applied in their experiments
and they used a constant feeding rate of 10%, which could be insuffi­
cient to induce an efficient masculinization of tilapias reared in BFT.
Baroiller and Cotta (2018), for instance, recommended feeding rates of
Table 2
Growth performance and survival for Nile tilapia larvae reared in clear water (control) or biofloc system (BFT), during 28 days of masculinization under different 17α-
methyltestosterone (MT) concentration in the ration.
Variables Control
(Clear water)
BFT CV
5×/day 5× ⋅ day− 1
MT concentration (mg ⋅ Kg− 1
feed)
8× ⋅ day− 1
MT concentration (mg ⋅ Kg− 1
feed)
60 60 90 120 150 180 60 90 120 150 180
BWf (g)**(1)
0.76 0.90 0.83 0.74 0.80 0.85 0.90 0.87 0.86 0.77 0.68 16.46
BMf (g)**(2)
110.19 155.59 158.58 139.10 150.33 156.37 171.16 158.40 160.52 138.13 121.59 18.43
SDf (Kg ⋅ m− 3
)**(3)
0.73 1.04 1.06 0.93 1.00 1.04 1.14 1.06 1.07 0.92 0.81 18.43
DWG (mg ⋅ day− 1
)**(4)
26.58 31.82 29.09 26.06 28.34 30.07 31.90 30.68 30.35 27.18 23.87 16.69
SGR (%⋅ day− 1
)* (5)
14.68 15.29 14.92 14.57 14.99 15.09 15.13 15.09 15.23 14.83 14.39 3.70
Survival (%)*(6)
77b
88ab
97a
95a
94a
93a
95a
93a
94a
91a
91a
6.29
BWf = Final body mean weight; BMf = Final mean biomass; SDf = Final stocking density; DWG = Daily weight gain; SGR = Specific growth rate (%/day).
*Means in the same line did not differ according to ANOVA and Tukey test (p > .05).
**Means with different letters in the same line differ according to ANOVA and SNK test (p < .05).
x1 = block (1 or 2, corresponding 0 or 1 in the model, respectively) and x2 = 17α-methyltestosterone concentration in the ration.
(1)Final body mean weight: y = 0.67 + 0.57 × 1–0.0011 × 2; R2
= 0.85.
(2)Final mean biomass: y = 128.66 + 95.09 × 1–0.21 × 2; R2
= 0.80.
(3)Final stocking density: y = 0.86 + 0.63 × 1–0.0014 × 2; R2
= 0.80.
(4)Daily weight gain: y = 23.55 + 20.28 × 1–0.04 × 2; R2
= 0.85.
(5)Specific growth rate: y = 14.15 + 2.47 × 1–0.0036 × 2; R2
= 0.88.
(6)Survival: The regression model was not significant (p > .05).
Fig. 2. Percentage of male, female, intersex, and undifferentiated animals of Nile tilapia submitted to different concentration of 17-α-methyltestosterone on the fish
food and feed frequency in control and BFT systems. The linear regression estimated by the transformed results from BFT treatments (y = 1.48–0.22 × 1–0.00098 × 2;
R2
= 0.45; where x1 = block (1 or 2, corresponding 0 or 1 in the model) and x2 = 17α-methyltestosterone concentration) presented a p-value = .051 for the coefficient
that describes the effect of dose treatments over the distribution of masculinization results.
R.Z. Costa e Silva et al.

7. Aquaculture 547 (2022) 737470
7
20% of biomass per day for the first week, 18% for the second, 16% for
the third week, and 15% for the fourth week. These feeding rates are
lower than those used in this study (30% of the fish weight for the first
week, 25% for the second, 20% for the third, and 15% for the fourth
week) and their effectiveness could also be tested in BFT.
The second strategy for statistical analysis was adopted to evaluate
the results only between BFT treatments, and access whether there are
differences in masculinization rate among the feeding frequency and
hormone concentration evaluated. Once again, there were no statistical
differences between the possible interactions. Also, the feeding fre­
quencies tested did not present a significant statistical effect. According
to the estimated regression, the increase in hormone concentration
reduced the percentage of males. However, the b regressor, which rep­
resents the effect of hormone concentration on the variation of mascu­
linization results was not significant (p = .0511) (Fig. 2).
In our study, we found an average of 5% of undifferentiated finger­
lings. For a better evaluation of our results, two different scenarios were
defined to describe the future of undifferentiated animals. In the first
scenario, the proportion of males from the undifferentiated fish was
inputted to be 50%; whereas in the second scenario, the proportion of
undifferentiated would be the same result obtained in the masculiniza­
tion of each corresponded treatment (e.g., in an experimental unit with
98% male, the count was: total male = male +0.98 × undifferentiated
animals). In both scenarios (Fig. 3), the regressors were significant and
similar to the regressor estimated from non-simulated data (− 0.00098
for non-inputted, − 0.00084 for situation 1, and − 0.00082 for situation
2). Therefore, the results suggest a reduction of masculinization due to
the increase in hormone concentration in the fish food. As in clear water
protocols, the hormone concentration treatments in BFT led to a paradox
sex reversal when the concentration exceeds a limit. Excessive hormone
concentration may promote the formation of females and intersex in­
dividuals and decrease in the growth of O. niloticus (Guerrero, 1975;
Cruz and Mair, 1994; Pandian and Sheela, 1995; Beardmore et al., 2001;
El-Sayed, 2006). In our study, as the hormone concentration increased
fewer male individuals were obtained, most likely due to the paradox
sex reversal effect (Fig. 2). In fact, as 60 mg ⋅ kg− 1
promoted the higher
masculinization rate, lower levels of MT concentration (Baroiller and
Cotta, 2018) should be tested for BFT in a further study to find out the
optimal MT concentration.
In conclusion, high concentrations of hormone in masculinization
protocols are not recommended due to the paradox sex reversal, even in
BFT systems. However, it is possible to achieve high masculinization
rates in Nile tilapia reared in BFT using MT from post yolk sac absorption
to 28 days of age, in a feeding rate of 30% for the first week, 25% for the
second one, 20% for the third and 15% for the fourth week. The results
of this study indicate that a masculinization rate equal or superior to
94% can be achieved, using fish feed enriched with MT in the concen­
tration of 60 mg ⋅ kg− 1
, the lowest concentration evaluated, with feeding
frequency of five times a day, therefore the cheapest and most effective
protocol.
Data availability
The data that support the findings of this study is available from the
corresponding author upon reasonable request.
Author contributions statement
Rodrigo Z. C. Silva - data collection, data analysis, interpretation of
data and article preparation.
Érika R. Alvarenga - contributed to the experimental design, data
analysis, interpretation of data and article preparation.
Sylvia V. Matta - contributed to the data analysis and interpretation
of data.
Gabriel F. O. Alves - contributed to the experimental design and
article preparation.
Ludson G. Manduca - contributed to the data collection and article
preparation.
Marcos A. Silva - contributed to the data analysis, interpretation of
data and article preparation.
Thomás T. Yoshinaga - contributed to the data analysis and inter­
pretation of data.
Arthur Francisco Araújo Fernandes - contributed to the data analysis
and article preparation.
Eduardo M. Turra - experimental design, data analysis, interpreta­
tion of data, article preparation and coordination.
Fig. 3. Two different scenarios of masculinization. In scenario 1, we estimated 50% of undifferentiated animals as female. In scenario 2, we expect to find the same
masculinization proportion identified in this work (for example, in an experimental unit with 98% of male, the count was: total male = male + (0.98 × undif­
ferentiated animals). In these estimations, the masculinization proportion data was arcsine transformed and a linear regression model was obtained: y scenario 1 =
1.51–0.22 × 1–0.00084 × 2; R2 = 0.55; y scenario 2 = 1.55–0.22 × 1–0.00082 × 2; R2
= 0.60, where x1 = block (1 or 2, corresponding 0 or 1 in the model) and x2 =
17α-methyltestosterone concentration.
R.Z. Costa e Silva et al.

8. Aquaculture 547 (2022) 737470
8
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Rodrigo Zhouri Costa e Silva reports financial support (scholarship)
was provided by Conselho Nacional de Desenvolvimento Científico e
Tecnológico.
Acknowledgments
This research received support from CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico).
References
Ahmad, I., Rani, B.A.M., Verma, A.K., Maqsood, M., 2017. Biofloc technology: an
emerging avenue in aquatic animal healthcare and nutrition. Aquac. Int. 25,
1215–1226. https://doi.org/10.1007/s10499-016-0108-8.
Alvarenga, É.R., Alves, G.F.O., Fernandes, A.F.A., Costa, G.R., Silva, M.A., Teixeira, E.A.,
Turra, E.M., 2018. Moderate salinities enhance growth performance of Nile tilapia
(Oreochromis niloticus) fingerlings in the biofloc system. Aquac. Res. 49, 2919–2926.
https://doi.org/10.1111/are.13728.
Alvarenga, E.R., Fernandes, A.F.A, Lopes, L.R., Soares, T.E., Alves, G.F.O.A., Costa, F.F.
B., Sales, S.C.M., Lima, G.K., Turra, E.M., 2020. Attempt to produce a Nile tilapia
tetraploid line by heat shock induction. Aquaculture 529, 735647.
APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American
Public Health Association, American Water Works Association, Water Environment
Federation, Washington, DC.
Arai, K., Fujimoto, T., 2018. Chromosome manipulation techniques and applications to
aquaculture. In: Wang, H.-P., Piferrer, F., Chen, S.-L. (Eds.), Sex Control in
Aquaculture. John Wiley & Sons Ltd., pp. 137–162
Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture
systems. Aquaculture 176, 227–235. https://doi.org/10.1016/S0044-8486(99)
00085-X.
Avnimelech, Y., 2009. Biofloc Technology: A Practical Guide Book. The World
Aquaculture Society, Baton Rouge, Louisiana. https://doi.org/10.13140/
2.1.4575.0402.
Azim, M.E., Little, D.C., 2008. The biofloc technology (BFT) in indoor tanks: water
quality, biofloc composition, and growth and welfare of Nile tilapia (Oreochromis
niloticus). Aquaculture 283, 29–35. https://doi.org/10.1016/j.
aquaculture.2008.06.036.
Baroiller, J.F., Cotta, H.D., 2018. Sex control in tilapias. In: Wang, H.-P., Piferrer, F.,
Chen, S.-L. (Eds.), Sex Control in Aquaculture. John Wiley & Sons Ltd., pp. 191–234
Baroiller, J.F., Cotta, H.D., Bezault, E., Wessels, S., Hoerstgen-Schwark, G., 2009. Tilapia
sex determination: where temperature and genetics meet. Comp. Biochem. Physiol.
A 153 (1), 30–38. https://doi.org/10.1016/j.cbpa.2008.11.018.
Barry, T.P., Marwah, A., Marwah, P., 2007. Stability of 17α-methyltestosterone in fish
feed. Aquaculture 271, 523–529. https://doi.org/10.1016/j.
aquaculture.2007.05.001.
Beardmore, J.A., Mair, G.C., Lewis, R.I., 2001. Monosex male production in finfish as
exemplified by tilápia: applications, problems, and prospects. Aquaculture 197,
283–301. https://doi.org/10.1016/S0044-8486(01)00590-7.
Bendschneider, K., Robinson, R.J., 1952. A new spectrophotometric method for the
determination of nitrite in sea water. J. Mar. Res. 11, 87–96.
Boyd, C.E., Tucker, C.S., Somridhivej, B., 2016. Alkalinity and hardness: critical but
elusive concepts in aquaculture. J. World Aquacult. Soc. 47, 6–41. https://doi.org/
10.1111/jwas.12241.
Brämick, U., Puckhaber, B., Langholz, H.-J., Hörstgen-Schwark, G., 1995. Testing of
triploid tilapia (Oreochromis niloticus) under tropical pond conditions. Aquaculture
137, 343–353. https://doi.org/10.1016/0044-8486(95)01104-8.
Crab, R., Defoirdt, T., Bossier, P., Verstraete, V., 2012. Biofloc technology in aquaculture:
beneficial effects and future challenges. Aquaculture 356-357, 351–356. https://doi.
org/10.1016/j.aquaculture.2012.04.046.
Cruz, E.M.V., Mair, G.C., 1994. Conditions for effective andron sex reversal in
Oreochromis niloticus (L.). Aquaculture 122, 237–248. https://doi.org/10.1016/
0044-8486(94)90513-4.
Daudpota, A.M., Abbas, G., Kalhoro, I.B., Shah, S.S.A., Kalhoro, H., Hafeez-UR-
Rehman, M., Ghaffar, A., 2016. Effect of feeding frequency on growth performance,
feed utilization and body composition of juvenile Nile tilapia, Oreochromis niloticus
(L.) reared in low salinity water. Pak. J. Zool. 48, 1–16.
David-Ruales, C.A., Betancur-Gonzalez, E.M., Valbuena-Villareal, R.D., 2019. Sexual
reversal with 17α-Methyltestosterone in Oreochromis sp.: comparison between
recirculation aquaculture system (RAS) and Biofloc technology (BFT). J. Agr. Sci.
Tech-Iran 9, 131–139. https://doi.org/10.17265/2161-6256/2019.02.007.
Di Rienzo, J.A., Casanoves, F., Balzarini, M.G., Gonzalez, L., Tablada, M., Robledo, C.W.,
2015. InfoStat Version 2015. Grupo InfoStat, FCA, Universidad Nacional de Cordoba,
Argentina. http://www.infostat.com.ar.
Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the
stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of
ammonia-nitrogen in aquaculture systems. Aquaculture 257, 346–358. https://doi.
org/10.1016/j.aquaculture.2006.03.019.
Ekasari, J., Angela, D., Waluyo, S.H., Bachtia, T., Surawidjaja, E.H., Bossier, P., De
Schryver, P., 2014. The size of biofloc determines the nutritional animals.
Aquaculture 427, 105–111. https://doi.org/10.1016/j.aquac ulture.2014.01.023.
Ekasari, J., Rivandi, D.R., Firdausi, A.P., Surawidjaja, E.H., Zairin Jr., M., Bossier, P., De
Schryver, P., 2015. Biofloc technology positively affects Nile tilapia (Oreochromis
niloticus) larvae performance. Aquaculture 441, 72–77.
El-Sayed, A.-F.M., 2006. Tilapia Culture. CABI Publishing, Wallingford, Oxfordshire.
https://doi.org/10.1079/9780851990 149.0000.
FAO, 2016. The State of World Fisheries and Aquaculture 2016. Contributing To Food
Security And Nutrition For All, Rome.
Farahmand, H., Razak, S.H.A., Hwang, G.L., Maclean, N., Rahman, M.A., 2007. Induction
of tetraploidy in transgenic tilapia (Oreochromis niloticus) using physical shocks. Iran.
J. Fish. Sci. 7, 27–46.
Fernandes, A.F.A., Silva, M.A., Alvarenga, E.R., Teixeira, E.A., Silva Junior, A.F.,
Alves, G.F.O., Turra, E.M., 2015. Morphometric traits as selection criteria for carcass
yield and body weight in Nile tilapia (Oreochromis niloticus L.) at five ages.
Aquaculture 446, 303–309.
Green, B.W., Rawles, S.D., Schrader, K.K., Gaylord, T.G., McEntire, M.E., 2019. Effects of
dietary protein content on hybrid tilapia (Oreochromis aureus × O. niloticus)
performance, common microbial off-flavor compounds, and water quality dynamics
in an outdoor biofloc technology production system. Aquaculture 503, 571–582.
https://doi.org/10.1016/j.aquaculture.2019.01.034.
Guerrero, R.D., 1975. Use of androgens for the production of all-male Tilapia aurea
(Steindachner). Trans. Am. Fish. Soc. 104, 342–348. https://doi.org/10.1577/1548-
8659(1975)104<342:UOAFTP>2.0.CO;2.
Guerrero, R.D., Shelton, W.L., 1974. An aceto-carmine squash method for sexing juvenile
fishes. Progress. Fish Cult. 36, 56. https://doi.org/10.1577/1548-8659(1974)36[56:
AASMFS]2.0.CO;2.
Hargreaves, J.A., 2013. Biofloc production systems for aquaculture. SRAC 4503, 1–12.
Hines, G.A., Watts, S.A., 1995. Non-steroidal chemical sex manipulation of Tilapia.
J. World Aquacult. Soc. 26, 98–102. https://doi.org/10.1111/j.1749-7345.1995.
tb00216.x.
Hussain, M.G., Rao, G.P.S., Humayun, N.M., Randall, C.F., Penman, D.J., Kime, D.,
Bromage, N.R., Myers, J.M., 1995. Comparative performance of growth, biochemical
composition and endocrine profiles in diploid and triploid tilapia Oreochromis
niloticus L. Aquaculture 138, 87–97. https://doi.org/10.1016/0044-8486(95)
01079-3.
Joshi, H.D., Tiwari, V.K., Gupta, S., Sharma, R., Lakra, W.S., Sahoo, U., 2019. Application
of nanotechnology for the production of masculinized Tilapia, Oreochromis niloticus
(Linnaeus, 1758). Aquaculture 511, 734206.
Lara-Flores, M., Olvera-Novoa, M.A., 2013. The use of lactic acid bacteria isolated from
intestinal tract of Nile tilapia (Oreochromis niloticus), as growth promoters in fish fed
low protein diets. Lat. Am. J. Aquat. Res. 41 (3), 490–497.
Luthada, R.W., Jerling, H.L., 2013. Effect of feeding frequency and feeding rate on
growth of Oreochromis mossambicus (Teleostei: Cichlidae) fry. Afr. J. Aquat. Sci. 38,
273–278. https://doi.org/10.2989/16085914.2013.779567.
Mair, G.C., Scott, A.G., Penman, D.J., Beardmore, J.A., Skibinski, O.F., 1995. Growth
performance trials of genetically male tilapia (GMT) derived from YY-males in
Oreochromis niloticus L.: on station comparisons with mixed sex and sex reversed
male population. Aquaculture 137, 313–322.
Makino, L.C., Nakaghi, L.S.O., Paes, M.C.F., Malheiros, E.B., Dias-Koberstein, T.C.R.,
2008. Effectiveness of methods of sexual identification in Nile tilapia (Oreochromis
niloticus) sexually reverted with hormone in diet with different diameters. Biosci. J.
25, 112–121.
Manduca, L.G., Silva, M.A., Alvarenga, E.R., Alves, G.F.O., Ferreira, N.H., Teixeira, E.A.,
Turra, E.M., 2021. Effects of different stocking densities on Nile tilapia performance
and profitability of a biofloc system with a minimum water exchange. Aquaculture
530, 735814. https://doi.org/10.1016/j.aquaculture.2020.735814.
Meurer, F., Bombardelli, A.R., Paixão, P.S., Silva, L.C.R., Santos, L.D., 2012. Feeding
frequency on growth and male percentage during sex reversion phase of Nile Tilapia.
Rev. Bras. Saúde Prod. Anim. 13, 1133–1142. https://doi.org/10.1590/S1519-
99402012000400024.
Naylor, R.L., Goldburg, R.J., Primavera, J.H., Kautsky, N., Beveridge, M.C.M., Clay, J.,
Troell, M., 2000. Effect of aquaculture on world fish supplies. Nature 405,
1017–1024. https://doi.org/10.1038/35016500.
Pandian, T.J., Sheela, G.S., 1995. Hormonal induction of sex reversal in fish. Aquaculture
138, 1–22. https://doi.org/10.1016/0044-8486(95)01075-0.
Pérez-Fuentes, J.A., Hernández-Vergara, M.P., Pérez-Rostro, C.I., Forgel, I., 2016. C:N
ratios affect nitrogen removal and production of Nile tilapia Oreochromis niloticus
raised in a biofloc system under high density cultivation. Aquaculture 452, 247–251.
https://doi.org/10.1016/j.aquaculture.2015.11.010.
Phelps, P.R., Popma, J.T., 2000. Sex reversal of tilapia. Tilapia Aquaculture in the
Americas. J. World Aquacult. Soc. 2, 34–59.
Popma, T.J., Green, B.W., 1990. Sex reversal of Tilapia in earthen ponds. In: Popma
(Ed.), Research and Development Series. International Center for Aquaculture,
Alabama Agriculture Experiment Station, Auburn University, Auburn, AL, p. 15.
R Core Team, 2016. R: A Language and Environment for Statistical Computing. R
Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/.
Rakocy, J.E., Bailey, D.S., Thoman, E.S., Shultz, R.C., 2004. Intensive tank culture of
tilapia with a suspended, bacterial-based, treatment process. New dimensions on
farmed tilapia. In: Bolivar, R., Mair, G., Fitzsimmons, K. (Eds.), Proceedings of the
Sixth International Symposium on Tilapia in Aquaculture. Manila, Philippines,
pp. 584–598.
R.Z. Costa e Silva et al.

9. Aquaculture 547 (2022) 737470
9
Rebouças, V.T., Lima, F.R.S., Cavalcante, D.H., Sá, M.V.C., 2016. Reassessment of the
suitable range of water pH for culture of Nile tilapia Oreochromis niloticus L. in
eutrophic water. Acta Sci. Anim. Sci. 8, 361–368. https://doi.org/10.4025/
actascianimsci.v38i4.32051.
Silva, M.A., Alvarenga, É.R., Alves, G.F.O., Manduca, L.G., Turra, E.M., Brito, T.S.,
Sales, S.C.M., Silva Júnior, A.F., Borges, W.J.M., Teixeira, E.A., 2018. Crude protein
levels in diets for two growth stages of Nile tilapia (Oreochromis niloticus) in a biofloc
system. Aquac. Res. 1–11. https://doi.org/10.1111/are.13730.
Singh, A.K., 2013. Introduction of modern endocrine techniques for the production of
monosex population of fishes. Gen. Comp. Endocrinol. 181, 146–155. https://doi.
org/10.1016/j.ygcen.2012.08.027.
Strickland, J.D.H., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis, , 2nd
ed.167. Fisheries research Board of Canada, Bulletin, Ottawa.
Turra, E.M., Oliveira, D.A.A., Valente, B.D., Teixeira, E.A., Prado, S.A., Melo, D.C.,
Silva, M.A., 2012a. Estimation of genetic parameters for body weights of Nile tilapia
Oreochromis niloticus using random regression models. Aquaculture 354, 31–37.
Turra, E.M., Oliveira, D.A.A., Valente, B.D., Teixeira, E.A., Prado, S.A., Alvarenga, E.R.,
Silva, M.A., 2012b. Longitudinal genetic analyses of fillet traits in Nile tilapia
Oreochromis niloticus. Aquaculture 356-357, 381–390.
Turra, E.M., Toral, F.B., Alvarenga, E.R., Raidan, F.S.S., Fernandes, A.F.A., Alves, G.F.O.,
Silva, M.A., 2016. Genotype×environment interaction for growth traits of Nile
tilapia in biofloc technology, recirculating water and cage systems. Aquaculture 460,
98–104.
Turra, E.M., Fernandes, A.F.A., Alvarenga, É.R., Alves, G.F.O., Manduca, L.G.,
Teixeira, E.A., Silva, M.A., 2018. Longitudinal analyses of correlated response
efficiencies of fillet traits in Nile tilapia. Animal 12, 1–9.
UNESCO, 1983. Chemical methods for use in marine environmental monitoring. In:
Manual and Guides 12. Intergovernmental Oceanographic Commission, Paris.
Wang, H.-P., Shen, Z.-G., 2018. Sex control in aquaculture: Concept to practice. In:
Wang, H.-P., Piferrer, F., Chen, S.-L. (Eds.), Sex Control in Aquaculture. John Wiley
& Sons Ltd., pp. 3–34
Wassermann, G.J., Afonso, L.O.B., 2003. Sex reversal in Nile tilapia (Oreocohromis
niloticus Linnaeus) by androgen immersion. Aquac. Res. 34, 65–71. https://doi.org/
10.1046/j.1365-2109.2003.00795.x.
Wedemeyer, G., 1996. Physiology of Fish in Intensive Culture Systems. Springer,
Chapman & Hall, New York, NY. https://doi.org/10.1007/978-1-4615-6011-1.
Yanbo, W., Wenju, Z., Weifen, L., Zirong, X., 2006. Acute toxicity of nitrite on tilapia
(Oreochromis niloticus) at different external chloride concentrations. Fish Physiol.
Biochem. 32, 49–54. https://doi.org/10.1007/s10695-005-5744-2.
R.Z. Costa e Silva et al.