Aquaculture 547 (2022) 737470

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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, E´ rika Ramos Alvarenga a, Sylvia Velloso Matta a,
Gabriel Francisco de Oliveira Alves a, Ludson Guimara˜es Manduca a, Marcos Antoˆnio Silva a, Thoma´s
Toshio Yoshinaga a, Arthur Francisco Araújo Fernandes b, Eduardo Maldonado Turra a, *
a
Escola de Veterina´ria da Universidade Federal de Minas Gerais, Av. Antˆonio 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

ARTICLEINFO
ABSTRACT
Key-words:
BFT Sexual control through hormone treatments is widely used in Nile tilapia due to its simplicity and high
Masculinization efficiency. However, possible impacts of hormone residues introduction on the environment are still a topic of
Sex control concern. One way to minimize the environmental impact of hormonal treatments is to use close aquaculture
Uniformity of fingerlings systems, such as the Biofloc Technology (BFT). However, BFT system can provide constant additional feed
Fish
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
—1
times a day. The control group consisted of fry raised in clear water tanks fed with 60 mg ⋅ kg hormone diet
provided five times a day. The experimental design was a factorial arrangement (5 2), plus control, on two
—1
random blocks, each block having two replicates per treatment. Stocking density applied was of 2 fry⋅ L
—1
(300 fry⋅ tank ). Water quality variables did not diverge between BFT systems and control group, except for
settablesolids, 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
—1
less proportion of males in contrast to 60 mg ⋅ kg groups and no differences were found between feed
frequencies tested. In conclusion, it was possible to achieve 94% masculinization rates in tilapia using 17 α-
—1
methyltestosterone at 60 mg ⋅ kg 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 population 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).

https://doi.org/10.1016/j.aquaculture.2021.737470
Received 17 May 2021; Received in revised form 16 August 2021; Accepted 8 September 2021
Available online 10 September 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
R.Z. Costa e Silva et al.
“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 determination 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
thermosensitivity has genetic components, it is possible to increase
the proportion 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
solutions for that are still in its beginning (Baroiller and DttaCotta,
2018). Most farmers use hormonal treatments, especially 17α-
methyltestosterone (MT), for obtaining male monosex populations
of Niletilapia. MT is preferentially chosen because it is a simple,
highly efficient, 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 tilapiaproducing 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
masculinization 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 prohibitive. 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 treateddirectly in systems where plankton
and/or microorganisms develop, and with possible loss of part of the
—1
feed, high dosages are often suggested (60 mg ⋅ Kg of feed),
—1
whereas, in indoor systems, lower dosages (30–45 mg ⋅ Kg 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 degradation. 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
2
Aquaculture 547 (2022) 737470
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 nutrients 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
—3
with more than 500 g body weight with 15 to 75 fish ⋅ m (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
—3
weight) can be produced at stocking densities of 400 individuals ⋅ m ,
—1
in moderately saline environments (4 to 8 mg ⋅ L ) 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
environmental impact, due to its capacity to recycle nutrients (Azim
and Little, 2008; Avnimelech, 2009). Nevertheless, based on the initial
hypothesis 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
masculinization should be tested. Corroborating this hypothesis, David-
Rualeset al. (2019) compared the sexual inversion of red tilapia
—1
induced by 17-α-methyltestosterone (60 mg ⋅kg of feed) in
Recirculating Aquaculture System (RAS) and BFT and found a
proportion of males of 91% and 64%, respectively. Therefore, we
aimed to investigate the concentration 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
University of Minas Gerais (Laborato´rio de Aquacultura LAQUA,
Escola de Veterin´aria/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
R.Z. Costa e Silva et al.
reproduction 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
density 2 fry ⋅ L ), as described by Lara-Flores and Olvera-Novoa
(2013).
The experiment was
designed in a factorial 2001; Baroiller and Cotta,× 2018).
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
combination 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 concentrations of
hormone were tested, 60, 90,
—1
120, 150 and 180 mg ⋅ kg on
the fish feed. The lower dose of
—1
60 mg ⋅ kg 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
—1
mg. kg 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,
—1
we evaluated if 60 mg ⋅ kg 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
-1
60 mg. kg of feed in clear
water with a frequency of five
times a day, which is the
protocol often used in outdoor
systems.
= = = 0.01 g, week 2 0.15 g, week
Results of
= different
masculinization rates between
Nile tilapia families have already
been obtained and presented in
—1
(stocking
the literature (Beardmore et al.,
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 evaporation and
— classified as intersex when
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
3 0.4 g and week 4 0.8 g),
according to results of previous
experiments. An expected ×
3
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
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 ⋅
Temperature, salinity, pH, and
dissolved oxygen (DO) were
measured three times per week
using the YSI 6920 V2 (Yellow
Springs Incorporated YSI, OH,
USA) multiparameter probe.
— Shelton (1974), validated by
TAN and N-NO2 were also
analyzed three times perweek
using spectrophotometry
(Biochrom Libra S22), according
to UNESCO (1983)
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
Parsons (1972), respectively.
During the experiment, when
ammonia exceeded 0.5 mg . L
1 2.4. Growth
, 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
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 difference
temperatures between
treatments, the water renewal
was made with water that was
Aquaculture 547 (2022) 737470
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 determination. 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 individually
the weighed and 60 were randomly
chosen for microscopic
analyses of gonads and sex
determination. These fish were
stored in Bouin liquid for 24 h
and then transferred to an
—1
L . alcohol solution at 70%. We
used the classification method
to define males, females and
undifferentiated fish using aceto-
carmin squash technic as
described by Guerrero and
Wassermann and Afonso
(2003) and reviewed by Makino
et al. (2008). Fish were
and
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,
and
21, and at the end of the
experiment.
—
performance and
survival
Growth performance was
evaluated based on the final
—1
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 BM f =
(BW15 × N15) + (BW21 × N21)
in
+ (BWf ×
Nf). BW15 is the body weight and
N15 is the number of fish
R.Z. Costa e Silva et al. Aquaculture 547 (2022) 737470
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 obtained as described by
Ekasari et al. (2015) by
[(BWf /BWi)1/t

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

4
 (Avnimelech,ammonia nitrogen) <
homogeneous, the results were analyzed by ANOVA and post-tested

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), Table 1

(Wedemeyer, 1996); pH = 5.5–9 (Rebouças et al., 2016);

depending on CV, < 10% by Tukey, > 10% and < 20% by SNK and

1.79
CV
above 20% by Duncan test. The values that presented p < .05 in
Shapiro Wilk or Bartlett test were submitted to Kruskal-Wallis test.

180
The performance variables were analyzed using two statistical
strategies. First, all treatments were compared using the mean, since

26.45
the control group is a qualitative treatment. In this comparison, if the
residuals of the response variable presented normal distribution with
zero mean, they would be evaluated by ANOVA and post test, as

150
described above. In a second statistical strategy, we considered only

t (p > .05). ***Means with different letters in the same line differ according to ANOVA and Duncan test (p < .05). *Means in the same line did not differ according to ANOVA and Tukey test (p > .05).
during 28 days of masculinization under different 17α-methyltestosterone (MT) concentration in the diet and two different feeding strategies (5 × ⋅ day and 8 × ⋅ day ).
—1
BFT treatments and analyzed the data by factorial ANOVA, including

26.82
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

120
were also fitted for all growth performance variables.
In order to compare body size uniformity between control and BFT,

26.73
—1
an individual measurement of weight was made in 75 individuals per

MT concentration (mg ⋅ Kg feed)

tank (150 individuals per treatment in each block) and the

—1
homogeneity of variance was tested by Bartlett and F-test to compare

90

—1
the two variances. Since the Bartlett and F tests do not consider the

60 6.45 26.78

(El-Sayed, 2006); DO (dissolved oxygen) > 4 mg ⋅ L

effect from blocks, the entire procedure was performed using results
from each block.
Masculinization data was transformed in arcsine of the square root

—1
to properly normalize the values and then submitted to ANOVA and

8× ⋅ day
Tukey tests. A linear regression model was fitted. For a better

180 6.49 26.44

evaluation of the treatment effects over masculinization, an analysis of
different scenarios was established: we assumed a first scenario
where 50% of undifferentiated 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

26.71
repeat itself with the remaining undifferentiated individuals. The results
considering the scenarios were then transformed in arcsine square
root and then submitted to linear regression analyses. Infostat (Di
150

Rienzo et al., 2015) and R (R Core Team, 2016) software were used
for the analysis.
26.76

3. Results and discussion

—1
Reference values for tilapia culture: Temperature = 27–32 ◦C (El-Sayed, 2006); nitrite <8 mg ⋅ L
Sexual control by hormone treatments is used in Nile tilapia
120

because this species can reproduce before reaching market size,

26.83

which generates many problems to tilapia farmers, such as

overpopulation, reduced growth, and die-off (Baroiller and Cotta,
MT concentration (mg ⋅ Kg feed)

2018; Bra¨mick et al., 1995; Farahmand et al., 2007; Hussain et al.,

—1

90

1995). Despite the environmental issues related to hormone use, it

remains widely used due to its simplicity and high efficiency (Baroiller
60 6.48 26.59

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
—1

generated by hormones. However, before assessing whether BFT

5× ⋅ day

would be a solution for reducing hormone residues, it is necessary to

6.48 26.54
BFT

establish an efficient masculinization protocol in this system, once its

high concentration and availability of live food in the water, above a
2009); salinity ≤ 8 g ⋅ L (Alvarenga et al., 2018)

traditional larvae culture system in ponds (and well above a clear water
Control (Clear water)

system), could interfere with the ingestion of food containing the daily
amount of androgen necessary for the masculinization 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
—1

systems, and established a protocol for masculinization of Nile tilapia

in BFT.
)* pH**

Water quality parameters were within the recommended range for

the development of tilapia (Table 1). As expected, TSS and SS were
—1
Temperature ( C)* DO (mg ⋅ L

different between treatments, with higher values found in BFT in

comparison to the control group. However, these differences did not
Variables

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 P´erez-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
—1
same dose (60 mg ⋅ kg of feed) in different systems and feed
frequencies (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
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
significant for growth performance variables and a higher feeding
frequency tested did not improve the growth nor survival of the
animals. Therefore, the linear models presented were composed only
by the effects of block and hormone concentration. All the performance
variables, 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 inefficient 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
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.)
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
17αmethyltestosterone (MT) concentration in the ration.

Variables Control
(Clear water) BFT CV

—1 —1
5×/day 5× ⋅ day ⋅ day
8×
—1 —1
MT concentration (mg ⋅ Kg feed) MT concentration (mg ⋅ Kg feed)

60 60 90 120 150 180 60 90 120 150 180

(1)
BWf (g)** 0.76 0.90 0.83 0.74 0.80 0.85 0.90 0.87 0.86 0.77 0.68 16.46
(2)
BMf (g)** 110.19 155.59 158.58 139.10 150.33 156.37 171.16 158.40 160.52 138.13 121.59 18.43
—3 (3)
SDf (Kg ⋅ m )** 0.73 1.04 1.06 0.93 1.00 1.04 1.14 1.06 1.07 0.92 0.81 18.43
—1 (4)
DWG (mg ⋅ day )** 26.58 31.82 29.09 26.06 28.34 30.07 31.90 30.68 30.35 27.18 23.87 16.69
—1 (5)
SGR (%⋅ day )* 14.68 15.29 14.92 14.57 14.99 15.09 15.13 15.09 15.23 14.83 14.39 3.70
(6) b ab a a a a a a a a a
Survival (%)* 77 88 97 95 94 93 95 93 94 91 91 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 x 2 = 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 x 2 = 17α-methyltestosterone concentration) presented a p-value = .051 for the coefficient
that describes the effect of dose treatments over the distribution of masculinization results.

the groups. Several masculinization protocols, including the use of MT
in diet, have been tested in the past decades. Literature extensively
mentions 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 hypothesis 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
—1
masculinization induced by MT (60 mg ⋅ kg 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 insufficient to induce an efficient masculinization of tilapias
reared 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 frequencies 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 represents the effect of hormone concentration
on the variation of masculinization results= was not significant (p .0511)
(Fig. 2).
In our study, we found an average of 5% of undifferentiated
fingerlings. 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 masculinization 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 individuals 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
—1
reversal effect (Fig. 2). In fact, as 60 mg ⋅ kg 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
Baroiller and Cotta (2018), for instance, recommended feeding rates of
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.
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
—1
concentration of 60 mg ⋅ kg , 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.
E´ rika 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.
Thoma´s T. Yoshinaga contributed to the data analysis and
interpretation of data.
Arthur Francisco Araújo Fernandes contributed to the data analysis
and article preparation.
Eduardo M. Turra experimental design, data analysis, interpretation
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 ×
undifferentiated 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 x 2 =
17α-methyltestosterone concentration.

Declaration of Competing Interest A 153 (1), 30–38. https://doi.org/10.1016/j.cbpa.2008.11.018.

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