Aquaculture 520 (2020) 734938

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Research paper

High percentages of larval tetraploids in the yellowtail tetra Astyanax T

altiparanae induced by heat-shock: The first case in Neotropical characins
Nivaldo Ferreira do Nascimentoa,b,⁎, Matheus Pereira-Santosa,c, Nycolas Levy-Pereirab,
Paulo Sérgio Monzanib, Daiane Niedzielskib, Takafumi Fujimotod, José Augusto Senhorinib,
Laura Satiko Okada Nakaghia, George Shigueki Yasuib
a
Aquaculture Center, Sao Paulo State University, Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil
b
Laboratory of Fish Biotechnology, National Center for Research and Conservation of Continental Fish, Chico Mendes Institute of Biodiversity Conservation, Rodovia Pref.
Euberto Nemésio Pereira de Godoy, Pirassununga, SP 13630-970, Brazil
c
Federal Rural University of Rio de Janeiro, BR-465 km 7, Seropédica, RJ 23897000, Brazil
d
Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, 041-8611 Hakodate, Japan

ARTICLE INFO ABSTRACT

Keywords: Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by
Fish inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization
Chromosome manipulation and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra
Heat-shock Astyanax altiparanae, triploids present sterility and increased carcass yield, suggesting the generation of tetra-
Polyploidy
ploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40° for
2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 °C were
compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping,
and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males
were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 ± 9.6%),
24 (16.0 ± 9.1%), and 26 mpf (10.3 ± 6.2%) in comparison with the control group (62.2 ± 7.7%). The flow
cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf pre-
sented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the
highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid
spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat
shock treatment at 26 mpf (40 °C for 2 min) is optimum for tetraploid induction in the A. altiparanae.
Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of
triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in
basic and applied sciences.

1. Introduction conventionally induced by temperature or pressure shocks few minutes

Triploid fish have attracted considerable attention in aquaculture
(Arai, 2001; Benfey, 2016) and when sterile, the deleterious effects of
early sexual maturation are avoided (Hulata, 2001; Taranger et al.,
2010). In such cases, the energy normally used for gonadal develop-
ment is deviated to somatic tissue in triploids, leading to increased
growth and carcass yield (Golpour et al., 2016; Morón-Alcain et al.,
2017; Nascimento et al., 2017b). Triploidization also reduces the en-
vironmental impacts caused by escape of farmed fish, since breeding
and introgression are avoided (Benfey, 2016). Triploids are
after fertilization in order to suppress the extrusion of the 2nd polar
body during meiosis (Piferrer et al., 2009). However, the induction of
triploid fish in commercial hatcheries may be challenging, due to lower
survival, the rise of abnormalities and the cost of equipment that is used
(Garcia et al., 2017; Peruzzi et al., 2007). In addition, several protocols
of induced triploidization are not 100% effective (Adamov et al., 2017;
Garcia et al., 2017).
In this scenario, an interesting alternative for mass production of
triploid fish is to use gametes from a tetraploid individual (e.g. when
normal eggs are fertilized with diploid sperm from a tetraploid male)

⁎
Corresponding author at: Aquaculture Center, Sao Paulo State University, Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil.
E-mail address: nivaldotec@yahoo.com.br (N.F. do Nascimento).

https://doi.org/10.1016/j.aquaculture.2020.734938
Received 29 May 2019; Received in revised form 31 December 2019; Accepted 7 January 2020
Available online 08 January 2020
0044-8486/ © 2020 Elsevier B.V. All rights reserved.
N.F. do Nascimento, et al. Aquaculture 520 (2020) 734938

(Nam and Kim, 2004; Weber et al., 2014; Weber et al., 2015). Such a
procedure avoids the deleterious effects of 2nd polar body retention,
resulting in increased survival (Chourrout et al., 1986; Lebeda and
Flajshans, 2015). Tetraploid fish can be artificially induced by sup-
pressing the cleavage of the zygote and also using thermal or pressure
shocks (Piferrer et al., 2009; Zhang and Onozato, 2004). However,
tetraploidization is difficult to achieve (Yoshikawa et al., 2008), and
viable lines are limited to a few species (Piferrer et al., 2009).
In the Neotropical region, for example, tetraploid induction is re-
ported only in a catfish species Rhamdia quelen. In this study, they re-
ported very low percentages of tetraploids, and no tetraploid lines were
established (Garcia et al., 2018). In this aspect, the yellowtail tetra
Astyanax altiparanae is an interesting candidate to establish such a
protocol, since this species has been used as a model organism for basic
and applied studies (Nascimento et al., 2017b; Pereira-Santos et al.,
2016; Piva et al., 2018; Siqueira-Silva et al., 2015). For this species, our
group has established protocols for production and rearing of triploid
fish. Firstly, to control the timing for fertilization, a protocol for in vitro
fertilization was developed (Yasui et al., 2015), which enabled studies
on early embryology and chromosome set manipulation, such as the
moment of the second polar body extrusion (Pereira-Santos et al.,
2016), triploid induction (Adamov et al., 2017) and first feeding within
triploids (Bertolini et al., 2018). Afterwards, it was observed that tri-
ploid female were sterile (Nascimento et al., 2017a) and presented in-
creased carcass yield (Nascimento et al., 2017b), suggesting that mass
production of triploid offspring of A. altiparanae could have potential in
aquaculture purposes. However, besides the efficacy of the previous
protocol using heat-shocking (~92% of triploids), no mass production
of triploid offspring (100%) were obtained (Adamov et al., 2017),
which can be achieved using tetraploid individuals. Additionally, as far
as we know, there is not a protocol to induce tetraploid individuals in
this species or even in other Neotropical characins. Therefore, the aim
of this recent study is to induce tetraploid fish in A. altiparanae using
temperature shock.
2. Material and methods
2.1. Ethics
This study was conducted in accordance with the Guide for the Care
and Use of Laboratory Animals at the Centro Nacional de Pesquisa e
Conservação da Biodiversidade Aquática Continental (CEUA/
CEPTA#0231.000070/2018–63).
(90 mm diameter) previously covered with polyvinylidene chloride
film.
2.3. Tetraploidization
Three mature couples of A. altiparanae were selected and induced to
reproduction as described previously (three repetitions). Oocytes from
each female (~ 8000) were inseminated using 90 μL of sperm from
different males, and the gametes were activated by adding 5 mL of
water. The fertilized eggs from each female (~ 8000) were then divided
in seven plastic containers in which the bottom contained a 100-μm
nylon mesh and was maintained in water with controlled temperature
at 22 °C. One container was kept intact (diploid control group), and the
others were subject to heat shock (40 °C; 2 min) at 16, 18, 20, 22, 24,
and 26 min post-fertilization (mpf) (~1100 eggs in each treatment).
These procedures were performed for each couple, giving rise to three
separated repetitions (no pooling of eggs). After heat-shocking, all
containers were transferred to water with constant aeration and con-
trolled temperature at 26 °C.
2.4. Development analysis and fish rearing
A small aliquot of about 150 eggs from every experimental group
was placed in a 90-mm Petri dish for observation of survival at 2-cell,
blastula, gastrula, and somite stages, as well hatching with normal and
abnormal larvae at this moment, using a stereomicroscope (Nikon SMZ
1500, Tokyo, Japan). Morphologies of normal and abnormal larvae are
described on Fig. 1. Digital images were obtained using a CCD camera
(Nikon Ds-Fi, Tokyo, Japan) with the Nis-Ar Elements software (Nikon,
Tokyo, Japan). At hatching, 60 larvae from each treatment were ran-
domly selected for ploidy analysis by flow cytometry.
Larvae from the higher rate of tetraploidization were reared to
adulthood (~six months). The larvae were reared in 40-L aquariums
with the temperature set at 28 °C and fed exclusively with Artemia
franciscana nauplii twice a day until 10 days post-hatching (dph). After
this period, the fish were fed with powdered commercial food
(4200 kcal kg−1 and 45% crude protein; Guabi, SP, Brazil). Tanks were
consistently monitored for cleaning and removal of dead fish. At 30
dph, the fish start to feed on commercial pellets (1 mm, 91.62% dry
matter, 45% crude protein, 8% crude fat, 2.8% crude fiber, and 12.10%

2.2. Broodstock induction

The fish (three adult couples of Astyanax altiparanae) used in this

experiment were previously maintained in 1000m2 earthen ponds at the
Centro Nacional de Pesquisa e Conservação da Biodiversidade Aquática
Continental/Instituto Chico Mendes de Conservação da Biodiversidade
(CEPTA/ICMBio), Pirassununga City, São Paulo State, Brazil (21°55′58″
S, 47°22′31″ W). The fish were fed twice a day with commercial diet
(4200 kcal kg−1 and 45% crude protein). Procedures for artificial fer-
tilization were performed according to Yasui et al. (2015). Adult males
(SL ~ 6 cm) and females (SL ~ 8 cm) were selected, anesthetized in
eugenol solution (100 mg L−1), and hormonally induced with a single
injection of pituitary gland extract from common carp fish (3 mg kg−1
body weight). Eight hours after, spawning behavior was observed
(males following the females), each fish were then taken for gamete
extraction. Males were anesthetized as described previously. Sperm was
collected by using a micropipette and immediately transferred to
1.5 mL tube containing 400 μL of modified Ringer's solution (NaCl
128.3 mM, KCl 23.6 mM, CaCl2 3.6 mM, MgCl2 2.1 mM). Sperm quality
was evaluated according to Yasui et al. (2015), and only samples with
progressive motility higher than 80% were used. Females were also Fig. 1. Morphological pattern of normal (A) and abnormal (B) larvae of yel-
anesthetized, and mature oocytes were stripped into a Petri dish lowtail tetra Astyanax altiparanae. Scale: 10 μm.

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N.F. do Nascimento, et al. Aquaculture 520 (2020) 734938

mineral matter) twice daily until apparent satiation. At four-months
age, all remaining fish were analyzed by flow cytometry (using a small
piece of the anal fin), and diploid and tetraploid individuals were
identified and maintained separately in 40 L aquariums as described
previously.
2.5. Blood smear and chromosome preparations
To confirm the flow cytometric analysis, 5 diploid and 5 adult tet-
raploid individuals were randomly collected from the aquariums. The
fish were anesthetized in eugenol solution (1 g.L−1) and had the blood
collected from caudal puncture using a syringe containing 1 drop of
EDTA (5%). Blood smears were prepared by placing a drop of blood on
a glass slide and dragging it from one side to another. The smears were
stained with the rapid panotic kit (Laborclin, Pinhais, Brazil) and ob-
served with an optic microscope (Nikon NI, Tokyo, Japan) at magnifi-
cation of 400×. The software Nis-Ar Elements (Nikon, Tokyo, Japan)
was used to take the microphotographs, and the open software Image J
(National Institutes of Health, USA, http://rsb.info.nih.gov/ij/) was
used for posterior analysis. Area (μm2), perimeter (μm), and both major
axis (μm) and minor axis (μm) were measured from nuclei of each
image and compared for the 2n (2143 nucleus) and 4n fish (1907 nu-
cleus). Circularity was calculated as: 4 π * (area)/(perimeter)2.
Afterwards, 3 diploid and 3 tetraploid fish were euthanized in eu-
genol solution (100 mg L−1) for chromosome preparation (using kidney
tissue fragments), which were obtained according to Gold et al. (1990)
followed by Giemsa staining.
2.6. Sperm analysis of tetraploid males
The sperm quality of two mature (~8 months) tetraploid and three
diploid males were evaluated. For motility analysis, 1 μL of sperm from
each male was pipetted on a Makler chamber (Selfi-Medical
Instruments, Haifa, Israel) and activated with a 30-fold dilution of
distillated water. Videos were captured with a microscope (Nikon-
Eclipse Ni, Tokyo, Japan) equipped with a CCD camera (Nikon DSRi2,
Nikon, Tokyo, Japan) with the NIS-Ar Elements software (Nikon,
Tokyo, Japan). The videos were analyzed using the Computer-Assisted
Sperm Analysis (CASA) in the ImageJ software (National Institutes of
Health, USA, http://rsb.info.nih.gov/ij/)(Wilson-Leedy and Ingermann,
2007) after standardization for the species. The parameters evaluated
was: motility (%), VCL (curvilinear velocity), VAP (average path velo-
city) and VSL (straight line velocity). Spermatozoa head length (μm) of
each male (n = 5) were also analyzed in the image J software.
Sperm viability was evaluated by flow cytometry, using the fluor-
escent dyes SYBR-14 and propidium iodide (PI) (live/dead sperm via-
bility kit; Molecular probes, Eugene, OR, USA)(see Flajšhans et al.,
2004). Samples of 150 μL at concentration of 3.6 × 106 spermatozoa
mL−1 were stained with SYBR-14 and PI according to manufacturer
instructions and incubated for 10 min. Afterwards, 10,000 events from
each samples were analyzed by flow cytometer (C6 Accuri; BD
Biosciences, San Jose, CA, USA). SYBR-14 was detected using the FL1
filter (530 ± 15 nm) and PI using FL3 filter (> 670 nm). Gating of the
forward-scatter (FSC) versus side-scatter (SSC) plots were performed to
exclude non-sperm events (debris). Subsequently, gated events were
views on a scatter plot (FL1 vs FL3) and membrane integrity assessed
based on the percentage sperm stained with SYBR-14 from those
stained with SYBR-14 and IP.
2.7. Fertilization of haploid oocytes with diploid spermatozoa
Diploid females (n = 2), tetraploid males (n = 2) and diploid
(n=2) males were selected and induced to reproduction as described in
Section 2.2. Oocytes from each female (~8000) were divided and in-
seminated with 90 μL of sperm from different males, and the gametes
were activated by adding 5 mL of water (Resulting in two separated
repetitions). A small aliquot from each treatment was placed in a 90-
mm Petri dish for observation of survival among development as de-
tailed in Section 2.4.
2.8. Flow cytometry
Samples used for flow cytometry were analyzed according to the
protocol developed by Xavier et al. (2017). The samples were lysed in a
detergent solution (9.53 mM MgSO4.7H2O, 47.67 mM KCl, 15 mM Tris,
74 mM Sucrose, and 0.8% of Triton X-100), filtered through 30 μm
nylon mesh. Then, the extracted nuclei were stained with the 4′, 6
diamidino-2-phenylindole dihydrochloride (DAPI). The DNA content
was then measured with a flow cytometer (CyFlow Ploidy, Analyzer,
Partec, GMBh, Germany). The ploidy of all samples was confirmed by
comparison with haploid control (spermatozoa from diploid males).
2.9. Statistics
Results are shown as mean ± standard error. The data were
checked for normality using the Liliefors test and analyzed by ANOVA.
Data expressed as percentages were arc sin (√%) transformed in order
to fit the assumptions of statistical variance homogeneity using the
Levene test (Brown and Forsythe, 1974). Analysis was performed using
the software STATISTICA v. 10.0 (Statsoft, Tulsa, U.S.A.) and sig-
nificance was set at P < .05.
3. Results
3.1. Early development of heat shocked eggs for tetraploid induction
Data on development of heat-shocked embryos are detailed in
Table 1. No differences between treatments were observed for survival

Table 1
Survival (%) of yellowtail tetra A. altiparanae in percentage (±SE) after heat shock treatment for induction of tetraploid fish.
Trataments (mpf) 2-cell (%) Blastula (%) Gastrula (%) Somite (%) Hatching (%) Normal (%) Ploidy

2n 4n 4n (%) Eggs

Control 97.5 ± 1.4 92.9 ± 3.6 75.1 ± 6.3 70.2 ± 7.7a 62.2 ± 7.7a 87.1 ± 4.5a 60 0 0.00 584
16 94.7 ± 4.0 90.6 ± 5.0 63.5 ± 9.7 46.9 ± 8.2ab 37.4 ± 7.4ab 50.4 ± 15.5ab 58 0 0.00 553
18 95.3 ± 3.3 90.9 ± 5.0 65.3 ± 8.5 47.0 ± 5.5ab 34.1 ± 1.4ab 62.9 ± 10.9ab 57 0 0.00 666
20 98.4 ± 0.3 92.2 ± 6.3 70.5 ± 7.2 48.4 ± 9.4ab 34.1 ± 3.8ab 37.9 ± 16.3ab 41 16 28.07 561
22 94.4 ± 3.5 88.5 ± 6.4 56.3 ± 12.0 35.5 ± 8.8ab 19.1 ± 9.6b 17.8 ± 6.7b 29 23 44.23 683
24 94.3 ± 4.4 89.0 ± 6.1 49.2 ± 14.0 24.6 ± 8.5b 16.0 ± 9.1b 21.7 ± 7.3b 17 23 57.50 681
26 92.9 ± 5.8 87.6 ± 7.1 34.3 ± 11.8 13.5 ± 7.1b 10.3 ± 6.2b 21.0 ± 7.9b 3 52 94.55 624
P-value 0.9952 0.9998 0.1340 0.0008 0.0007 0.0009 –

Fertilized eggs were heat shocked at 16, 18, 20, 22, 24 and 26 mpf. After treatment, the eggs were incubated at 26 °C until hatching. Dada were obtained from three
different egg sources (families). Distinct superscript letters within a column indicates statistical differences by the Tukey multiple range test (ANOVA; P < .05). SE:
standard error; mpf: minutes post-fertilization.

3
N.F. do Nascimento, et al. Aquaculture 520 (2020) 734938

Fig. 2. Blood smears and metaphase chromosomes of

diploid (left) and tetraploid (right) individuals of
yellowtail tetra Astyanax altiparanae. Erythrocytes
nuclei from diploids (A) presented small size in
comparison with tetraploids (B). The chromosome
numbers are 50 (C) and 100 (D) in diploid and tet-
raploid, respectively. Scale for A, B, C and D: 10 μm.

rates at the 2-cell, blastula, and gastrula stages. However, at the somite individuals had 100 chromosomes (Fig. 2 C,D).
stage, the control group presented higher survival, and the 24 and 26
mpf treatments showed lower survival rate. Similar results were ob- 3.3. Analysis of diploid spermatozoa from tetraploid males
served for hatching rates, presenting significant higher values for con-
trol group and lower for the 22, 24, and 26 mpf treatments, respec- Diploid spermatozoa from tetraploid males present significantly
tively. After hatching, the percentage of normal larvae was significantly increased head (3.00 ± 0. 09 μm) when compared with haploid sper-
lower in embryos heated shocked at 22, 24, and 26 mpf than the control matozoa (1.92 ± 0.05 μm)(Fig. 3). Additionally, results of sperm
group. analysis showed that haploid spermatozoa from diploid males present
The flow cytometric analysis showed that the control group and the higher motility rate (Table 3).
treatments heat shocked at 16 and 18 mpf presented only diploid in-
dividuals (Table 1). However, in treatments from 20 to 26 mpf, tetra- 3.4. Early development of haploid eggs fertilized with diploid spermatozoa
ploid individuals were observed with the highest percentage at 26 mpf from tetraploid males
(94.6%) (Table 1).
Data on development are detailed in Table 4. Higher survival rates
were observed in the control group (haploid eggs fertilized with haploid
3.2. Blood smear and chromosome counting
spermatozoa) at the 2-cell, blastula, gastrula, somite and Hatching
stages. At hatching, flow cytometry showed that all eggs fertilized with
In comparison with diploid fish, erythrocyte nuclei from tetraploid
diploid spermatozoa were triploid (100%) (Table 4).
fish presented significantly increased area, perimeter, and major axis;
however, significantly lower circularity were observed (Fig. 2 A,B;
4. Discussion
Table 2). For minor axis, no differences were observed. Additionally,
while diploids presented 50 metaphase chromosomes, tetraploid
In the present study, high percentages of tetraploids were success-
fully obtained in A. altiparanae by heat shocking. Those results are very
Table 2
interesting for aquaculture, since tetraploids can be used as a source of
Average nuclear measurements (±SE) of erythrocytes from diploid and tetra-
ploid A. altiparanae. diploid gametes for mass production of sterile triploids without the
negative effects of the shock treatment, such as lower survival and
Parameters 2n 4n P-value higher abnormalities rates (Garcia et al., 2017; Lebeda and Flajshans,
2
Area (μm ) 16.15 ± 0.89a
23.34 ± 0.82b
0.0018 2015). In A. altiparanae, previous studies showed that triploid females
Perimeter (μm) 15.46 ± 0.39a 19.58 ± 0.49b 0.0003 are sterile (Nascimento et al., 2017a), presenting increased carcass
Major axis (μm) 5.40 ± 0.13a 7.63 ± 0.19b 0.0000 yield (Nascimento et al., 2017b), which supports mass production of
Minor axis (μm) 3.78 ± 0.12 3.91 ± 0.05 0.6144 triploid fish using tetraploid individuals.
Circularity 0.84 ± 0.01a 0.79 ± 0.01b 0.0002
Nuclei measured 2143 1907 –
Our previous study on early development in A. altiparaane showed
that the first cleavage takes place at 43 min post-fertilization (at 22 °C),
Distinct letter in the same line indicate significant difference (ANOVA, and pronuclei fusion occurs within 10 mpf (Dos Santos et al., 2016).
P < .05). Therefore, the timing for heat shock was standardized based on a

4
N.F. do Nascimento, et al. Aquaculture 520 (2020) 734938

Fig. 3. Spermatozoa from a diploid (A) and tetraploid (B) male of yellowtail tetra Astyanax altiparanae. Scale for A and B: 10 μm.

Table 3 Here we show that erythrocytes nuclei measurement is an effective tool

Sperm parameters from diploid and tetraploid males of A. altiparanae.
Parameters 2n 4n
a b
Motility (%) 84.70 ± 4.75 59.59 ± 0.01
VCL (μm/s) 112.35 ± 11.61 87.85 ± 0.49
VAP (μm/s) 102.52 ± 10.29 74.96 ± 0.19
VLS (μm/s) 75.11 ± 7.10 56.67 ± 0.55
Concentration 6.8 × 108 ± 1.2 × 108 1.2 × 107 ± 9.7 × 106
Viability⁎ 95.96 ± 1.07 88.75 ± 1.15
Different superscript letters denote statistical differences by the tukey test
(ANOVA, P < .05). MOT: motility; VCL, curvilinear velocity; VAP, average path
velocity; VSL, straight-line velocity. ⁎Percentage of live cells.
relative unit of embryological age (t0 or tau zero)(Gomelsky, 2003),
which show the duration of one mitotic cycle. Therefore, as in other
species the successful heat shock is nearly at pro-metaphase of the first
cleavage (Fujimoto et al., 2013; Sakao et al., 2006), with the concept of
“tau zero” it is possible to “predict” the possible time to initiate the
heat-shock. Additionally, the heat shock used was similar to the pro-
cedure for triploidization in A. altiparanae (40 °C for 2 min), which
resulted in increased survival (Adamov et al., 2017) and ensured that
our method almost achieved 100% of tetraploids.
The decreased survival observed during development of tetraploid-
induced fish are commonly reported in other species (Sakao et al.,
2006). After hatching, several studies showed no survival at first
feeding (Gil et al., 2016; Haniffa et al., 2004; Sakao et al., 2006). Lower
survival of heat-shocked groups are probably due to the presence of
mosaicism, aneuploidy or high homozygosity (Fujimoto et al., 2013;
Pandian and Koteeswaran, 1998; Zhang and Onozato, 2004). In this
study, both normal and abnormal tetraploid larvae were analyzed and
no mosaicism or aneuploidy were observed; therefore, the decreased
survival can be addressed to factors other than chromosomal altera-
tions, which need to be investigated in the future.
Several authors has successful used the erythrocytes nuclei mea-
surements to confirm polyploidy in fishes (Boron, 1994; Flajšhans et al.,
2011) and since most protocols for triploid or tetraploid induction are
not 100% effective, mechanisms for ploidy confirmation are essential.
to confirm tetraploidy in A. altiparanae. Among erythrocytes nucleus
p-value measurements, some authors stated that the axes are generally con-
sidered the best prediction of ploidy (Kenanoğlu et al., 2012). Flajšhans
et al. (2011), on the other hand, showed that area and perimeter were
0.0001
0.1827 the most accurate ploidy predictors. In our study, except for the minor
0.1171
0.1491
axis, all other parameters were significantly different among diploid
0.2137 and tetraploid fish A. altiparanae.
0.0586 Tetraploid fish were also identified by flow cytometry (a well-re-
cognized, effective, and accurate technique for nuclear DNA content
analysis) (Allen, 1983; Xavier et al., 2017) and chromosome counts by
karyotype (Allen Jr et al., 1982). Therefore, the combined methods
confirmed the efficacy of the current protocol. Additionally, in the case
that flow cytometry is not available, the blood smear or the cytogenetic
method could be an interesting alternative.
When mature, tetraploid males of A. altiparanae produce diploid
spermatozoa with viability and motility. Despite the survival rate
among development was lower than the control group, such results
indicated that tetraploid of A. altiparanae are an important alternative
for mass production of triploid fish, as observed in other studies (Nam
and Kim, 2004; Weber et al., 2014; Weber et al., 2015).
In conclusion, this study determines a protocol to produce highest
percentages of tetraploid in A. altiparanae, the first in Neotropical
Characins. The procedures were optimized using temperature shock at
26 mpf (40 °C for 2 min), followed by incubation at 26 °C. Additionally,
tetraploid males produce viable diploid spermatozoa, which can be
used to mass production of triploid fish. Such a protocol is innovative
for the species and highly applicable for basic and applied sciences.
Declaration of Competing Interest
None.
Acknowledgements
The authors are grateful to Sao Paulo Research Foundation
(FAPESP) for the financial support of this research (Young Investigators

Table 4
Survival (%) of yellowtail tetra A. altiparanae in percentage (±SE) after fertilization with haploid and diploid spermatozoa (from tetraploid males).
Trataments 2-cell (%) Blastula (%) Gastrula (%) Somite (%) Hatching (%) Normal (%) Ploidy

2n 3n 3n (%) Eggs

1n Spermatozoa 97.05 ± 1.98a 97.05 ± 1.98a 96.56 ± 1.49a 95.59 ± 0.52a 95.59 ± 0.52a 95.22 ± 3.30 40 0 0.00 245
2n Spermatozoa 37.08 ± 2.76b 37.08 ± 2.76b 36.18 ± 2.85b 35.77 ± 2.44b 35.77 ± 2.03b 88.53 ± 8.53 0 40 100.00% 225
P-value 0.0082 0.0082 0.0047 0.0016 0.0012 0.5702 –

Dada were obtained from two different egg sources (families). Distinct superscript letters within a column indicates statistical differences by the Tukey multiple range
test (ANOVA; P < .05). SE: standard error.

5
N.F. do Nascimento, et al.
Award Grant #2010/17429-1; Young Researcher Scholarship #2011/
11664-1 and the PhD Scholarship #2016/12383-0) and CNPq
#471140/2012-0. We also acknowledge CEPTA/ICMBio for kindly
providing the facilities and experimental fish.
References
Adamov, N.S., Nascimento, N.F., Maciel, E.C.S., Pereira-Santos, M., Senhorini, J.A.,
Calado, L.L., Evangelista, M.M., Nakaghi, L.S.O., Guerrero, A.H.M., Fujimoto, T.,
2017. Triploid induction in the yellowtail tetra, Astyanax altiparanae, using tem-
perature shock: tools for conservation and aquaculture. J. Word Aquac. Soc. 48,
741–750.
Allen, S.K., 1983. Flow-cytometry - assaying experimental polyploid fish and shellfish.
Aquaculture. 33, 317–328.
Allen Jr., S.K., Gagnon, P.S., Hidu, H., 1982. Induced triploidy in the soft-shell clam:
cytogenetic and allozymic confirmation. J. Hered. 73, 421–428.
Arai, K., 2001. Genetic improvement of aquaculture finfish species by chromosome ma-
nipulation techniques in Japan. Aquaculture 197, 205–228.
Benfey, T.J., 2016. Effectiveness of triploidy as a management tool for reproductive
containment of farmed fish: Atlantic salmon (Salmo salar) as a case study. Rev. Aquac.
8, 264–284.
Bertolini, R.M., Senhorini, J.A., Nascimento, N.F., Pereira-Santos, M., Nakaghi, L.S.O.,
Peres, W.A.M., Silva, R.C., Yasui, G.S., 2018. First feeding of diploid and triploid
yellowtail tetra Astyanax altiparanae: An initial stage for application in laboratory
studies. Aquac. Res. 49, 68–74.
Boron, A., 1994. Use of erythrocyte measurements to detect natural triploids of spined
loach Cobitis taenia (L.). Cytobios 78, 197–202.
Brown, M.B., Forsythe, B.F., 1974. Robust test for the equality of variances. J. Am. Stat.
Assoc. 69, 364–367.
Chourrout, D., Chevassus, B., Krieg, F., Happe, A., Burger, G., Renard, P., 1986.
Production of second generation triploid and tetraploid rainbow trout by mating
tetraploid males and diploid females—potential of tetraploid fish. Theor. Appl. Genet.
72, 193–206.
Dos Santos, M.P., Yasui, G.S., Xavier, P.L.P., de Macedo Adamov, N.S., do Nascimento,
N.F., Fujimoto, T., Senhorini, J.A., Nakaghi, L.S.O., 2016. Morphology of gametes,
post-fertilization events and the effect of temperature on the embryonic development
of Astyanax altiparanae (Teleostei, Characidae). Zygote. 24, 795–807.
Flajšhans, M., Pšenička, M., Rodina, M., Těšitel, J., 2011. Image cytometric measurements
of diploid, triploid and tetraploid fish erythrocytes in blood smears reflect the true
dimensions of live cells. Cell Biol. Int. 35, 67–71.
Fujimoto, T., Sakao, S., Oshima, K., Yamaha, E., Arai, K., 2013. Heat-shock-induced tet-
raploid and diploid/tetraploid mosaic in pond loach, Misgurnus anguillicaudatus.
Aquac. Int. 21, 769–781.
Garcia, S., Silva, B.C., Yasui, G.S., Bernardes Júnior, J.J., Amaral Júnior, H., Zaniboni
Filho, E., 2017. Triploid induction in Rhamdia quelen (Siluriformes, Heptapteridae) by
double temperature shocking. LAJAR 45.
Garcia, S., Júnior, H.A., Yasui, G.S., Liebl, F., Souto, L.I.M., Zaniboni-Filho, E., 2018.
Tetraploidia em Rhamdia quelen (Quoy e Gaimard, 1824) por choque térmico duplo
(quente e frio). Bol. Inst. Pesca 43, 257–265.
Gil, H.W., Kong, H.J., An, C.M., Kim, B.-S., Lim, S.-G., Park, I.-S., 2016. Cytogenetic study
of diploid and induced tetraploid in Korean rose bitterling, Rhodeus uyekii.
SpringerPlus 5, 186.
Gold, J., Li, Y., Shipley, N., Powers, P., 1990. Improved methods for working with fish
chromosomes with a review of metaphase chromosome banding. J. Fish Biol. 37,
563–575.
Golpour, A., Siddique, M.A.M., Siqueira-Silva, D.H., Pšenicka, M., 2016. Induced sterility
in fish and its potential and challenges for aquaculture and germ cell transplantation
technology: a review. Biologia 71, 853–864.
Gomelsky, B., 2003. Chromosome set manipulation and sex control in common carp: a
review. Aquat. Living Resour. 16, 408–415.
Haniffa, M.A., Sridhar, S., Nagarajan, M., 2004. Induction of triploidy and tetraploidy in
stinging catfish, Heteropneustes fossilis (Bloch), using heat shock. Aquac. Res. 35,
937–942.
Aquaculture 520 (2020) 734938
Hulata, G., 2001. Genetic manipulations in aquaculture: a review of stock improvement
by classical and modern technologies. Genetica 111, 155–173.
Kenanoğlu, O.N., Ergün, S., Soytaş, N., Akı, C., Yılmaz, S., Tapan, F., 2012. Determination
of Triploidy in rainbow trout, Oncorhynchus mykiss using erythrocyte measurements.
Mar. Sci. Tech. Bull. 1, 17–19.
Lebeda, I., Flajshans, M., 2015. Production of tetraploid sturgeons. J. Anim. Sci. 93,
3759–3764.
Morón-Alcain, E., Mendia, A.C., Muñoz, L.H., Boaglio, A.C., Cerutti, P.A., Hernández,
D.R., López, P.A., Vigliano, F.A., 2017. Effects of heat and cold shock-induced tri-
ploidy on productive parameters of silver catfish (Rhamdia quelen) late-hatched in the
reproductive season. Aquaculture 473, 303–309.
Nam, Y.K., Kim, D.S., 2004. Ploidy status of progeny from the crosses between tetraploid
males and diploid females in mud loach (Misgurnus mizolepis). Aquaculture 236,
575–582.
Nascimento, N.F., de Siqueira-Silva, D.H., Pereira-Santos, M., Fujimoto, T., Senhorini,
J.A., Nakaghi, L.S.O., Yasui, G.S., 2017a. Stereological analysis of gonads from di-
ploid and triploid fish yellowtail tetra Astyanax altiparanae (Garutti & Britski) in la-
boratory conditions. Zygote 25, 537–544.
Nascimento, N.F., Pereira-Santos, M., Piva, L.H., Manzini, B., Fujimoto, T., Senhorini,
J.A., Yasui, G.S., Nakaghi, L.S.O., 2017b. Growth, fatty acid composition, and re-
productive parameters of diploid and triploid yellowtail tetra Astyanax altiparanae.
Aquaculture 471, 163–171.
Pandian, T.J., Koteeswaran, R., 1998. Ploidy induction and sex control in fish.
Hydrobiologia 384, 167–243.
Pereira-Santos, M., Yasui, G., Xavier, P., de Macedo, A.N., do Nascimento, N., Fujimoto,
T., Senhorini, J., Nakaghi, L., 2016. Morphology of gametes, post-fertilization events
and the effect of temperature on the embryonic development of Astyanax altiparanae
(Teleostei, Characidae). Zygote (Cambridge, England) 24, 795–807.
Peruzzi, S., Anne, K., Raul, P., Goran, K., 2007. Thermal shock induction of triploidy in
Atlantic cod (Gadus morhua L.). Aquac. Res. 38, 926–932.
Piferrer, F., Beaumont, A., Falguiere, J.-C., Flajshans, M., Haffray, P., Colombo, L., 2009.
Polyploid fish and shellfish: production, biology and applications to aquaculture for
performance improvement and genetic containment. Aquaculture 293, 125–156.
Piva, L.H., de Siqueira-Silva, D.H., Goes, C.A.G., Fujimoto, T., Saito, T., Dragone, L.V.,
Senhorini, J.A., Porto-Foresti, F., Ferraz, J.B.S., Yasui, G.S., 2018. Triploid or hybrid
tetra: which is the ideal sterile host for surrogate technology? Theriogenology 108,
239–244.
Sakao, S., Fujimoto, T., Kimura, S., Yamaha, E., Arai, K., 2006. Drastic mortality in tet-
raploid induction results from the elevation of ploidy in masu salmon Oncorhynchus
masou. Aquaculture 252, 147–160.
Siqueira-Silva, D.H., Silva, A.P., Ninhaus-Silveira, A., Verissimo-Silveira, R., 2015. The
effects of temperature and busulfan (Myleran) on the yellowtail tetra Astyanax alti-
paranae (Pisces, Characiformes) spermatogenesis. Theriogenology 84, 1033–1042.
Taranger, G.L., Carrillo, M., Schulz, R.W., Fontaine, P., Zanuy, S., Felip, A., Weltzien, F.-
A., Dufour, S., Karlsen, O., Norberg, B., Andersson, E., Hausen, T., 2010. Control of
puberty in farmed fish. Gen. Comp. Endocrinol. 165, 483–515.
Weber, G.M., Hostuttler, M.A., Cleveland, B.M., Leeds, T.D., 2014. Growth performance
comparison of intercross-triploid, induced triploid, and diploid rainbow trout.
Aquaculture 433, 85–93.
Weber, G.M., Hostuttler, M.A., Semmens, K.J., Beers, B.A., 2015. Induction and viability
of tetraploids in brook trout (Salvelinus fontinalis). Can. J. Fish. Aquat. Sci. 72,
1443–1449.
Xavier, P.L., Senhorini, J.A., Pereira-Santos, M., Fujimoto, T., Shimoda, E., Silva, L.A., dos
Santos, S.A., Yasui, G.S., 2017. A flow cytometry protocol to estimate DNA content in
the yellowtail tetra Astyanax altiparanae. Front. Genet. 8, 131.
Yasui, G.S., Senhorini, J.A., Shimoda, E., Pereira-Santos, M., Nakaghi, L.S.O., Fujimoto,
T., Arias-Rodriguez, L., Silva, L.A., 2015. Improvement of gamete quality and its
short-term storage: an approach for biotechnology in laboratory fish. Animal 9,
464–470.
Yoshikawa, H., Morishima, K., Fujimoto, T., Arias-Rodriguez, L., Yamaha, E., Arai, K.,
2008. Ploidy manipulation using diploid sperm in the loach, Misgurnus anguillicau-
datus : a review. J. Appl. Ichthyol. 24, 410–414.
Zhang, X., Onozato, H., 2004. Hydrostatic pressure treatment during first mitousis does
not supress the first cleavage but the second one. Aquaculture 240, 101–103.