Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 11 - 23, 2017
Doi: 10.20950/1678-2305.2017.11.23
ADHESIVENESS NEUTRALIZATION IN EGGS OF Pseudoplatystoma fasciatum
(TELEOSTEI: SILURIFORMES: PIMELODIDAE)
Daniel Guimarães FIGUEIREDO-ARIKI
1
; Rafael Yutaka KURADOMI
1
; Thiago Gonçalves DE
SOUZA
1
; Patrick HAINFELLNER
1
; Fabio PORTO-FORESTI
2
; Sergio Ricardo BATLOUNI
1
ABSTRACT
The aim of this study was to remove the adhesiveness of Pseudoplatystoma fasciatum eggs using
solutions of urea for 10, 30, 60 and 90 min (treatments T0-T3) or, in urea for 10 min and washed in
tannin (T4). In the control group, eggs were kept in water. After two experiments, we observed
that eggs of control group presented the best embryo viability rates, even maintaining egg
adhesiveness, being better than the other treatments. The T4, had the worst embryo viability rates.
We observed that embryos of the T4 treatment presented reduced growth and formed a separate
group when analyzing morphological parameters (egg diameter, total egg area, embryo area and
yolk sac area) by multivariate analysis. Concluding, the control group, free of chemicals, provided
the best results and is considered the best alternative for the immediate conservation and
aquaculture production of P. fasciatum.
Keywords: induced spawning; sticky eggs; urea; tannin
NEUTRALIZAÇÃO DA ADESIVIDADE EM OVOS DE Pseudoplatystoma fasciatum
(TELEOSTEI: SILURIFORMES: PIMELODIDAE)
RESUMO
O objetivo deste estudo foi neutralizar a adesividade de ovos de Pseudoplatystoma fasciatum a partir
da utilização de soluções de ureia por 10, 30, 60 ou 90 min (tratamentos T0-T3) ou em ureia por 10
min e lavados em tanino (T4). No controle, os ovos foram mantidos apenas em água. Após dois
experimentos realizados, no grupo controle foram observadas as melhores taxas de viabilidade
embrionária, mesmo mantendo a adesividade dos ovos, sendo melhor que os demais tratamentos.
No T4 foram verificadas as piores taxas de viabilidade embrionária. Foi observado que os embriões
do tratamento T4 apresentaram crescimento reduzido e formaram um grupo separado após análise
dos parâmetros morfológicos (diâmetro do ovo, área total do ovo, área do embrião e área do saco
vitelínico) por análise multivariada. Concluiu-se que o grupo controle, livre de produtos químicos,
proporcionou os melhores resultados, sendo considerado a melhor alternativa para a conservação
imediata e produção aquícola de P. fasciatum.
Palavras-chave: desova induzida; ovos adesivos; ureia; tanino
Original Article/Artigo Científico: Recebido em 15/12/2016 Aprovado em 19/05/2017
1
Universidade Estadual Paulista (UNESP), Centro de Aquicultura (CAUNESP). Via de Acesso Prof. Paulo Donato
Castellane, s/n. CEP: 14884-900 Jaboticabal SP - Brazil. e-mails: dmx0802@hotmail.com; rafael_yk@yahoo.com.br;
thiago.desouza@ig.com.br; tricktreck@hotmail.com; batlouni@caunesp.unesp.br (corresponding author)
2
Universidade Estadual Paulista (UNESP), Faculdade de Ciências, Departamento de Ciências Biológicas. Av. Luiz
Edmundo Carrijo Coube, 14-01 CEP: 17033-360 Bauru SP Brazil. e-mail: fpforesti@fc.unesp.br
* Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Grant 1085142).
12 FIGUEIREDO-ARIKI et al.
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 11 - 23, 2017
INTRODUCTION
The Pseudoplatystoma fasciatum, Pseudoplatystoma
corruscans and their hybrids are large Pimelodidae
catfishes endemic of the Neotropical region
referred as “sorubim” that have excellent flavor,
low fat content meat and are among of the most
expensive and highly appreciated freshwater fish
throughout several of South American countries
(HONORATO et al., 2014). The aquaculture
production of sorubim has increased by 30.1% in
just one year, from 15.71 million kg in 2013 to
20.43 million kg in 2014 (IBGE, 2015).
Despite all their zootechnical qualities, the
production of these species presents an obstacle,
which is the fry production difficulty due to the
high mortality of eggs in incubators. In this
concern, it is known that many factors can
affect egg and gamete quality, including the
reproductive management provided to the
breeders throughout the year and also by genetic
factors recently explored by transcriptome and
proteomic techniques (BOBE and LABBÉ, 2010;
ALIX et al., 2015). It is important to know the
optimal conditions that allow an adequate
embryonic development, because factors, such as
time and husbandry practices for spawning
induction, gamete handling post striping, egg post
ovulatory aging, the range of water physiological
temperatures, salinity, oxygen concentrations are
known to affect the embryonic development and
fish egg quality (WU, 2009; BOBE and LABBÉ,
2010; MYLONAS et al., 2010). For species with
adhesive eggs, it is known that adhesiveness must
be neutralized to prevent the formation of clusters
of eggs that results in high mortality rates caused
by suffocation and fungal growth (DOROSHOV
et al., 1983). For this purpose the application of an
urea solution and then a tannin solution is the
most common protocol (KOWTAL et al., 1986;
RIBEIRO, 2001; BOUCHARD and ALOISI, 2002;
KUJAWA et al., 2010). The adhesiveness
neutralization may be obtained using only tannin
solution (ZARSKI et al., 2015) or by using specific
enzymes (GELA et al., 2003; LINHART et al.,
2003a, b; LINHART et al., 2004; CARRAL et al.,
2006). However, the combination of urea and
tannin is practiced because it seems to prolong
the effect of removing the adhesiveness (KOWTAL
et al., 1986). The combination of substances and
the time of exposure need to be that resulting in
best adhesiveness removal and highest fertility
and hatching success. Protocols have been
standardized for some fish species during the last
decades, in which the exposure to urea (with
different concentrations) varies from 15 to 60 min,
while the tannin (also in different concentrations)
exposure ranges from fast baths up to 5 minutes
(SIDDIQUE et al., 2016).
On the other hand, the use of chemical
protocols for neutralizing adhesiveness has been
reported, in some cases, to impair the embryonic
development and reduce fertility and hatching
rates (LINHART et al., 2000; DEMSKA-ZAKÉZ
et al., 2005; FELEDI et al., 2011). The use of
different substances (concentrations and time of
exposure) has to be specifically determined for
species with adhesive eggs, especially P. fasciatum
or any species that needs adhesiveness removal so
the eggs may be used in hatcheries.
However, mainly because the vast majority of
tropical fish of commercial importance are egg
scatterers that do not have adhesive eggs, there is
no information about these aspects in these
species. Concerning P. fasciatum, because of the
absence of efficient protocols to neutralize the egg
adhesiveness, as well as other factors, interspecific
breeding is often accomplished using oocytes
of P. corruscans (which are not adhesive) (RIZZO
et al., 2002; RIZZO and GODINHO, 2003) that are
fertilized with sperm of P. fasciatum. This
crossbreeding results in the production of the
hybrid fish "pintachara" (PORTO-FORESTI et al.,
2008; PRADO et al., 2011) and eliminates the
problem of egg adhesiveness. On the other hand,
the production of “pintachara” contributes to a
problem that has attracted increasing attention:
the introduction of fertile hybrids in the wild
because of escapes in fish farms. Specifically for
the case of hybrids from the Pimelodidae, the
major risk is the fertility of these hybrids, which
lacks a reliable morphological identification, and
may cause atypical crosses with wild fish and
generate genetic contamination (HASHIMOTO
et al., 2015). Interspecific hybrids of Neotropical
fishes are produced for several reasons. In some
cases, hybrids can be produced because of the lack
of technology for pure species (HASHIMOTO
et al., 2015), such as the case of the absence of
a protocol for neutralize egg adhesiveness in
P. fasciatum.
Adhesiveness neutralization in eggs of Pseudoplatystoma fasciatum… 13
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 11 - 23, 2017
Thus, the aim of this study was to evaluate
different protocols (using chemical treatments)
for neutralizing the adhesiveness of P. fasciatum
eggs, to find the most appropriate treatment
which should combine both adhesiveness
neutralization and promote adequate reproductive
rates and thereby contributing to the development
of a methodology for the reproduction of this
species in captivity for conservation and
aquaculture purposes.
MATERIAL AND METHODS
Maintenance and Animal Care
This study consisted of two experiments
conducted separately over two distinct breeding
seasons in the Centro de Aquicultura da
Universidade Estadual Paulista - UNESP
(CAUNESP), Jaboticabal, SP, Brazil (21º15’17”S,
48º19’20”W). The genetic identity of the P. fasciatum
used was confirmed through this analysis,
performed at the Fish Genetics Laboratory of
Unesp, campus of Bauru, SP, Brazil. The animals
of the breeding stock of CAUNESP were
microchipped (ANIMALTAG - Korth RFID Ltda.,
São Carlos, SP) to ensure the identification of each
animal and traceability of fingerlings from the
breeding. From each animal a caudal fin fragment
was collected and stored in 100% alcohol for
molecular analysis. DNA extraction was conducted
using the Genomic DNA Purification Kit according
to the manufacturer's protocol (Promega). The
amount of DNA was assessed with a molecular
marker pattern (Invitrogen Low DNA Mass
Ladder) by 1% agarose gel electrophoresis.
Samples were identified using the Polymerase
Chain Reaction (PCR) of regions of the nuclear
genes RAG2, Globin, EF1, 18S and sequences of
mitochondrial gene 16S, and the Prt microsatellite
marker. Data analysis was performed by
comparing the bands after PCR using standard
samples of the two species analyzed (P. fasciatum
and P. corruscans) previously identified.
Fish were kept in earthen ponds (10 x 20 x 1.5 m)
and fed 6 d/week in two portions, equivalent to
5.0% of the total weight in the warm months
(September-February) and 1.0% in the cold
months (March-August), at 09:00 h and 17:00 h
with commercial extruded feed (moisture content
[max.] 8.0%; crude protein [min.] 32.0%; ethereal
extract [min.] 6.5%; fibrous matter [max.] 7.0%;
ash [max.] 10.0%; calcium [max.] 1.2%;
phosphorus [min.] 0.6%; according to the
manufacturer). Water parameters measured using
a YSI model 55 oximeter and a YSI model 63
multiparameter probe (Yellow Spring Instruments,
Yellow Springs, OH, USA) indicated average
dissolved oxygen levels of 3.70 ± 1.12 mg L
-1
,
temperature of 23.35 ± 3.98 ºC, pH of 7.38 ± 0.24,
conductivity of 84.68 ± 11.18 μS cm
-1
and
transparency of 0.60 m (measured at 09:00 h using
a Secchi disk).
Individuals suited for induced spawning
were kept in plastic tanks (1000 L) with constant
water circulation, artificial aeration and an
average water temperature of 26.1 ± 0.4 ºC. In both
experiments, males received a single dose of carp
pituitary extract (CPE) (2.0 mg kg
-1
), and females
received two doses (0.5 and 5.0 mg kg
-1
, with a 12
h interval between doses). Females were selected
by external characteristics (swollen abdomen) and
males were chosen according to the color and
fluidity of the milt extruded after gentle pressure
on the abdomen (LEONARDO et al., 2004). Three
females and four males were used in both
experiments. At the time of ovulation, females
were removed from the tank, and the location
near the genital papilla was dried with paper
towels for dry strip spawning. Just prior to
fertilization, a 150 mL oocyte pool was made
using 50 mL of oocytes of each female (all three
females ovulated in both experiments). Semen
was collected from the males ten minutes before
fertilization and stored separately in plastic falcon
tubes inside the refrigerator at 4.0 ºC. Just before
fertilization, a 4 mL semen pool was made using
1.0 mL of semen of each male (all four males
spermiated in both experiments). The insemination
dose used for all treatments was 2.21 x 10
5
spz
oocyte
-1
, which was based on insemination doses
successfully used for other reophilic species in
captivity (BOMBARDELLI et al., 2006; SHIMODA
et al., 2007; DE SOUZA et al., 2015). In both
experiments, the pooled semen was mixed with
the pooled oocytes and after that, the gametes
were activated according to each treatment, using
either the urea solution or hatchery water. Neither
of the pooled samples contained urine or fecal
contamination.
14 FIGUEIREDO-ARIKI et al.
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 11 - 23, 2017
Treatments
The treatments were applied during the
process of activation of gametes, fertilization
and ova hydration (AFH). The solutions used
were based on the protocol for carp (Cyprinus
carpio) described by RIBEIRO (2001), which
consists of a modified “Woynarovich solution”
(WOYNAROVICH and WOYNAROVICH, 1980)
or “urea solution”, composed of 40 g of sodium
chloride and 30 g of urea (Synth, Diadema, Brazil)
dissolved in 10 L of hatchery water (4 g L
-1
or 0.4%
of NaCl and 3g L
-1
or 0.3% of urea); and a tannin
solution, composed of 5 g of powder tannin or
tannic acid (Tanac, Montenegro, Brazil) also
dissolved in 10 L of hatchery water (0.5 g L
-1
or
0.05% of tannin). The solutions were prepared
30 min prior to use.
Experiment 1
The pooled samples of oocytes and semen
were divided and subjected to five distinct
conditions for AFH in individual plastic bowls,
representing four treatments and a control group
(Table 1). After applying the treatments, 4 mL of
eggs (245 ± 17 eggs in 1 mL of eggs) were placed
into each incubator (cone-shaped, with a volume
of 2.5 L and constant water flow of 0.5 L min
-1
),
with 3 replicates per treatment. The urea solution
in the T1, T2 and T3 was slowly renewed at 30
min intervals.
Table 1. Control and treatments used in this study for adhesiveness neutralization in eggs of
Pseudoplatystoma fasciatum. In experiment 2, the oocytes were fertilized in the same manner as in experiment 1.
*AFH stands for the process of activation, fertilization and hydration of the gametes and eggs.
Experiment 2
In the Experiment 2, we repeated the same
treatments and introduced a new one (T0), in
which the urea solution was used during
fertilization and hydration for 10 min (instead of
water as in C) (Table 1).
Sampling and reproductive rates
At 5 h and at 15 h (gastrula and almost fully
formed embryo respectively) post-fertilization
(HPF), 100 eggs from each replicate (300 eggs per
treatment) were collected and analyzed under
a stereoscopic microscope M50 (Leica Microsystems,
Wetzlar, Germany), where viable eggs (with
embryonic development) were counted to determine
the fertilization (FR) and hatching rates (HR)
respectively. Then, the samples were fixed in 2.5%
glutaraldehyde in 0.05 M phosphate buffer (pH
7.2) and refrigerated. The FR and HR were
calculated using the following formula: (viable
eggs/total eggs) x 100.
Morphometric analysis
In both experiments, 15 HPF embryonated
eggs (previously fixed), randomly selected per
treatment, were photographed using a stereoscopic
microscope Leica M50 with IC80 HD stereoscopic
microscope capture system (Leica Microsystems,
Wetzlar, Germany). Analyses were conducted
with Leica application suite software 4.3 (Leica
Microsystems Limited, Copyright 2003-2013). The
average diameter (D), total area of the egg (TA),
area of the embryo (EA) and area of the yolk sac
(SA) were measured using the freeware program
Image J (v 1.46r, Wayne Rasband, National
Institutes of Health, USA) (Figure 1). To determine
Treatment
identification
Treatment description
Experiment 1
Experiment 2
C
AFH* with hatchery water (10 min.)
AFH with hatchery water (10 min.)
T1
AFH with urea solution (30 min.)
AFH with urea solution (30 min.)
T2
AFH with urea solution (60 min.)
AFH with urea solution (60 min.)
T3
AFH with urea solution (90 min.)
AFH with urea solution (90 min.)
T4
AFH with urea solution (10 min.) +
washing with tannin solution (15 sec.)
AFH with urea solution (10 min.) +
washing with tannin solution (15 sec.)
T0
AFH with urea solution (10 min.)
Adhesiveness neutralization in eggs of Pseudoplatystoma fasciatum… 15
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 11 - 23, 2017
D, the average horizontal and vertical diameters
were considered for determining an average
(Figures 1b, c). The areas were automatically
calculated by the program (Figures 1d-f). All
measurements were taken in triplicate, and the mean
values were used in the analysis. In order to assess
the proportion in morphometric traits, three indexes
were defined: EA/SA, EA/TA and SA/TA.
Figure 1. Step by step morphometric analysis of a Pseudoplatystoma fasciatum egg. (a) Contrast adjustment; (b)
vertical and (c) horizontal diameter (used to calculate D); (d) total area of the egg (TA); (e) area of the
embryo (EA); and (f) area of the yolk sac (SA).
Adhesiveness removal analysis
The adhesiveness removal was evaluated by
visual estimation of the eggs and the arrangement
in each treatment (adhered and not adhered eggs).
This subjective visual method was used to avoid
higher mortality rates by premature manipulation.
The results were expressed by the percentage of
eggs that remained adhered to the incubator or to
other eggs, and the percentage of eggs that were
not adhered.
Sperm analysis
At the time of extrusion of the sperm, part of
it was separated to determine the time of sperm
motility, membrane integrity and sperm
concentration. Only samples that were free of
contaminants such as feces and urine were used.
In each experiment, freshly stripped sperm was
collected and stored at 4.0 ºC until the sperm of all
males were stripped. The sperm of each male were
analyzed separately but the results presented as
mean ± standard deviation and the sperm used to
fertilize the eggs was a pool from all males used.
Time of motility
In both experiments the spermatozoa
activation was tested using deionized water as a
control, and urea solution to test the possibility
that the activation solution would interfere in the
gamete quality. Observations of sperm motility
were conducted at room temperature (23.0 ± 2.0ºC)
using three replicates per sample, soon after milt
collection, with the aid of a light microscope
under 40 times magnification. The duration of
sperm motility was subjectively evaluated as the
time elapsed from activation until 50.0% of the
spermatozoa maintained forward swimming
activity using a stopwatch. For this assessment,
1.0 µL of semen from each breeder was applied to
a glass slide, proceeded activation using 20.0 µL of
deionized water and 20.0 µL of urea solution (in
different glass slides), and the stopwatch was
16 FIGUEIREDO-ARIKI et al.
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 11 - 23, 2017
concomitantly started while a cover slip was placed
over the solution, similar to the methodology used
by DE SOUZA et al. (2015) and KURADOMI et al.
(2016).
Membrane integrity
The percentage of live spermatozoa (sperm
survival) was evaluated for each male based on
the integrity of the membrane determined
according to the penetration capabilities of eosin-
nigrosin dyes (5.0% eosin and 10.0% nigrosin) in
live cells (unstained) and dead cells (pink stained).
A total of 200 cells per slide were counted on a
microscope under 100 times magnification. The
percentage of live cells was calculated as the
number of live spermatozoa divided by the
number of total cells evaluated multiplied by 100.
Concentration
The sperm concentration was estimated
according to standard methods (sperm cells mL
-1
milt) using a counting chamber similar to that
described by BUYUKHATIPOGL and HOLTZ
(1984). For this purpose, a sample of sperm was
fixed in buffered formaldehyde saline solution
(1:1000).
Statistical analysis
The experimental design was completely
randomized, with five treatments, three replicates
in experiment 1 and six treatments with three
replicates in experiment 2. The normality and
homoscedasticity of all the variables were
confirmed for all variables. For the FR and HR, the
data were subjected to arcsin transformation, and
one-way ANOVA test, and if found significant,
we applied the Fisher least significant difference
test. For the morphometric measurements, the
one-way ANOVA was also used, and if found
significant, we applied the Tukey honestly
significant difference test. Principal components
analysis (PCA) was performed using morphometric
indexes (EA/SA, EA/TA and SA/TA) from all
four five groups (C, T1, T2, T3 and T4) of
experiment 1. All the results are represented as
mean ± standard error (SE) and were considered
as significantly different when p<0.05. All statistical
analysis was performed using the software
Statistica 7.0 (StatSoft Inc., Tulsa, OK, USA).
RESULTS