JOURNAL OF THE Vol. 45, No.

1
WORLD AQUACULTURE SOCIETY February, 2014
doi: 10.1111/jwas.12089

Use of ACP-104 at Different Dilutions for Sperm

Cryopreservation of Dourado, Salminus brasiliensis
Ana Carolina V. Zanandrea, Marcos Weingartner and
Evoy Zaniboni-Filho1
Laboratório de Biologia e Cultivo de Peixes de Água Doce (LAPAD), Universidade Federal de
Santa Catarina, Rodovia SC 406, no 3532, 88066-000, Florianópolis/SC, Brasil

Abstract
The dourado, Salminus brasiliensis, is an important fish in South American river basins. The
fish-farming potential and concerns for its conservation in watersheds has stimulated studies with
this species. This study compared the effect of different dilutions of two cryoprotectant solutions
on the quality of cryopreserved dourado sperm (duration of motility/motility rate) post-thawing
using three different activating solutions. The cryoprotectant solution ACP-104 was compared with
the cryoprotectant solution most commonly used for dourado, which consists of a mixture of
dimethyl sulfoxide, glucose, egg yolk, and distilled water. The semen was mixed following dilutions
of semen : cryoprotectant solution: 1:3, 1:9, 1:15, 1:21, and 1:27. The cryopreserved samples were
activated using distilled water, 0.45% NaCl or 1% NaHCO3 . The results showed differences in
thawed semen using the different activating solutions and cryoprotectant solutions. The glucose-
based cryoprotectant not only resulted in sperm motility equal to or better than that produced by
ACP-104, but the quality was not affected by the dilution. However, the sperm quality improved with
increasing dilutions for ACP-104. Under the conditions tested, the standard cryoprotectant solution is
recommended for the cryopreservation of dourado semen because it requires a lower solution volume
and, therefore, reduced storage space for the same volume of semen.

The dourado, Salminus brasiliensis, is a fish
that inhabits many rivers in South America
and is a member of the Characidae family.
The fish-farming potential of S . brasiliensis
and concerns for its conservation in watersheds,
where it is threatened by extinction, has stim-
ulated interest in this species (Weingartner and
Zaniboni-Filho 2010).
The cryopreservation of sperm and its main-
tenance in gene banks is an important practice
to conservation of biodiversity and to protect-
ing endangered species, and has the potential
to reduce the production costs of aquaculture.
The viability of cryopreserved sperm relies on
the use of cryoprotectant solutions that prevent
cryoinjuries to the spermatozoa during freezing
and thawing processes (Viveiros et al. 2009).
According to Viveiros and Godinho (2009),
the fertilization rates expected for cryopre-
served semen from Brazilian fish species vary
1 Corresponding author.
© Copyright by the World Aquaculture Society 2014
from 31 to 100% compared to fresh semen.
For dourado, Viveiros et al. (2009) observed
a significant reduction in the hatching rate
achieved using cryopreserved semen (17–23%)
compared with fresh semen (60%). An inves-
tigation of alternative products and processes
will be necessary to improve the viability of
sperm following cryopreservation.
The use of coconut water “in natura” for
the cryopreservation of tambaqui, Colossoma
macropomum, semen produced satisfactory
results once the osmolarity was adjusted (Farias
et al. 1999). However, even with advances
in the processing of coconut water, once this
solution is wrapped and stored, changes in its
quality can still occur. To ensure a reliable
standard, powder coconut water was developed.
The use of ACP® (powdered coconut water)
as a component in cryoprotectant solutions
has been tested for birds (Rondon et al. 2008)
and mammals (Mota-Filho et al. 2011; Silva
et al. 2012). ACP-104 is a product that was

82
 SPERM CRYOPRESERVATION OF DOURADO SALMINUS BRASILIENSIS
developed specifically for teleost fish (Viveiros
et al. 2008).
The evaluation of sperm motility post-
thawing in semen cryopreserved using
ACP-104 in curimba, Prochilodus lineatus;
piava, Leporinus obtusidens; and piracanjuba,
Brycon orbignyanus, produced results similar
to the control (Viveiros et al. 2008); however,
ACP-104 has not been tested in dourado.
The analysis of sperm motility has been
widely used as an indicator of sperm quality
(Viveiros and Godinho 2009; Bobe and Labbé
2010; Fauvel et al. 2010). Several studies have
reported satisfactory results for the motility
of post-thawed cryopreserved semen from
South American characiforms (Cruz-Casallas
et al. 2004; Melo and Godinho 2006; Oliveira
et al. 2007; Taitson et al. 2008), including the
dourado (Coser et al. 1984; Carolsfeld et al.
2003; Viveiros et al. 2009).
In post-thawing analysis, the external
medium used to activate the semen can affect
the characteristics of the sperm (Yasui et al.
2012), such as sperm motility. Viveiros and
Godinho (2009) reported that distilled water,
0.3% NaCl and 1% NaHCO3 are the activation
media most commonly used to induce sperm
motility.
In this study, different tests were per-
formed to determine the best conditions for
maintaining the motility of cryopreserved
sperm from S . brasiliensis, dourado, using
induced animals and non-induced animals. A
cryoprotectant solution based on ACP-104 was
compared with the most commonly utilized
cryoprotectant solution for the species, and
both solutions were evaluated at different
ratios of semen : cryoprotectant solution and
tested with the three most commonly utilized
activating solutions.
Materials and Methods
Male Selection and Sperm Collection
First generation captive-bred S . brasiliensis,
dourado, males, which were descendants of
wild fish caught in the upper Uruguay River
(Brazil), were used in this study. The fish
were 5-yr-old and were maintained in ponds
83
in which they were fed daily with commercial
feed containing 40% crude protein.
Male fish that released semen following the
application of slight abdominal pressure were
selected and taken to the laboratory where they
were maintained in circular 1000-L tanks. The
water was renewed constantly at rate of 17
times a day and was maintained at 26 C and
7.0 mg/L dissolved oxygen.
The selected males weighed an average of
1,024 ± 97.13 g, measured 460 ± 11.08 mm
in length and were divided into two groups:
hormonally induced and non-induced. The hor-
monal induction was performed by the admin-
istration of two intramuscular injections of carp
pituitary extract (CPE) (Danúbio Aquacultura,
Blumenau, Santa Catarina, Brazil), containing
the equivalent of 0.4 and 4 mg CPE/kg at an
interval of 12 h between applications, according
to Weingartner and Zaniboni-Filho (2010).
For semen collection, the male fish were
removed from the tanks, and their urogenital
regions were dried using paper towels. The
semen was collected by applying abdominal
pressure in the caudal direction, avoiding con-
tamination of the sperm with urine and feces.
The semen was collected in Falcon tubes, and
then the non-motile sperm were confirmed and
sperm quality was assessed immediately. To
avoid the variability in sperm quality inher-
ent among males (Labbé et al. 2001; Rurangwa
et al. 2004), semen from one male from each
group was cryopreserved for use in all exper-
iments. The fresh semen collected from the
hormonally induced group exhibited motility
duration of 51 sec, 90% intensity and a sperm
concentration of 1.27 × 1010 sperm/mL. The
fresh semen collected from the non-induced
group exhibited motility duration of 42 sec,
90% intensity and a sperm concentration of 1.32
× 1010 sperm/mL.
Comparison of Cryoprotectant Solutions
at Different Dilutions
Different dilutions of the cryoprotectant solu-
tion and semen were evaluated for two cryopro-
tectant solutions. The first cryoprotectant solu-
tion was a generic formula that was developed
84 ZANANDREA ET AL.
for migratory South American fish (Carols-
feld et al. 2003), consisting of 10% (15 mL)
dimethyl sulfoxide (DMSO), 5% (7.5 g) glu-
cose, one chicken egg yolk and 135 mL of dis-
tilled water, and it was designated the standard
cryoprotectant solution, CP, in this study. The
second cryoprotectant solution was ACP-104-
350 mOsmol (ACP Biotecnologia, Fortaleza,
Ceará, Brazil), which consisted of powdered
coconut water (3.74 g) and methylglycol (10%
v:v) diluted in distilled water to a final volume
of 50 mL, and was designated CA in this study.
The collected semen was mixed with the CP
and CA cryoprotectant solutions at the follow-
ing dilutions: 1:3, 1:9, 1:15, 1:21, and 1:27
(semen : cryoprotectant solution). After dilu-
tion, sperm samples were immediately aspirated
into 0.5 mL straws and immediately frozen in
a nitrogen vapor vessel (Taylor-Wharton dry
vapor shipper, CP model 300, Harsco Corp.,
Theodore, AL, USA).
Thawing and Examination of Sperm Motility
The samples were analyzed between 7 and
20 d after freezing. The thawing was conducted
by exposing the straws to room temperature
(25 C), and each treatment was evaluated by
activating the samples using three different acti-
vating solutions. The activating solutions were
distilled water, 0.45% NaCl and 1% NaHCO3 ,
and each analysis was conducted in triplicate
(three straws) for each treatment combination.
The post-thawing sperm motility was mea-
sured subjectively after checking the non-motile
sperm samples. The samples were analyzed
on slides under a light microscope (100×), as
described by Carolsfeld et al. (2003). To evalu-
ate the effect of the different treatments, was
measured the motility duration (the motility
time in seconds) and the motility rate (the sperm
vigor in percentage).
Statistical Analysis
Statistical analyses were performed using
Statistica software (StatSoft Inc., Tulsa, OK,
USA). After an arcsine transformation and
observed data normality, a factorial ANOVA
using the cryoprotectant and activators as
Table 1. Duration of motility and motility rate of
cryopreserved sperm from hormonally induced and
non-induced dourado, Salminus brasiliensis.
Duration of
Males motility (sec) Motility rate (%)
Induced 37.80 ± 24.07 32.67 ± 21.56
Non-induced 34.49 ± 22.47 29.11 ± 21.29
factors was applied, followed by Tukey’s
test for significant differences between means
(P < 0.05) (Zar 1996). All data are presented
as the means ± standard deviation. To evaluate
the effect of the different dilutions of the
cryoprotectant solutions, linear regression
analysis was utilized.
Results
The motility results of the cryopreserved
semen obtained from the hormonally induced
and non-induced males, considering all cry-
oprotectant solutions, all dilutions used and also
all activators used, are shown in Table 1.
Increasing the dilution of the semen in the
ACP-104 cryoprotectant solution increased
the sperm quality (the motility rate and the
duration of motility); however, there were no
significant changes in sperm quality using the
standard cryoprotectant solution at different
dilutions. Linear regression demonstrated
a significant relationship between the dilu-
tion and samples preserved using the ACP
cryoprotectant solution (Fig. 1).
The tested solutions successfully activated
the cryopreserved sperm, with the exception
of the samples cryopreserved in ACP at a 1:3
dilution and activated with distilled water or
1% NaHCO3 . The results showed differences in
thawed semen using the different activator solu-
tions and the cryoprotectant solutions (Table 2).
Using the standard cryoprotectant solution,
the sodium chloride activating solution resulted
in the longest duration of motility, while
distilled water resulted in the shortest. For the
motility rate, with the exception of the ACP
samples activated with sodium chloride and
sodium bicarbonate, there were no differences
among the analyzed combinations.
 SPERM CRYOPRESERVATION OF DOURADO SALMINUS BRASILIENSIS 85

A CP CA Linear (CA) maturation of sperm in the testicles and sperm

70
ducts. In this study, it was observed that the
60
use of hormonal induction allowed for a higher
Duration of motility (s)

50 flow of dourado semen, which facilitated the

40 process of cryopreservation.
30 For matrinxã, Brycon amazonicus, males
20
induced using carp pituitary extract exhibited
y = 2.0833x + 1.2722
a higher semen volume and decreased sperm
10
r² = 0.8745 concentration without any changes in motility
0 (Pardo-Carrasco et al. 2006). Variations in
3 9 15 21 27
Dilution of cryopreserved solution (1:x) the quality and quantity of semen may occur
during the reproductive period of the fish;
B CP CA Linear (CA)
50 therefore, sperm analysis can be used to help
determine the optimal time point for semen
Motility rate (%)

40
collection (Rurangwa et al. 2004).
30
Viveiros et al. (2010a) compared the ACP-
20 104-300 mOsmol solution with a control cry-
10 y = 1.5944x + 2.8863 oprotectant solution based on glucose and
r² = 0.8438 methylglycol for the cryopreservation of sperm
0
3 9 15 21 27 from P . lineatus, another South American
Dilution of cryopreserved solution (1:x) characiform. The results demonstrated that
Figure 1. Duration of motility (A) and motility rate (B) the ACP-104 solution diluted at a ratio of
of the cryopreserved sperm from dourado, Salminus 1:9 (semen : cryoprotectant solution) resulted
brasiliensis, in different dilutions of standard (CP) and in improved sperm motility; however, the
ACP-104 (CA) cryoprotectant solutions expressed as the sperm velocity and fertilization rate remained
mean value obtained in the activators for a dilution in
unchanged, suggesting that ACP-104 is suit-
both induced and non-induced males.
able for the cryopreservation of semen from
this species. The use of ACP-104 combined
Table 2. Duration of motility and motility rate of with methylglycol and diluted at a ratio of
cryopreserved sperm from dourado, Salminus brasiliensis,
activated using different activating solutions.1
1:9 resulted in inferior sperm quality for
S . brasiliensis when compared to the standard
Activating Cryoprotectant Duration of cryoprotectant solution consisting of DMSO
solution solution motility (sec) Motility rate (%)
and glucose. Only when ACP-104 was diluted
NaCl 0.45% CP 51.70 ± 18.25a 36.67 ± 13.22ab to 1:15 or greater did the motility rate and dura-
CA 33.93 ± 23.20bc 24.67 ± 17.17bc
NaHCO3 1% CP 42.70 ± 16.62ab 41.33 ± 13.58a
tion of motility reach values similar to those
CA 26.89 ± 23.80bc 13.00 ± 12.08c obtained using the standard solution.
Distilled water CP 24.90 ± 15.66c 28.00 ± 18.83ab The recommended dilution of semen with
CA 36.77 ± 29.40abc 41.67 ± 32.17ab
cryoprotectant solution varies between dif-
(CP: standard cryoprotectant solution; CA: ACP-104).
1 Different superscripts indicate significant differences using ferent studies and for different species. For
Tukey’s mean separation test (P < 0.05). example, Carolsfeld et al. (2003) recom-
mended using a dilution between 1:3 and 1:5
(semen : cryoprotectant solution) for South
Discussion American fish. Although a variation in sperm
According to Bobe and Labbé (2010), quality was observed when using different
spawning inducers are very efficient in dilutions of the ACP solution, the sperm quality
increasing semen volume and reducing semen remained unchanged using different dilutions of
density, which suggests that inducers maintain the standard solution. However, Viveiros et al.
or increase sperm motility and implies that (2009) reported that the highest semen motility
hormonal stimulation is favorable for the for dourado was obtained using a combination
86 ZANANDREA ET AL.
of the cryoprotectant solution with DMSO at a
dilution of 1:5 (semen:cryoprotectant solution)
compared to a dilution of 1:10.
Yasui et al. (2012) observed a reduction
in the fertilization rates for loach, Misgurnus
anguillicaudatus, as the concentrations of
calcium, potassium, and magnesium increased
and specified that a high concentration of
potassium should be avoided in cryoprotectant
solutions. ACP-104 is derived from coconut
water, which is composed of several minerals,
including higher concentrations of potassium
and calcium, as well as lower concentrations
of other minerals (Viveiros et al. 2010a). This
high concentration of minerals in ACP, in
comparison with the standard solution, may
influence the effectiveness of the solution and
thus require a higher dilution to achieve the
maximum spermatozoa motility.
The existing studies using ACP for the
cryopreservation of fish semen demonstrate
its potential (Viveiros et al. 2008; Viveiros
et al. 2010a); however, the results remain
inconclusive and more research is necessary to
understand its role. For the standard solution
consisting of DMSO, glucose, and egg yolk,
the results obtained for dourado are similar
to those observed for other South American
fish, such as yamu, Brycon siebenthalae
(Cruz-Casallas et al. 2004), matrinxã, Brycon
cephalus (Ninhaus-Silveira et al. 2006), and
cachama blanca, Piaractus brachypomus
(Ramirez-Merlano et al. 2011). However, for
other South American fish species, such as
B . orbignyanus (Maria et al. 2006), P . lineatus
(Felizardo et al. 2010), and piraputanga, Brycon
insignis (Viveiros et al. 2010b), the cryop-
reserved sperm quality post-thaw was higher
when using methylglycol or methanol rather
than DMSO as the intracellular cryoprotectant,
independent of the extracellular cryoprotectant.
The composition of the activating solution
for cryopreserved semen can change the
quality of motility. When the activation of
cryopreserved sperm using 1% NaHCO3 or
distilled water was compared, the use of water
resulted in lower sperm motility for P . lineatus
(Murgas et al. 2007) and dourado cryopre-
served semen (Carolsfeld et al. 2003). In this
study, it was also observed that the duration of
motility using sodium bicarbonate and sodium
chloride was better than that obtained with
distilled water for samples cryopreserved using
the standard solution. However, for this same
cryoprotectant solution, the motility rate was
similar for all tested activators. Fertilization
tests are recommended to evaluate the effect
of these solutions on the interaction between
the oocyte and spermatozoa (Rurangwa et al.
2004; Viveiros and Godinho 2009).
This study indicates that different activator
solutions affected the sperm quality of the
dourado cryopreserved semen for both cryopro-
tectant tested. The use of ACP-104 is indicated
for dourado sperm cryopreservation only at
dilutions of semen to cryoprotectant solution
greater than or equal to 1:15. However, for the
standard cryoprotectant solution all dilutions
presented similar results. On the basis of these
conditions, the results indicate that the use of
the standard cryoprotectant solution developed
by Carolsfeld et al. (2003) for dourado sperm
cryopreservation is recommended. The use of
the standard cryoprotectant not only resulted
in sperm motility equal to or better than
that when using the ACP-104, but the sperm
motility was not affected by the dilution factor;
therefore, the costs of cryopreservation are
reduced because a lower dilution requires less
space and less cryoprotectant solution to store
the same volume of semen.
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