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CryoLetters 33 (2), 125-133 (2012)
© CryoLetters, businessoffice@cryoletters.org
INCREASING STORAGE CAPABILITY OF PACU (Piaractus
mesopotamicus) EMBRYOS BY CHILLING: DEVELOPMENT OF A
USEFUL METHODOLOGY FOR HATCHERIES MANAGEMENT
D. C. Fornari1, R. P. Ribeiro1, D. P. Streit Jr2, L. Vargas1, L. C. Godoy2*, C. A. L.
Oliveira1, M. Digmayer1, J. M. Galo1 and P. R. Neves1
1
PeixeGen Research Group, Maringá State University, Department of Animal Science,
Maringá, Brazil.
2
Aquam Research Group, Federal University of Rio Grande do Sul, Department of
Animal Science, Porto Alegre, Brazil.
*Corresponding author
email: godoyaqua@yahoo.com.br
Abstract
Cryopreservation of fish gametes has been studied extensively in the last few
decades, but the successful cryopreservation of fish embryos remains elusive. However,
recent studies using short-term chilling techniques have shown that it is possible to store
embryos at low temperatures with no significant loss in viability. Information on
cryopreservation of Neotropical freshwater fish embryos has so far been very limited in
the literature. In the present study, chilling protocols for storage of pacu embryos at 8°C for up to 24 h were studied using different concentrations of sucrose in methanol.
Embryos tolerated the subzero temperature for up to 6 h with no adverse effects (P >
0.05). After 12 h chilling, hatching rate of 64.0 ± 3.5% was recorded. Low temperature
storage of pacu embryos by chilling is detailed here for the first time. Further studies are
needed to extend the storage time and to improve the hatching rate.
Keywords: Cryopreservation, chilled embryos, Neotropical fish, Brazilian fish farming,
breeding program
INTRODUCTION
Cryopreservation of fish gametes has been studied extensively in the last three
decades, and the successful cryopreservation of the spermatozoa from many species,
including salmonid, cyprinids, silurids, acipenseridae, anastomids and characids, is well
documented (6, 15, 17, 22, 23, 27, 29). During the last decade, fish sperm
cryopreservation has become one of the most effective tools for reproduction
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management in fish farming. In aquaculture, frozen fish semen is frequently used for
artificial insemination of stripped mature oocytes when fresh semen is not available.
However, successful cryopreservation of fish embryos has not yet been achieved
(11, 12, 30, 31) mainly due to their low membrane permeability, large size, high lipid
content, a thick chorion, complex structure and a high sensitivity at low temperatures (3,
10, 18, 23, 25, 30). Although successful freezing of fish embryo remains elusive, a
recent study carried out by Streit Jr. et al. (21) using a chilling technique showed that is
possible to store pacu (Piaractus mesopotamicus) embryos at subzero temperatures for
6 h without significant loss of hatching rates.
Unlike freezing technique where specific cooling rates can be achieved by using
liquid nitrogen (LN), and once frozen in liquid nitrogen samples can be stored for
extended periods of time; the chilling technique consists of exposing cryoprotected fish
embryos to subzero temperatures (normally using a refrigerator) followed by short
storage periods (3, 10). This method is very useful for hatchery management since it
allows synchronizing the development of embryos collected from different spawning
events (16) and optimizes the use of hatchery facilities.
Previous studies performed by Streit Jr. et al. (21) on toxicity of several
cryoprotectants (CPAs) to pacu embryos showed that methanol was the best permeable
cryoprotectant, as also reported for other fish species (2, 8, 10, 13, 28), due to its low
toxicity and good permeation through the embryo membranes (7, 21). However, there is
no information regarding methanol use for longer period of exposure at subzero
temperature and its effects on pacu embryos viability.
Pacu (Piaractus mesopotamicus) was used in this study because it is the second
most reared native fish in Brazil, especially in the Midwest and Southeast areas of the
country, contributing to 5.9% of the 209,812 tonnes produced by aquaculture in 2007
(14). Easy adaptation to captivity, rusticity, fast growth rate, good feed conversion rate
and good/exotic flavor make pacu highly popular and there is a strong commercial
interest in this species (1, 21).
In the present study, a series of experiments were designed in order to develop a
protocol for pacu embryos storage at -8°C in different concentrations of methanol and
sucrose.
MATERIALS AND METHODS
Broodstock care and egg production
The study was carried out at the Hydrology and Aquaculture Station - Duke Energy
International, Salto Grande, São Paulo State (Brazil), in collaboration with both
PeixeGen and Aquam research groups.
Pacu (Piaractus mesopotamicus) broodstock were randomly sampled at gonadal
maturation stage. Mature females were visually identified by external characteristics
such as enlarged and soft abdomen and a reddish and swollen gonadal papilla. Mature
males were identified by a soft abdomen and releasing of milt when the abdomen was
gently squeezed. Selected breeders were transferred to a 1,000-L tank following
hormone injection. Females were injected intraperitoneally with a commercial carp
pituitary crude extract (CPE) twice at a 12 h interval at concentrations of 0.5 and 5.0 mg
kg-1 CPE respectively. Males received a single injection of 2.5 mg kg-1 CPE at the same
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time the second injection was applied to females. The water temperature and fish
behaviour were monitored every hour. The time of ovulation after the second injection
is temperature dependent and for pacu the spawning took place 240 h-degrees
(accumulated thermal unit) after the second injection. Both mature oocytes and fresh
semen were stripped into the same beaker (dry method) by gentle abdominal massage.
Tank water was then added to activate spermatozoa motility and to induce fertilization.
Selection of embryos
Eggs were incubated in 7-L open-flow conical hatcheries at 27 ± 0.8°C and the
sequence of events was monitored in order to follow embryonic development. Three
random samples (~800 embryos for each sample) were collected after the blastoporous
closing stage, (6 h post-fertilization, Fig 1A) for assessing the fertilization rate. Dead
embryos (Fig 1B) were discarded and healthy embryos (judged by chorion morphology)
were selected for the experiments. Pacu embryos normally reach the hatching stage 18
h post-fertilization (Fig 1C).
Figure 1. (A) pacu embryo at blastoporous closing stage (75% epiboly movement); (B)
dead embryo; and (C) live larva. Digital images obtained using a stereomicroscope (x
30 magnification).
Studies on the effect of chilling on Pacu embryos survival
Groups of 100 viable embryos were placed in 6-mL glass vials and exposed to
cryoprotectant solutions containing 9% methanol and four different concentrations of
sucrose (8.5, 17.1, 25.5 and 34.0%, Table 1). Methanol concentration of 9% (2.8 M)
was used according to Streit Jr. et al. (21) which indicated this concentration was most
effective in protecting pacu embryos at blastoporous closing stage. Embryos chilled in
tank water were used as chilled controls (Table 1).
The vials containing embryos and cryoprotectant solutions were sealed and cooled
gradually by immersion in an ice-water bath at 15°C for 10 min. Vials were then
transferred to another ice-water bath at 5°C for 10 min before storage in a refrigerator at
-8°C for 6, 12 and 24 h. This chilling protocol was adapted from Ahammad et al. (3).
At the end of each treatment time period, the sealed vials were transferred from the
refrigerator to 3-L open-flow conical hatcheries at 27 ± 0.8°C and acclimated for 2 min.
The embryos were then released into the hatcheries to complete embryonic
development.
Embryos cultured at 27 ± 0.8°C were used as natural controls. For all experiments,
11 replicates (~1100 embryos in total) were used for each treatment and each
experiment was repeated 3 times over a 4-week period.
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Table 1. Composition of the four solutions used in pacu embryos chilling study
Cryoprotectants concentration (%)*
Sucrose
Methanol
8.50
9.00
Treatments
C1
C2
17.10
9.00
C3
25.50
9.00
C4
34.00
9.00
-
-
Chilled control**
Values are on the w/v basis.
**
CPAs-free chilled control.
*
Hatching assessment
When embryonic development was completed (18 h post-fertilization), both control
and treated groups were carefully removed from the hatcheries to determine the
hatching rate by counting live and dead larvae/embryos under a stereomicroscope. Only
larvae with vigorous mobility and swimming capability were considered as live larvae.
The hatching rate was calculated as follows:
Hatching rate (%) 
number of live larvae
x100
total number of embryos
Data analyses
The experimental design was completely randomized in a 5 x 3 factorial
arrangement (five chilling solutions and three storage times) with 11 replications. Oneway analysis of variance followed by Tukey’s post-hoc test was used for statistical
analysis (P < 0.05). All tests were conducted after the confirmation of homogeneity of
variances (Levene’s test) and normality of the data distribution (Kolmogorov-Smirnov’s
test). All the analyses were performed using Proc GLM of SAS software (SAS Institute,
Cary, NC, USA, 2003).
RESULTS
The hatching rate of the control groups, which were selected and transferred
directly to hatcheries without undergoing any cooling treatment was of 93.4 ± 2.1%. No
embryos survived after chilling in tank water (CPAs-free treatment) following exposure
at -8°C in all experiments (Fig 2).
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Figure 2. Hatching rate of pacu embryos after exposure at -8°C for 6, 12 and 24 h in
9% methanol + 8.5% (C1), 17.1% (C2), 25.5% (C3), and 34.0% (C4) sucrose, and tank
water (chilled control). Error bars represent standard errors of means, * represents
significant differences (P < 0.05) between room temperature control and treated
groups. Bars labeled with the same letter do not differ significantly from each other
within the same storage time (P > 0.05).
After 6h exposure, high survival rates were obtained for embryos exposed to -8°C
in methanol plus sucrose and no significant differences in the hatching rate between the
control group and chilled groups were observed (Fig 2). Significant differences (P <
0.05) were observed between the control and treated groups after 12 h storage. Embryos
chilled in C4 showed the lowest hatching rate (30.7 ± 3.5%) after12 h storage (Fig 2).
However, there were no significant differences among groups treated in C1, C2 and C3
after 12 h (59.3 ± 4.0%, 64.0 ± 3.5%, and 51.5 ± 3.6% respectively). The results showed
that hatching rate after exposure to subzero temperature decreased in a time-dependent
manner.
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The results from this experiment showed that pacu embryos chilled in C1 treatment
tolerated -8°C for up to 12 h without their viability being compromised (P > 0.05) when
compared with those after 6 h storage (Fig 3). However, the hatching rate recorded after
12 h (59.3 ± 4.0%) showed significant difference (P < 0.05) from the control group (Fig
2).
Despite the significant differences in hatching rates between 6 and 12 h treatment in
C2 group, the hatching rate of chilled embryos reached 64.0 ± 3.4% after 12 h at -8C°.
C1 (8.5% sucrose + 9% methanol) was the only solution that produced 26.0 ± 3.5%
hatching rate of pacu embryos after 24 h storage at -8°C (Fig 3).
Figure 3. Hatching rate of pacu embryos after exposure at -8°C for 6, 12 and 24 h in
9% methanol + 8.5% (C1), 17.1% (C2), 25.5% (C3), and 34.0% (C4) sucrose, and tank
water (chilled control). Error bars represent standard errors of means, * represents
significant differences (P < 0.05) between room temperature control and treated
groups. Bars labeled with the same letter within each treatment do not differ
significantly throughout the storage time (P > 0.05).
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DISCUSSION
Reproduction management is very important in aquaculture and one of the limiting
factors to the reproduction success is the quality of gametes. One of the important
criteria of the quality of a gamete is its ability to fertilize or to be fertilized, and the
subsequent development into a normal embryo (5). However, the quality of gametes can
also be defined differently depending on the specific biotechnological applications such
as their use for cryobanking, nuclear transfer or androgenesis. Hatching rate is one of
the common criteria for assessing the ability of the fertilized egg to successful
development which can be monitored in most fish species. In this study, the control
group of embryos showed high hatching rates, showing the high quality of the gametes
used for this study (5). The similarities (P > 0.05) of the hatching rates between the
control and chilled groups 6 h after exposure to subzero temperature indicated the
efficiency of chilling solutions in maintaining the viability of pacu embryos under these
conditions.
When pacu embryos were chilled in CPAs-free solutions, total mortality were
observed at storage times, it is possible that the yolk syncytial layer and other
embryonic structures such as chorion and blastoderm have been damaged. According to
Fornari et al. (9) the blastoderm cells, the chorion and the yolk syncytial layer had the
most damaged structures at low temperatures and resulting none viable embryos.
However such injuries were possibly prevented in other treatments due to the addition
of methanol as a permeating CPA in combination of sucrose.
Studies have shown that the use of sugars as non-permeable CPAs provide
additional protection to membranes from the consequences of dehydration in
mammalian embryos (4) and optimizes the performance of permeable CPAs when used
in combination. Other studies also showed that the addition of sucrose (0.5 M) in
methanol (2 M) increased the survival of mrigal (Cirrhinus mrigala), catla (Catla catla)
and rohu (Labeo rohita) embryos (2) at 4°C.
According to Dinnyés et al. (8), the beneficial effect of sucrose may be related to a
moderate level of dehydration that helps to protect the cell membrane at low
temperatures. Nevertheless, the protective effect of sucrose is decreased when higher
concentrations are used. Under these conditions sucrose may induce extreme
dehydration and become toxic therefore leading to high mortality of the embryos. This
could be verified by results obtained from the present study that after 12 h storage at 8C° hatching rates were significantly decreased for embryos chilled in C4 when
compared with those chilled in C1, C2 and C3.
In the present study we improved the storage life of pacu embryos at subzero
temperature when compared the results obtained by Streit Jr et al. (21). The hatching
rate recorded in C2 treatment (64.0 ± 3.4%) after 12 h storage at -8°C was similar to
that achieved by Streit Jr. et al. (69.2%, 21) after 6 h storage, using the same chilling
solution (9% methanol + 17.1% sucrose). The higher hatching rate observed in our
study may due to the quality of the gametes as in Streit Jr. et al.’s (21) study the
hatching rate of the control group was 80.0% whilst the hatching rate of the control
group in the present study was 93.4%.
The protocol reported in the present study for successful short-term chilling storage
of pacu embryos using methanol and sucrose has important implications as this simple
low temperature storage technique can be adopted for reproduction management and
131
especially in the remote areas of Brazil where facilities are very limited. It can be used
for synchronization the development of embryos from different spawning dates or by
delaying the embryonic development when the amount embryos produced are higher
than expected by fish farmer who may not have enough facilities to cope under these
circumstances. Considering the above mentioned situation, a hatching rate of 64.0%
after 12 h storage at -8°C could be very useful.
Moreover, the method developed in the present study makes the transportation of
large amount of cooled embryos in small containers over long distances possible by
using small volumes of solutions and eliminate the use of oxygen cylinders. For
breeding programs, the exchange of fish embryos among hatcheries is an important way
of sourcing new genes and the chilling protocols developed in the present study can be
very valuable for facilitating this.
Further studies are needed in order to extend the storage time and to improve the
hatching rate.
Acknowledgements: We would like to thank Miss Fernanda de Mello for her statistical
support. We are also grateful to the anonymous reviewers for many suggestions which
have helped in improving the manuscript. The research was partly funded by CAPES
Foundation, Ministry of Education of Brazil and by Hydrology and Aquaculture Station
- Duke Energy International, São Paulo State, Brazil.
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Accepted for publication 16/12/2011
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