Vol.5, No.7A, 1-6 (2013) Natural Science
http://dx.doi.org/10.4236/ns.2013.57A001
Artificial reproduction of neotropical fish: Extrusion
or natural spawning?
David Augusto Reynalte-Tat aje¹*, Carolina Antonieta Lopes¹, Sunshine de Ávila-Simas¹,
Juan Ramon Esquivel Garcia², Evoy Zaniboni-Filho1,3
1Departamento de Aquicultura, Laboratório de Biologia e Cultivo de Peixes de Água Doce, Universidade Federal de Santa Catarina,
Centro de Ciências Agrárias, Florianópolis, Brazil; *Corresponding Author:
2Estação de Piscicultura Panamá, Paulo Lopes, Brazil
3Programa de Pós-Graduação em Aquicultura, Departamento de Aquicultura, Universidade Federal de Santa Catarina, Centro de
Ciências Agrárias, Florianópolis, Brazil
Received 5 June 2012; revised 5 July 2013; accepted 12 July 2013
Copyright © 2013 David Augusto Reynalte-Tataje et al. This is an open access article distributed under the Creative Commons At-
tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-
erly cited.
ABSTRACT
In captive conditions, most neotropical migra-
tory species do not have the necessary incen-
tive to complete gonadal maturation and spawn-
ing, which require induction techniques, usually
with the use of hormones. Numerous studies
have therefore focused on finding an effective
hormonal treatment to induce reproduction for
each species. A standard treatment was suc-
cessfully developed for most of these species,
which consists of two doses of Carp Pituitary
Extract (CPE), totaling 5.5 mgCPE/kg of fish.
However, the best strategy for the subsequent
fertilization of gametes is still unclear. This stu-
dy was conducted with five species of com-
mercial interest that do not reproduce naturally
in captivity: pacu Piaractus mesopotamicus,
piracanjuba Brycon orbignyanus, curimbatá
Prochilodus lineatus, dourado Salminus brasil-
iensis and piauçu Leporinus macrocephalus.
Ninety-nine broodstocks were used, consisting
of males and females of the five species to
compare two techniques for spawning and fer-
tilization: extrusion followed by the dry method
and natural spawning. All fish were induced with
two hormone doses, i.e., 0.5 and 5.0 mgCPE/kg,
and were subjected to one of the two types of
fertilization. The results were compared using
the fertilization rate, number of oocytes pro-
duced, relative fecundity and the broodstocks'
survival rate on days following spawning. All
species responded positively to spawning for
extrusion, and on ly S. brasiliensis did not spawn
through the natural spawning method. The
natural spawning technique provided a higher
fertilization rate of eggs and a greater brood-
stock survival rate (P < 0.05) for all species, ex-
cept S. brasiliensis. It was concluded that the
natural spawning technique can increase the
production of viable eggs and reduce the mor-
tality of species during breeding, except for S.
brasiliensis.
Keywords: Induced Breeding; Spawning; Fish
Farming; Broodstocks; Induction Techniques
1. INTRODUCTION
Currently, the formation of wild fish broodstocks that
have adequate genetic variability is hampered due to a
number of factors, such as pollution, deforestation and
dam construction, which are promoting the reduction of
natural stocks and, in some situations, the extinction of
neotropical fishes’ large-sized stocks. Accordingly, the
maintenance and survival of broodstocks in hatchery
stations, government research agencies and private re-
search are considered of great importance [1]. However,
several causes of broodstock mortality have not yet been
overcome in the traditional management protocols to
produce fingerlings of neotropical species; one of these
is the absence of induction techniques for reproduction,
which allows a high production of offspring without the
high risk broodstock sacrifice.
Most commercial neotropical freshwater fish species
have migratory habits and depend on the migration to
complete gonadal maturation and spawn [2]. These spe-
cies are generally characterized by having high values of

D. A. Reynalte-Tat aje et al. / Natural Science 5 (2013) 1-6
2
total length, absolute fecundity, perivitelline space, as
well as low values of hydrated egg diameter and duration
of embryogenesis. Conversely, because these species
cannot breed in captivity, numerous studies have been
performed to develop hormonal induction techniques that
promote their final maturation and spawning.
In South America, most procedures for induced spaw-
ning use the extrusion technique to strip the gametes,
followed by the dry method for fertilization. This proce-
dure is traditional and widespread in the reproducetive
sector. However, recent studies with neotropical fish spe-
cies have demonstrated that natural spawning, which has
no human interference during gamete release, reduces
fish mortality and increases the egg fertilization rate [3].
The present study aims to compare results obtained
through traditional spawning with those obtained through
natural spawning for five neotropical species of com-
mercial importance.
2. MATERIALS AND METHODS
2.1. Execution Place
This study was conducted at the Panamá fish hatchery,
located in the city of Paulo Lopes, and at Estação Pis-
cicultura São Carlos, located in the city of São Carlos;
both sites are in the state of Santa Catarina (Brazil). The
data was analyzed and processed at the Laboratório de
Biologia e Cultivo de Peixes de Água Doce (LAPAD) in
Universidade Fe de ra l de Santa Catarin a.
2.2. Broodstocks Selection
A total of 99 broodstocks were used, consisting males
and females of five species. These fish were kept in tanks
on land with an area between 800 and 2000 m2. The
broodstock selection was based on the external charac-
teristics of gonadal maturation described by Woy-
narovich a n d Horváth [4] ( Table 1).
Ta bl e 1 . Total broodstocks used for each species with the age
(years) and weight (grams) listed.
Species Total
broodstock s Broodstock s number Age (years) Weight (g)
Female Male
Brycon
orbignyanus 15 5 10 4 1.100 - 2.200
Leporinus
macrocephalus 24 12 12 3 800 - 1.200
Piaractus
mesopotamicus 18 6 12 2 - 4 1.400 - 3.600
Prochilodus
lineatus 18 6 12 1 - 4 900 - 3.200
Salminus
brasiliensis 24 8 16 2 - 4 1.600 - 4300
After selection, broodstocks were sexed and placed in
tanks of 1000-liters with an open water system, where
they remained until the hormonal induction.
2.3. Hormonal Induction
Carp Pituitary Extract (CPE) was used because it is the
hormone most often used by the productive sector. For
hormonal application, broodstocks were removed from
the tank with the help of a net, placed in plastic bag s and
weighed. Finally the fish were intraperitoneally injected
with two hormone doses (0.5 mgCPE/kg and 5.0 mg-
CPE/kg) at intervals of 12 hours between doses, as rec-
ommended by Woynarovich and Horváth [4].
2.4. Spawning Technique
2.4.1. Natural Spawning
After the second hormone dose, males and females
were grouped in (2:1) ratios, except for L. macrocepha-
lus, which was maintain ed at a ratio of (1:1) in 100 0-liter
tanks. The tanks were partially covered to prevent fish
stress. The fish were under constant observation to evalu-
ate behavior and note the spawning time. Spawning al-
ways occurred after two or three fish maintained a syn-
chronized swimming and their bodies became close and
laterally aligned. In the P. lineatus and L. macrocephalus
species, the emission of sounds by males during spawn-
ing was observed. After spawning, the eggs were re-
moved with a plastic bucket that had a mesh retention of
0.5 mm to concentrate the eggs. This bucket was in-
stalled at the outlet of the water tank, and the eggs were
taken to the incubator. During transfer, a 500-mL beaker
was used to measure the eggs by decanting the eggs into
the beaker and measuring the volume marked . The num-
ber of eggs per milliliter was determined by collecting
four 3-mL samples of fully hydrated eggs from the
bucket retainer using a 10-mL pipette; the samples were
then counted on a pe tri plate [5]. This counting was con-
ducted for each spawning.
2.4.2. Dry Method (Spawning f or E x tr usion)
After the last hormonal application, fish were con-
tinuously removed once the females begin to release the
oocytes. The female was removed carefully using a towel.
The extrusion was performed by collecting oocytes in a
dry plastic becker. In the sequence, the sperm, which
were also obtained by extrusion, was added directly over
the freshly collected oocytes. After 1 to 2 minutes of
mixing the gametes, water was added to promote the
activation of sperm and fertilization. Once the eggs be-
gan to hydrate, they were transferred to incubators ac-
cording to the recommendations of Ihering and Azevedo
[6]. A small quantity of oocytes (± 0.5 g) was removed
before mixing with semen; these oocytes were weighed

D. A. Reynalte-Tat aje et al. / Natural Science 5 (2013) 1-6 3
and fixed in 4% buffered formaldehyde solution for later
quantification. During reproduction, the water tempera-
ture remained between 24.0˚C and 27.5˚C, the pH be-
tween 6.5 and 8.2 and the dissolved oxygen between 5.6
and 9.8 mg/L. The experimental design is shown in
Table 2.
2.5. Degree-Hour
The time interval between the last decisive application
and ovulation was calculated using degree-hours, which
consists of multiplying the elapsed time between the last
hormone injection and multiplying the spawning by the
mean water temperature during this period, which is
recommended by Woynarovich and Horváth [4].
2.6. Fertilization Rate
The fertilization rate was calculated nine hours after
fertilization by analyzing 260 eggs per incubator, ac-
cording to the recommendations of Zaniboni-Filho and
Barbosa [7], by observing the eggs using a stereoscopic
microscope at 10 x. The total number of eggs produced
per broodstock weight was calculated by standardizing
the values of the total number.
2.7. Relative Fecundity
The relative fecundity rate was quantified for the total
number of eggs produced, and this value was divided by
the total weight of the fish. This calculation was done
with the aim of standardizing the data extrusion and
natural spawning.
2.8. Statistical Analysis
The number of females that spawned for each treat-
Ta b le 2. Experimental design utilized to compare the different
types of spawning for reproduction of five neotropical species.
N = Natural; E = Extrusion.
Spawning
types Broodstocks number Male doses Female doses
Species
Female Male (mgCPE/kg) (mgCPE/kg)
N 2 4 0.4 0.5 - 5.0
Brycon
orbignyanus E 3 6 0.4 0.5 - 5.0
N 6 6 0.4 0.5 - 5.0
Leporinus
macrocephalus E 6 6 0.4 0.5 - 5.0
N 3 6 0.4 0.5 - 5.0
Piaractus
mesopotamicus E 3 6 0.4 0.5 - 5.0
N 3 6 0.4 0.5 - 5.0
Prochilodus
lineatus E 3 6 0.4 0.5 - 5.0
N 3 6 0.4 0.5 - 5.0
Salminus
brasiliensis E 5 10 0. 4 0.5 - 5. 0
ment was determined, as well as the degree-hour value
for spawning, fertilization rate, relative fecundity, num-
ber of eggs produced and the survival of broodstocks
after 10 days. All analyses used a univariate analysis of
variance (ANOVA) with a 5% significance level.
3. RESULTS
All tested fish species spawned via extrusion. All fe-
males of L. macrocephalus and P. mesopotamicus spa-
wned regardless of the technique used; in natural spawn-
ing, only S. brasiliensis did not spawn into the tank.
We found a variation in degree-hour between species,
with the shortest spawning time observed for B. orbign-
yanus and the longest observed for P. mesopotamicus.
We observed a trend of higher degree-hour values for
natural spawning than for spawning by extrusion, but this
tendency was only statistically confirmed for P. mesopo-
tamicus (Table 3).
The survival of broodstocks after spawning varied ac-
cording to the species, with the lowest values observ ed in
B. orbignyanus spawning extrusion. In general, higher
survival rates were observed with the technique of natu-
ral spawning, where survival values ranged between
66.7% and 100% (Table 3).
For L. macrocephalus and P. lineatus, the numbers of
eggs produced by both spawning methods showed no
statistically significant difference (P > 0.05). The fertili-
zation rate parameter showed the greatest difference be-
tween natural spawning and extrusion; all species show-
ed a higher rate of egg fertilization with natural spawning
and extrusion; all species showed a higher rate spawning
compared to extrusion (P < 0.05) (Table 4).
Table 3. Mean values and standard deviation (±) of de-
gree-hour and survival percentage values for spawning females
and survival of broodstocks for different species assessed. N =
Natural; E = Extrusion.
Broodstock
Survival (%)
Species Spawned
type
Spawned
Females
(%)
Degree-
Hour (DH)1 F M
N 100 188 ± 27 100 100
Brycon
orbignyanus E 66 142 ± 29 0 33
N 66 193 ± 16 100 100
Leporinus
macrocephalus E 100 202 ± 26 67 67
N 100 298 ± 21 100 100
Piaractus
mesopotamicus E 100 262 ± 15 67 83
N 66 184 ± 32 67 100
Prochilodus
lineatus E 66 225 ± 16 33 100
N 0 - 67 100
Salminus
brasiliensis E 75 151 ± 18 60 80

D. A. Reynalte-Tat aje et al. / Natural Science 5 (2013) 1-6
4
Table 4. Mean values and standard deviation (±) of the total
weight of oocytes spawned, the total volume of eggs per
spawning, the total number of eggs produced per kilogram per
spawning and the fertilization rate (%) per treatment. N = Natu-
ral; E = Extrusion.
Specie Spawning
type Total
weight Total volume Total Number Fertiliz ation
Rate (%)
N - - - 98.7 ± 8.4a
Brycon
orbignyanus E - - - 15.4 ± 14.7b
N -
3362 ±
2249 193225 ± 112914a 94.5 ± 3.3a
Leporinus
macrocephalus E 129 ± 58 - 264136 ±
102148a 25.8 ± 25.3b
N - - - 97.5 ± 12.1a
Piaractus
mesopotamicus E - - - 42.8 ± 15.3b
N -
4542 ±
1149 298149 ± 141811a 96.1 ± 2.4a
Prochilodus
lineatus E 143 ± 15 - 326129 ±
224175a 75. 4 ± 11.7b
N - - - -
Salminus
brasiliensis E 161 ± 22 - 267496 ±
118121 40.4 ± 9.1
4. DISCUSSION
Among the spawning induction techniques used for
neotropical migratory fish, the use of extrusion is well
documented [4,7,8], as it has been used to produce such
commercially important neotropical species as dourado,
S. brasiliensis; matrinxã, B. cephalus; tambaqui C. ma-
cropomum and pacu, P. mesopotomicus. This type of
technique is widely used due to its low operational man-
agement, high control of production and its status as a
well-disseminated technique. Moreover, according to
Bermudez, et al. [9], performing fertilization by extru-
sion reduces egg handling, which is considered an im-
portant factor in embryo development, reduces the
chance of fungi appearance, increases the fertilization
rate and increases the quality of future larvae.
However, a disadvantage of extrusion is that it may
increase the mortality rate of broodstock used, which is
generally positively related to the species-specific degree
of tolerance for handling. Females and males of Brycon
siebenthalae show high mortality rates after induction
[10]. Catfish (Rhamdia quelen) and piauçu (L. macro-
cephalus) commonly exhibit high mortality rates a few
days after spawning by extrusion [11]. For B. orbign-
yanus, up to 100% broodstock mortality rates have been
observed [12]. The overall broodstock mortality rates
after extrusion can exceed 50% in neotropical freshwater
species. In this study, we observed that in the four evalu-
ated species survival rates of individuals increased with
natural spawning. The survival percentage of spawning
by extrusion was low, especially for B. orbignyanus,
which exhibited more than 80% mortality of broodstock
males and 100% of brood stock females.
Survival after extrusion was related to species behave-
ior. L. macrocephalus presented most docile behavior in
handling and had the highest survival rates after extru-
sion; the converse was observed for B. orbignyanus.
Many hormonally induced fish begin to spawn in the
presence of active males after ovulation. In literature,
this phenomenon is referred to either as “induced spawn-
ing” or “hormonal induced spawning”. Landinez [13]
believe that hormonal indu ction followed by fish spawn-
ing occurring naturally in tanks without extrusion is a
“semi-natural” spawning ; however, there are problems in
accepting this terminology. The term “induced spawn-
ing” is conducive to erroneous interpretations and may
even be mistaken for spawning by extrusion because the
extrusion process is also a form of spawning induced by
a handler. However, the name “semi-natural spawning”
seems confusing. Perhaps the most appropriate term is
“natural spawning” because the act of spawning is only
regulate d by breedin g.
Saldaña and Ascon [14], working on tambaqui C.
macropomum, observed high fertilization rates (above
90%) with the natural spawning technique. Similar re-
sults were found by Sirol, et al. [15] in P. mesopotamicus
and by Varela, et al. [11] in R. quelen. In this study, the
technique of natural spawning resulted in the spawning
of more than 50% of females of different species, result-
ing in fertilization rates above 95%. This value exceeds
the general average fertilization rate of 54.3% obtained
via extrusion. Fertilization rates differed by an even
greater margin, with values of 7.5% for extrusion versus
86.1% for natural spawning. All values of fertilization
rates via extrusion were lower than with natural spawn-
ing.
According Bromage, et al. [16], determining the exact
time of ovulation to induce female egg production is very
important to obtain high-quality eggs, which is funda-
mental to achieving h igh rates of subseq uent fertilization .
The appearance of female eggs before or after the mo-
ment of ovulation can result in low fertilization rates and
low-quality larvae [17,18]. The ex act time of ovulation in
females may vary according to the species, with the
largest time intervals observed for cold water fish such as
trout Oncorhynchus mykiss (4 - 6 days) [18] and Clupea
harengus (14 days) [19] and smaller time intervals ob-
served for subtropical and tropical fish as Carassius au-
ratus (2 - 3 hours) [20] and Prochilodus platensis (1 hour)
[21]. Therefore, because the species evaluated in this
study are neotropical, the period in which the eggs are
viable may be assumed to be relatively short. In natural
spawning, individuals adjust and synchronize the release
of gametes across a great range of maturity levels, thus
ensuring a high fertilization rate. Conversely, with
spawning by extrusion, the possibility of making mis-
takes when selecting the female to be extruded in-

D. A. Reynalte-Tat aje et al. / Natural Science 5 (2013) 1-6 5
creases due to the short optimum maturation window.
This phenomenon is demonstrated in this study by the
relatively low fertilization rates and the large variation in
values.
According to Bermudez, et al. [9], natural spawning
creates the possibility that broodstocks may not be en-
tirely prepared for spawning and that the female gamete
may release without a corresponding release of the male
semen, causing a subsequent loss of eggs. Another possi-
bility is that females do not release their gametes because
they are not ready. In this study, we observed successful
spawns with high fertilization rates, which indicate that
males responded to female spawning. However, it was
found that some female piauçu (L. macrocephalus) and
curimbatá (P. lineatus) did not spawn, indicating that
their eggs were not ready. The absence of spawning in
dourado (S. brasiliensis) in a natural system may be an
indication that fish were not ready or that using carp pi-
tuitary extract in this system is not effective for this spe-
cies.
In this study, we observed higher average egg produc-
tion with extrusion. However, no significant differences
(P > 0.05) in egg production were found between treat-
ments for the different species evaluated.
Knowledge about aquaculture species of neotropical
migratory fish is in its initial stages, and many studies are
still needed to better understand the reproduction of these
fish. The results of this study indicate that two investi-
gated methodologies can be useful to obtain gametes
from these species. The extrusion method offers greater
practicality and less handling in spawning. Furthermore,
the natural spawning method achieves a higher survival
of broodsto cks and high fertilization rates. Extrusion can
be important in large-scale reproductions and in cases
where the financial and environmental costs of brood-
stocks are not high. Natural spawning is an important
technique that could be applied for fish that are able to
spawn in a tank containing males and that have high
economic and environmental broodstock values. In case
of an endangered fish such as piracanjuba (B. orbign-
yanus), the species has a high environmental value due to
its absence in nature and to the need to conserve its ge-
netic load. The natural spawning method is therefore
recommended for reproduction to reestablish a popula-
tion where 1) matrices with high genetic variability are
used, and 2) the loss of individuals during reproduction
could have inc al c ul abl e val ue.
5. CONCLUSION
Research perspectives related to spawning techniques
improvements have continuingly been developed; how-
ever, there is still limited knowledge regarding the neo-
tropical migratory fish reproductive biology. Moreover
the spawning techniques currently used still need im-
provement. Thus, some crucial aspects still need to be
evaluated, for example, the specific moment in which
fish is ready to spawn and the use of other hormones for
induction of natural spawn ing.
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