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Theriogenology 77 (2012) 1570 1574

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Effect of timing of hormonal induction on reproductive activity in

lambari (Astyanax bimaculatus)
V.O. Felizardoa,*, L.D.S. Murgasa, E.S. Andradea, P.A. Lpezb, R.T.F. Freitasc,
M.R. Ferreiraa
b

a
Department of Veterinary Medicine Federal University of Lavras, Lavras, Brazil
Science Veterinary University, National University of Rosrio (FCV-UNR), Santa Fe, Argentina
c
Department of Animal Science, Federal University of Lavras, Lavras, Brazil

Received 3 May 2011; received in revised form 11 November 2011; accepted 22 November 2011

Abstract
The objective was to evaluate the influence of the timing of hormonal induction, using gonadorelin or common carp pituitary extract
(CPE), on the reproductive activity of female Astyanax bimaculatus. Fish (N 44) were weighed, measured, and acclimatized to
experimental conditions with a photoperiod of 12 h:12 h light:dark (L:D) for 10 days. Ovulation was induced with a single dose of CPE
(6 mg/kg) or gonadorelin (80 g/kg), given at 12:00 (halfway through the light phase (LP) or 24:00 (halfway through the dark phase
(DP), in a 2 2 factorial design. The time of ovulation was calculated in degree hours and daily motor activity was recorded using a
photocell. The fish were killed and the liver and gonads were weighed for calculation of gonadosomatic (GSI) and hepatosomatic (HSI)
indexes, respectively. Absolute fecundity (AF), absolute fecundity relative to weight (AFRW) and length (AFRL), diameter of oocytes
(mM), and percentage of oocytes with the germinal vesicle in a peripheral position (PPGV) were recorded. All females responded
(ovulated). The female Astyanax bimaculatus had twilight motor activity rhythm. Females given CPE at 12:00 had a higher (P 0.05)
percentage of oocytes with the germinal vesicle in a peripheral position compared with the group that received gonadorelin in the same
period (95 6 vs. 79 21%, mean SD). The absolute fecundity relative to weight was higher in groups induced at 12:00, regardless
of the hormone used (LP: 805 448 and 700 214, for CPE and gonadorelin, respectively; dark phase: 580 396 and 529 105,
P 0.05). Both times used for hormonal induction with CPE and gonadorelin were suitable for inducing reproduction in lambari,
although induction with CPE in LP had the best results.
2012 Elsevier Inc. All rights reserved.
Keywords: CPE; Daily rhythm; Gonadorelin; Oocytes; Induction of ovulation; Fish

1. Introduction
Daily biological rhythms are associated with environmental fluctuations that have a periodicity of 24 h
(circadian rhythm), entrained to the cycle of light and

* Corresponding author. Tel.: 1 55 35 9111 5177; fax: 1 55 35

3829 1735.
E-mail address: viviofbio@yahoo.com.br (V. de Oliveira Felizardo).
0093-691X/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.11.025

dark. Light and temperature cycles play a crucial role in

seasonal rhythms, such as those involved in fish reproduction [1,2]. For example, in teleost fish, ovulation
and spawning usually occur at certain times of day,
depending on the photoperiod [3]. Peaks of plasma
GnRH concentrations vary among species of fish according to the time of day. This hormone is responsible
for final maturation of oocytes [4]; therefore, time of
day is one of the most important environmental variables affecting induced spawning. Muniz et al. [5] dem-

V.O. Felizardo et al. / Theriogenology 77 (2012) 1570 1574

onstrated the influence of photoperiod on the ovulation

process in tambaqui (Colossoma macropomum) induced with luteinizing hormone releasing hormone
(LHRH-gonadorelin). This was linked to plasma concentrations of sex steroids present during early stages of
hormonal induction.
Common carp pituitary extract (CPE) is widely used
to induce final maturation in Brazilian migratory fish,
achieving acceptable results for several species [6] including Prochilodus lineatus [7] and Brycon orbignyanus [8]. However, the high cost of the CPE has encouraged studies on alternative hormones, e.g., ovaprim,
hCG, GnRH, and GnRH analogues [9,10]. Gonadorelin
(GnRH) in the form of lyophilized diacetate tetrahydrate has been used [5], but there are apparently no
reports of using this hormone to induce ovulation in fish
that have daily patterns of activity.
Astyanax bimaculatus (lambari) is widely distributed across South America [11], and is popularly referred to as lambari, yellow tail piaba, or yellow tail
lambari. Reproductive activity begins once individuals
reach 5 cm in length [12], and total or split spawning
may occur, depending on environmental conditions
[13]. Due to its small size, ease of manipulation in the
laboratory and dependence on environmental conditions for reproduction, A. bimaculatus can be used as a
model for other large tropical species. This species was
used in tests of reproductive induction with CPE by
Sato et al. [14]. However, there is little if any knowledge of the daily rhythm of motor activity and the
influence of the timing of hormone application on induced breeding of lambari. Thus, the objectives of this
study were to determine reproductive parameters of
female lambari (A. bimaculatus) induced with gonadorelin or CPE at two times of the day, and to determine
the daily rhythm of motor activity of this species.
2. Materials and methods
The experiments were performed in the laboratory
of Physiology and Pharmacology in the Department of
Veterinary Medicine, Federal University of Lavras
(UFLA), during December 2009. A total of 44 adult
female lambari, weighing 7.9 2.4 g and measuring
8.4 0.8 cm in length, were obtained from the aquaculture station of UFLA. Females suitable for hormonal
induction were selected according to external features,
e.g., swollen abdomen and a swollen and reddish genital pore, as described [14].
After selection, the fish were kept in 20-L glass
aquaria (11 fish per aquarium) with a system of water

1571

recirculation, in which water from the aquaria was

captured and passed through a mechanical and biological filter of 250 L for later use; this system maintains
water quality. A photoperiod of 12 h:12 h light:dark
(LD) was maintained using fluorescent lamps. The light
intensity was 1173 lux, measured by a digital light
meter Model LDR-208 (Instrutherm, So Paulo, So
Paulo, Brazil). During the experimental period, the water temperature, dissolved oxygen concentration, and
pH were maintained at 27 1 C, 6 0.5 mg/L, and
6.5 0.5, respectively. Throughout the experimental
period, the fish were fed commercial extruded feed
(40% crude protein) pellets (2 mm in diameter), at a
rate of 2% of body weight, in two daily portions,
provided during the light period.
During Experiment 1, daily motor activity of female
lambari was determined, whereas in Experiment 2, reproductive end points were determined in females induced with gonadorelin or CPE given during the light
or dark phase (2 2 factorial).
2.1. Experiment 1
Motor activity was recorded daily for 10 days, before performing the hormonal induction, using an infrared photocell (Model E3S-[SCAP]AD[R]62, Omron, Tokyo, Japan) installed on the exterior of each
aquarium [15], each containing 11 fish. The photocell
was activated by movement as fish passed in front of
the photocell and records were sent to and stored in a
computer connected to the photocell. A chart was created from the numerical data, showing the average
daily motor activity over 10 days, with a trend line
every 10 min. The type of motor activity (diurnal,
nocturnal, or crepuscular) was determined by observing
the time of maximum movement of fish within the
photoperiod and performing a descriptive analysis of
the data, according to methodology previously described [15,16].
2.2. Experiment 2
Fish were acclimatized to experimental conditions
for 10 days. After 24 h of fasting, hormonal induction
was induced with a single, im injection at the base of
the pectoral fin, at two times, as follows: groups 1 and
2 received 80 g/kg of gonadorelin at 12:00 (mid light
[ML]) and 24:00 (mid dark [MD]), respectively,
whereas groups 3 and 4 received CPE at a dose of 6
mg/kg, at the same times as groups 1 and 2, respectively. The experiment was conducted in a randomized
design in a 2 2 factorial design (two hormones and
two times of application), with 11 replicates, where

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V.O. Felizardo et al. / Theriogenology 77 (2012) 1570 1574

each female was considered an experimental unit. The

hormonal application at night was done using a red
light, because this was not perceived as light by the fish.
Ovulation was confirmed by the observation of
swimming behavior characteristic of females at the
time of ovulation, involving lateral and circular movements, as described [14]. After ovulation, fish were
killed using the anesthetic benzocaine (2 g dissolved in
5 mL ethanol for each liter of water). A ventral incision was made to remove and weigh the gonads
(GW) and liver (LW). The gonadosomatic (GSI) and
hepatosomatic (HSI) indexes were calculated using
the following equations: GSI (GW/total weight
[TW]) 100; HSI (liver weight/TW) 100 [17].
After weighing the gonads, oocytes (0.1 g) were
collected to determine the following: absolute fecundity (AF) GW number of oocytes in 0.1 g total
number of eggs produced per fish); absolute fecundity relative to weight (AFRW) AF/TW total
number of oocytes per gram of sample); and absolute
fecundity relative to length (AFRL) AF/total
length (TL) the number of oocytes per centimeter
of animal.
The diameter (DIAM) of 10 oocytes per female was
measured following their immersion in Gilson solution
(5 mL alcohol 60%, 44 mL distilled water, 0.7 nitric
acid 80%, 1 g of mercuric chloride, and 0.9 mL of
glacial acetic acid). The peripheral position of the germinal vesicle (PPGV) % was determined by examining
25 oocytes immersed in Serra solution (60 mL of 90%
ethanol, 30 mL of formaldehyde, and 10 mL of glacial
acetic acid) for each female. The diameter and PPGV
were assessed using an optical microscope at magnification 40.
The degree hours (DH C) at which each spawning
was extruded was determined according to Silva [18].
Data were subjected to two-way ANOVA, using the
computational package SAEG (Sistema para Anlises
Estatsticas, Verso 9.1: Fundao Arthur BernardesUFV-Viosa, 2007), following a statistical model with
two main effects (hormone and time of application),
and the interaction between these factors. When there
was a significant (P 0.05) main effect or interaction,
means were compared using the F test (5% probability).
3. Results and discussion
Female lambari had a twilight motor rhythm activity
(Fig. 1), with a small peak in activity at the end of the
light period and the beginning of the dark period. This

Fig. 1. Daily locomotor activity of lambari (Astyanax bimaculatus),

in a photoperiod of 12 h:12 h light:dark (LD). White and dark bars at
the top of the chart indicate light and dark periods, respectively.

preference for activity at certain times of day seems to

be species-specific, and may be associated with genetics, adaptations to habitats (food availability, predation,
etc.) or sensorial factors e.g., better vision for capturing
food [19].
All female lambari responded to hormonal induction
with ovulation and fertile oocytes. There was a hormone versus induction period interaction (P 0.05) for
diameter, PPGV and degrees hours of ovulation, and
only for effect of the period of application for AFRW.
However, none of the other variables were significantly
affected by the factors studied (Table 1).
Fish that received hormonal induction with CPE during ovulation in ML had more DH than those induced in
MD (DH 333.9 vs. 240.1 C, P 0.05; Table 1). The
results obtained from the fish that received hormonal induction with CPE in ML differed from those given gonadorelin at the same time (DH 333.9 vs. 250.3 C, P
0.05). However, for groups treated with gonadorelin, there
were no differences (P 0.05) in the degree hours of
ovulation, regardless of the time of hormonal induction
(ML: DH 250.3 C; MD: DH 240.3 C). The difference
between the ML groups in DH for ovulation had no
negative effect on the reproductive characteristics of the
specimens, because there were no differences (P 0.05)
in the relevant end points (Table 1), with the exception of
the AFRW.
The release of oocytes from females that were
induced with CPE in ML began at 333.9 DH (11 h)
after application of a single dose of the hormone, at
a water temperature of 27 C. These results seemed
similar to those reported by Sato et al. [14], who
recorded spawning at 331 degree hours (12.8 h)
using the same procedures at a water temperature of
26 C. This difference in time of spawning of lambari was probably due to the temperature of the

V.O. Felizardo et al. / Theriogenology 77 (2012) 1570 1574

Table 1
Mean ( SD) of several end points of tetra that underwent
hormonal induction with two hormones and two periods of
hormone application (2 2 factorial, N 44 fish).
End point

Diameter
(m)*
PPGV (%)*
DHO*
AF
AFRL
GSI (%)
HSI (%)
AFRW

Period of
application

Hormone
Carp pituitary
extract

Gonadorelin

LP

1066.5 142.2

989.2 78.6

DP
LP
DP
LP
DP
LP
DP
LP
DP
LP
DP
LP
DP
LP
DP

1135.4 49.1a
95 6a
86 26
333.9 30a,A
240.1 13B
7443 6501
5241 4660
818 625
594 484
18.0 7.9
14.0 9.3
1.0 0.6
1.2 1.0
752.5 330.0A
554.5 250.0B

932.7 98.8b
79 21b
93 8
250.3 14b
240.3 11
5290 1769
4119 1170
636 201
486 119
14.2 3.3
10.6 2.4
1.9 1.6
1.3 0.9

Within an end point and row, means without a common superscript

(a,b) differed (P 0.05). Within an end point and column, means
without a common superscript (A,B) differed (P 0.05).
AF, absolute fecundity; AFRL, absolute fecundity on length; AFRW,
absolute fecundity relative to weight; DHO, degree hours of ovulation; DP, dark period; GSI, gonadosomatic index; HSI, hepatosomatic index; LP, light period; PPGV, peripheral position of the
germinal vesicle.
* Interaction (P 0.05) of hormone and period of hormone application.
Main effect (P 0.05) of period of hormone application.

water, which is known to affect spawning in various

species, through modulation of physiological processes and endocrine regulation [20].
The GSI is a measure of the percentage of gonads
relative to the total weight of the individuals and varies
according to the species, type of spawning, time of
year, environmental conditions, and management. It is
influenced by physiological capacity and environmental conditions [21]. In the present study, the largest GSI
(29.6%) as in the group given CPE at ML. This was
similar to the value of 20.6% reported by Sato et al.
[14] for this species. The lowest GSI (2.99%) occurred
in females given CPE in MD. This minimum value was
probably due to the lack of steroid synthesis, which is
the main factor causing increased deposition of vitellogenin in the oocyte and, consequently, determining
ovarian weight during reproductive development [22].
There was no difference (P 0.05) between the HSI
in females induced with CPE or gonadorelin (Table 1).
This index represents the percentage of the liver weight

1573

relative to the total weight of individuals. The HSI

indicates the reproductive period, during which it is
associated with other factors, e.g., the GSI [21]. The
liver synthesizes and secretes vitellogenin during gonadal maturation in females, and is necessary for
oocyte maturation [23].
The AF of lambari females ranged from 720 to 18
157 oocytes. Sato et al. [14] found a similar number of
AF in the same species (range, 11 086 31 720). The
AFRW was higher (P 0.05) in animals treated in ML,
regardless of the hormone used (ML: 752.5 330.0 vs.
MD: 554.5 250.0, P 0.05, Table 1), compared with
the group that were induced in MD. However, it is
notable that, whereas hormonal induction did not influence the quantity of oocytes, it affected maturation of
oocytes already produced.
Muniz et al. [5] observed that the timing of hormonal application influenced the ovulatory process in
tambaquis (Colossoma macropomum). These authors
suggested that the best results regarding ovulation were
achieved by giving the hormone between 0600 and
0800, with a second treatment at approximately 1800 to
2000. This timing promoted increasing blood concentrations of estradiol.
Sex steroids and other gonadal factors exert both
positive and negative feedback on the hypothalamus,
pituitary, and gonad itself [24], being responsible for
both the induction of oocyte maturation and the deposition of vitellogenin in the oocyte. This directly influences the weight of the ovary during reproductive development [22], and is responsible for the higher GSI in
animals induced in ML. There is probably an additive
effect of sex steroids and melatonin, because the latter,
which is synthesized at night, influences the release of
gonadotropin hormones, such as FSH and LH, which in
turn are responsible for gametogenesis and the maturation of gametes [25,26].
The apparent disparity between the observations
made by Muniz et al. [5] and the current study may be
due to the timing of induction, which in this study took
place in ML or MD, rather than at the beginning of the
day. Moreover, different species of fish may exhibit
specific responses to hormonal induction.
All lambari females undergoing hormonal induction with CPE or gonadorelin responded positively,
so the protocols used can be considered satisfactory
for artificial propagation of this species. However, it
was noteworthy that protocols in which CPE was
given at ML had an increased (P 0.05) percentage
of oocytes with PPGV, compared with those that
received gonadorelin.

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V.O. Felizardo et al. / Theriogenology 77 (2012) 1570 1574

A higher percentage of oocytes with PPGV was

recorded in fish induced in ML using CPE compared
with the group that received gonadorelin (PPGV: 95
6% vs. 79 21%, P 0.05) in the same period. Based
on PPGV values (Table 1), oocytes were ready to be
fertilized, which is the main indicator of oocyte viability [17].
In conclusion, both CPE and gonadorelin were effective in inducing female lambari reproduction, regardless of the timing of application. Lambari has a
crepuscular activity rhythm with increased peak activity earlier in the day. The behavioral rhythmicity displayed by tetra may influence the physiological response to hormonal induction required to trigger the
reproductive event.

Acknowledgments
Funding was provided by FAPEMIG, CAPES, and
CNPQ. Tortuga Zootcnica Land Co. provided the gonadorelin.

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