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Theriogenology 77 (2012) 1709 1716


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Effect of GnRHa, pimozide and Ovaprim on ovulation and plasma


sex steroid hormones in African catfish Clarias gariepinus
S.M. Sharaf*
Animal Production and Fish Resources Dept., Faculty of Agriculture, Suez Canal University, 41522 Ismailia, Egypt
Received 14 May 2011; received in revised form 11 November 2011; accepted 9 December 2011

Abstract
Nine groups each of four fish were injected with a single intramuscular dose of the following preparations: Physiological saline
(0.9% NaCl) as a control group, 0.5 ml kg1 Ovaprim, 20 and 40 g kg1 BW of GnRHa, 8 and 16 mL kg1 pimozide tablets
and the following combination of GnRHa with pimozide (GP): 20 g 4 mg, 30 g 8 mg and 40 g 16 mg kg1 BW.
The primary oocyte diameter (POD) before hormone administration ranged from 943.3 to 1071.0 m. The latency periods (LP)
were in the range of 9.0 to 12.0 h after injection. The highest ovulation ratio (OR) was observed in groups Ovaprim, GP(30
8) and GP(40 16). Other treatments were effective for ovulation, the ovulation ratio in Groups G(40) and GP(20 4) were
significantly higher than G(20) treatment. The ovulation index (OI) was in the range 62 to 77% and showed significant differences
among groups. There was no significant difference in fertilization ratio (FR) among Ovaprim, GP(30 8) and GP(40 16)
groups, while there were significant difference between the previous group and G(20) and G(40) groups. Control, P8, P16 showed
negative results in all the parameters LP, OED, OR, OI and FR. Levels of sex steroids were analyzed on 6 and 12 h after initiation
of treatments. A significant increase in plasma E2 with GP(30 8) injection was observed 6 and 12 h after injection, while there
were no significant increase between all the other groups 6 h after injection. Treatments with GP(20 4) resulted in a significant
increase in plasma T concentration in females compared with control after 6 h. In contrast, plasma T and E2 concentrations were
lower during the combined GP(20 4), GP(30 8) and GP(40 16) after 12 h than after 16 h of injection. The combined
treatments (GnRHa PIM) are better compared with Ovaprim which gave the same results, they have some advantages, such as
reliable response and low cost. Ovaprim is more than 3 to 5-fold of the cost of (GnRH PIM). Therefore, this method could be
useful tool for commercial catfish breeders to ensure spawning success.
2012 Elsevier Inc. All rights reserved.
Keywords: Clarias gariepinus; GnRHa; Pimozide; Ovaprim; Sex steroids; Ovulation

1. Introduction
In nature, African catfish, Clarias gariepinus, has a
discontinuous annual reproductive cycle with alternate
periods of resting, pre-spawning and breeding, regulated by cyclically active gonadotrophes [1]. The breeding season correlates with periods of maximal rainfall
* Corresponding author. Tel.: 201224173750.
E-mail address: safaa_sharaf@agr.suez.edu.eg (S.M. Sharaf).
0093-691X/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.12.019

and a pre-spawning LH surge takes place at least once


during this period [2]. Spawning occurs usually during
the scotophase, after rain in recently inundated marginal areas.
In captivity, catfish are kept under constant environmental conditions. Throughout the year, their pituitaries contain large and densely granulated gonadotrophes, storing large amounts of LH [3].
Under laboratory and fish-farming conditions, the
natural cues are difficult to mimic. Over the last few

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S.M. Sharaf / Theriogenology 77 (2012) 1709 1716

decades, hormonal manipulations to induce final oocyte


maturation and spawning have made possible the control of reproduction in cultured fishes and have contributed significantly to the expansion and diversification
of the aquaculture industry [4].
Although Clarias gariepinus complete vitellogenesis within the first year of captivity, final maturation
does not occur unless gravid females are induced to
spawn by hormonal manipulation [5]. The introduction
of GnRH analogues has proven to be efficient in inducing maturation and spawning in many fish species [6
13]. Likewise, an antidopaminergic drug, pimozide, has
also been found to be highly effective for stimulating
the spawning process of fishes mainly in cyprinids and
catfishes [14,15]. These methods stimulate secretion of
endogenous gonadotropin (Gth) [4,16].
Many fish exhibit this dopaminergic inhibitory action and, therefore, require the addition of a DA antagonist to facilitate the release of LH in response to
GnRHa [17,18]. The GnRHa and domperidone are the
most popular compounds for induction of ovulation and
spermiation in various fish species [18 21].
The addition of a dopamine receptor antagonist
(DA) to potentiate the response to GnRHa depends on
the presence of a dopaminergic inhibitory tone in the
target species [16,17,22]. Induction of spawning in fish
using GnRHa together with DA, such as metoclopramide, domperidone (DOM) and pimozide, is known as
the Linpe method [17]. The success of using GnRHa
alone or in combination with DA has been described in
several species, such as common carp (Cyprinus carpio) [2326], catfish (Heteropneustes fossilis) [27],
Indian major carps, such as rohu (Labeo rohita)
and mrigal (Cirrhinus mrigala) [28], nase (Chondrostoma nasus) [29] (Szabo, et al., 2002), pearl mullet
(Chalcalburnus tarichi) [30,31], rainbow trout (Oncorhynchus mykiss) [14], lake trout (Salvelinus namaycush) [32] and sockeye salmon (Oncorhynchus nekra)
[9]. The form of GnRHa, the type of DA, the species of
fish and environmental factors may affect the ovulatory
response [4]. For these reasons it is necessary to examine the response in each species under local conditions.
2. Materials and methods
2.1. Broodstock selection and maintenance
Experiments were conducted at the Animal and Fish
Production Department, Suez Canal University, Ismailia, Egypt. Adult female catfish Clarias gariepinus
(selected by certain external morphologic characteristics) were captured from fish farms [14]. Ovarian biop-

Table 1
Substances and doses applied to stimulate ovulation in Clarias
gariepinus females (n 4).
Treatment groups
Control
Ovaprim
G(20)
G(40)
P8
P16
GP(20 4)
GP(30 8)
GP(40 16)

Substance

Dosage

Saline (0.9%NaCl)
Ovaprim
GnRHa
GnRHa
PIM
PIM
GnRH PIM
GnRH PIM
GnRH PIM

20 kg1
40 kg1
8 mg
16 mg
20 kg1 4 mg
30 kg1 8 mg
40 kg1 16 mg

sies were taken, only fish having more than 60% of the
oocytes with a migrating germinal vesicle were selected
for ovulation experiment. Thirty-six female fish weighing 500 to 1000-g body weight (BW) were selected.
Before injection fish were individually weighed and
assigned to nine groups. No food was provided during
the experiment.
2.2. Hormones and experimental design
Nine groups of four fish each were injected a single
intramuscular dose into the dorsal muscle above lateral
line with different preparations as follows: Physiological saline (0.9% NaCl) was injected as control group,
Ovaprim (Syndel Laboratory, Limited, Canada) 0.5 ml
kg1 which contains the synthetic GnRH analog and
domperidone, 20 and 40 g kg1 BW of GnRH {42 g
kg1 buserelin acetate (Pyr-HisTrpSerTyr-D-Ser(But)-LeuArgPro ethylamide) and 10 mg benzyl alcohol), synthetic, Receptal, Intervet, Germany}, 8, 16
ml kg1 pimozide tablets (Orap Forte, Cilag, Belgium)
were powdered and then dissolved in physiological
saline and dimethyl sulfoxide, respectively [33] and the
following combination of GnRH with pimozide: 20 g 4
mg, 30 g 8 mg and 40 g 16 mg kg1 BW
(Table 1).
After injection fish were placed in well-aerated tanks
with recirculated water and temperature of 28 1 C.
The first examination for ovulation was carried out 6 h
after injection and repeated every hour. So when ovulation was observed, the eggs were stripped manually
after the latency period (mean time between injection
and ovulation [23]) and weighed from each female
separately, the weight being expressed as percentage of
female BW. The number of eggs released was calculated following the gravimetric method [34]. Assessment of ovulation was carried out by determining the
ovulation success (number of ovulated females/number
of injected) 100% and by ovulation index (OI) weight

S.M. Sharaf / Theriogenology 77 (2012) 1709 1716

of stripped egg mass/(weight of stripped egg mass


remnant ovaries) 100% [29]. The OI can be a rapid
and suitable index for ovulation estimation, but the fish
must be sacrificed after ovulation for calculating the
remnant ovaries.
Ovulation occurred within 9 to 12 h after injection.
Eggs were manually stripped from females and collected in a dry plastic plate. Egg quality was estimated
morphologically through a dissecting microscope, on a
1.0-mL aliquot and was defined as viable when they
were perfectly spherical, translucent and lacked vittelline space [35]. Batches of approximately 700 viable
eggs (0.5 g) were transferred to Petri dish to be fertilized.
Artificial insemination was carried out with mixed
milt obtained from the macerated testes of several
killed mature males. The spermiation was stimulated
with 0.5 ml kg1 ovaprim, and were fertilized in a Petri
dish as reported by [36] the fertilized eggs were then
spread into 3-liter plastic bowl containing 2 L of clean
water for incubation.
Fertilization success was determined under a dissecting microscope 24 h after fertilization, when eggs
were at the stage of gastrulation [37]. Dull, unfertilized
dead eggs were separated from transparent, living ones.
2.3. Steroid analysis
Fifty-four female fish (3 for each treatment were
sampled 6 and 12 h after injection with saline (control
group), ovaprim, 20 and 40 g kg1 BW of GnRHa, 8
and 16 mL kg1 PIM and finally GnRHa with pimozide
groups: 20 g 4 mg, 30 g 8 mg and 40 g 16
g kg1 BW) for steroid analysis. After being anesthetized in a solution of 100 ppm MS222 (Sigma), samples
were taken from the heart by using heparinized disposable syringes and the plasma was separated by centrifugation at 3000 rpm for 10 min at 4C and stored at
20 C until further steroid analysis. Plasma levels of
estradiol-17 (E2) and testosterone (T) were measured
by radioimmunoassay (RIA) using methods described
by [38,39] for determining T and E2, respectively. The
inter- and intraassay variation were 3.2 and 5.4% for E2
and 3.5 and 4.8% for T.
2.4. Statistical analysis
Body weight, oocyte diameter, OI, fertilization and
hatching rate, E2 and T concentrations are presented as
mean standard error. The obtained data were subjected to one-way ANOVA. Differences between means
were tested at the 5% probability level using Duncans
test [40]. All the statistical analyses were done using

1711

SPSS program version 10 (SPSS, Richmond, USA) as


described by [41].
3. Results
The oocyte diameter before hormone administration
showed no significant difference between fish (ANOVA
0.05) and ranged from 943.3 to 1071.0 m.
The latency periods were in the range of 9.0 to
12.0 h after injection. Fish in Ovaprim group showed
the shortest (9.9 0.2) latency period. While the longest period (11.6 0.2 h) was observed in Group
G(20). The latency period was (10.5 0.4 h) in the
group GnRHa with pimozide (GP)(30 8) which was
not significantly different compared with GP(20 4)
and GP(40 16) groups (P 0.05).
No fish ovulated in the control group after injecting
physiological saline. The lowest ovulation ratio (20%)
in the hormone treated groups was observed in Group
G20. The highest ovulation ratio (100%) was observed
in Ovaprim group, GP(30 8) and GP(40 16). Other
treatments were effective for ovulation, the ovulation
ratio in Group G(40) and GP(20 4) were significantly
higher than G [20] treatment and there was a significant
difference between the two groups (Table 2).
At the time of ovulation, the largest egg diameter
was (1239 115.9 m) in fish that were treated with
GP(40 16) which was significantly higher than
Group G(20) (P 0.05) but not significantly different
compared with Ovaprim group, G [40], GP(40 4) and
GP(30 8).
The ovulation index was in the range 62 to 77% and
showed significantly differences among groups (Table
2). The lowest OI were in G(20), G(40) and GP(20
4) groups, while the other groups showed higher OI
values (P 0.05).
Fertilization ratio in treated fish was in the range of
66 to 84% (Table 2). There was no significant difference in fertilization success among Ovaprim, GP(30
8) and GP(40 16) groups (P 0.05), while there
were significant difference between the previous group
and G(20) and G(40) groups.
Treatments of control, P8, P16 showed negative
results in all the parameters latency periods (LP), OED,
OR, OI and fertilization ratio (FR).
Levels of sex steroids; Figs. 1 and 2 show the plasma
estradiol and testosterone concentrations measured 6
and 12 h after injection. In control, the evolution of
plasma steroid levels over time showed that in females
both E2 and T were maintained stable from 6 to 12 h. A
significant increase in plasma E2 with GP(30 8)

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S.M. Sharaf / Theriogenology 77 (2012) 1709 1716

Table 2
The effect of different treatments on latency period (h), ovulatory egg diameter (mm), ovulation ratio (%), ovulation index (%), Fertilization
success (%) and hormone cost of Clarias gariepinus.
Treatment
groups

Body weight (g)

Primary oocyte
diameter (m)

Latency
period (h)

Ovulated egg
diameter (m)

Ovulation
ratio%

Ovulation
index%

Fertilization
ratio%

Cost a (LE)

Control
Ovaprim
G[20]
G[40]
P8
P16
GP(20 4)
GP(30 8)
GP(40 16)

836.93 147.08
929.33 64.0
727.63 49.01
780.47 87.17
708.93 63.62
677.10 73.04
710.40 90.21
895.77 72.16
682.13 54.04

986.67 24.04
1033.3 44.1
1071.0 72.3
1026.7 89.7
966.7 60.1
973.3 99.6
943.3 64.4
1010.0 51.3
983.3 44.1

0
9.9c 0.23
11.6a 0.2
11.5a 0.24
0
0
11ab 0.36
10.5bc 0.37
11ab 0.41

0
1140.9ab 64.3
1050.5b 28.8
1190.5ab 21.9
0
0
1154.0ab 55.0
1154.2ab 39.1
1239.9a 115.9

0
100a
20d
60c
0
0
90b
98a
100a

0
73.1a 1.6
62.1b 1.2
66.8b 1.8
0
0
62.2b 2.2
73.4a 2.6
77.1a 2.5

0
84.0 0.6
77.3 2.8
79.3 1.2
0
0
81.0 1.5
81.5 0.6
83 1.7

0
30.0
2.5
5.0
2.0
4.0
3.5
5.75
9.0

The same superscript in the same column is not significantly different at P 0.05.
a
$1.0 LE 6.0.

4. Discussion
The present study showed the effect of administration of synthetic hypothalamus hormones combined
with the antidopaminergic antagonist PIM on spawning
in C. gariepinus. The failure of fish to ovulate after
treatment with GnRHa or PIM and the control vehicles
may suggest that plasma gonadotropin (Gth) levels of
those fish had remained low. A surge in Gth can initiate
final events of oocyte maturation and ovulation in C.
macrocephalus, as in C. gariepinus [42]. In other fish,
administration of LHRHa or PIM resulted in higher Gth
levels than the controls, but the ability to cause oocyte
maturation and ovulation depended on the dose, species
[14,17,19] and the maturational stage of the oocytes
14

C
va
O

on
tr
ol

d
C

cd

cd
C

)
P(
40
+1
6)

P(
30
+8
G

P(
20
+4

P(
16
)

P(
8)

G
(4
0)

G
(2
0)

va

B
cd

)
P(
40
+1
6)

bcd

cd

P(
30
+8

abcd

AB

abc

bc
bcd
A

P(
20
+4

bcd

P(
16
)

cd

abcd
B

AB

ab

P(
8)

AB

AB

G
(4
0)

AB a

G
(2
0)

AB

12 h

10
T (ng/L)

12 h

6h

12

6h

10
9
8
7
6
5
4
3
2
1
0

on
tr
ol

E2 (ng/L)

injection was observed 6 and 12 h after injection


(Fig.1), while there were no significant increase between all the other groups 6 h after injection. Plasma E2
concentration in all treated groups after injection were
higher than those in control group. Although, plasma T
level varied with plasma E2 concentration, the plasma
level of T was lower than that of measured for plasma
E2 during the experiment. Treatments with GP(20 4)
resulted in a significant increase in plasma T concentration in females compared with control after 6 h in
contrast, plasma T and E2 concentrations were lower
during the combined GP(20 4), GP(30 8) and
GP(40 16) after 12 h than after 6 h of injection.

Treatments

Fig. 1. Plasma levels of estradiole (E2) in female Clarias gariepinus


breeders, 6 h. (closed column, capital letters) and 12 h (open column,
lowercase letters) after treatment with saline (control), ovaprim,
GnRH(20), GnRH(40), PIM(8), PIM(16) and the combined
GnRHaPIM (204), (308) and (4016). Vertical bars represent
standard error of triplicate measurements. Different letters indicate
significant differences (P 0.05) among treatments. Data are expressed as mean SEM.

Treatments

Fig. 2. Plasma levels of estradiole (T) in female Clarias gariepinus


breeders, 6 h (closed column, lowercase letters) and 12 h (open
column, capital letters) after treatment with saline (control), ovaprim,
GnRH(20), GnRH(40), PIM(8), PIM(16) and the combined
GnRHaPIM (204), (308) and (4016). Vertical bars represent
standard error of triplicate measurements. Different letters indicate
significant differences (P 0.05) among treatments. Data are expressed as mean SEM.

S.M. Sharaf / Theriogenology 77 (2012) 1709 1716

[24]. The present results confirm the current view that


PIM antagonizes the action of DA on GTH cells and
thus facilitates the stimulation of Gth release by
LHRHa [43 46].
However, fish respond differently, when induced
with GnRH or their analogue(s), in terms of elevation
in the gonadotropin levels [31,47], steroid levels
[48,49] ovulation [50 52] latency period [46,53,54]
and spawning [55,56]. The variation in response can be
attributed to the species of the fish, amino acid sequence and purity of the GnRH analogue [47,51], dose
of the GnRH and presence of weak or strong dopaminergic inhibitory mechanism [52]. The present results
showed that the oocyte diameters before hormone administration ranged from 943 to 1070 m in comparison, the oocyte diameters of several species of catfish
were C. gariepinus (700 1006 m) [57], C. batrachus
(1009 1590 m) [58] and C. macrocephalus (1490
1590 m) [15]. The observed Latency period for C.
gariepinus in the present work were shorter than that
for Heteropneustes fossilis [27], common carp [24,
26,59] and other cyprinids [60], probably because of
the high temperature (2729 C) during the spawning
season of C. gariepinus. However, at a similar temperature the latency period in carp spawned by carp pituitary extract was 9 h while that in GnRH MET
(metoclopramide) treated carp was 14 h [23]. Therefore, different latency periods can be related to species
specific [61]. In this study the shortest latency period
was observed in Ovaprim group and GP(30 8),
probably because the dose of the previous injections
were more appropriate, while the dose of G(20), G(40),
GP(20 4) and GP(40 16) were lower than optimal.
The egg diameters at ovulation ranged from 1050 to
1239 m in C. gariepinus this agreed with [62], while
that in C. batrachus, ranged from 1240 to 1290 m
[58]. In comparing the preinjection oocyte diameters
and the ovulatory egg diameter, it can be seen that
oocyte generally enlarge during final oocyte maturation
because of hydration.
Following fertilization, the eggs of most fishes absorb more water and the chorion (egg membrane) separate from the cortex which results in the appearance of
the previtelline space [63].
The groups with low OI values that received GnRH
alone was insufficient for massive ovulation in the
ovary of C. gariepinus and it was necessary to use the
combination of GnRH and PIM in appropriate dose as
in GP(30 8), GP(40 16) and Ovaprim. It also
indicates that the existence of dopamine inhibition on

1713

Gth release from the pituitary of C. gariepinus, these


results agree with [61].
As a result, it is proposed to combine GnRHa with a
dopamine antagonist, such as domperidone, pimozide
or metoclopramide for successful spawning induction
in cyrinid fish [4], C. macrocephalus [15,64], D. labrax
[65] and C. gariepinus [42]. Fertilization success did
not show any significant differences among groups.
Suggesting that inducing ovulation with Ovaprim, GnRHa alone or combined with PIM did not have any
adverse effect on egg viability. Similar results were
obtained in common carp [23,25,59], pearl mullet [30],
kutum [18] and loach [63]. Control and PIM treatments
were negative in LP, OED, OR, OI, and FR.
On another hand, no effects of PIM were seen in
females, indicating the absence or weak expression of a
DA inhibition in females. The results of the present
study demonstrate that treatment with GnRHa, alone or
in combination with PIM could increase the amount of
sex steroids and subsequent ovarian development in
Clarias gariepinus. Treatment with GnRHa presumably stimulates the secretion of gonadotropic hormones, which in turn may increase the sex steroids and
induce the development of ovarian lobules, this was
well established that GnRHa in Sparus aurata [66,67].
In the present study, treatment with GnRHa found to
promote sex steroid production, elevate plasma E2 and
T levels. These results agree with these previously
reported for Dicentrarchus labrax by [65] where the
treatment of GnRHa induced an increase of plasma sex
steroids. The sharp increase in plasma E2 and T could
occur because of a high aromatase activity in the ovary
at the moment of GnRH administration. However, present results are in contrast to the situation in salmonids
where the treatment of GnRHa induced a decrease of
plasma sex steroids [68]. Previous studies have shown
that GnRHa stimulates Gth secretion in the S. aurata
and that dopamine may not inhibit GnRHa-stimulated
Gth release [66]. This is consistent with the present
study where injection of GnRHaPIM induced greater
responses to plasma steroid levels, comparing fish
treated with GnRHa alone. GnRHa has also been
shown to act directly on ovarian follicles to modulate
vitellogenesis [69]. The PIM treatment alone had no
effect on basal FSH or LH transcript levels and the
cotreatment did not modify further the GnRHa effects,
neither in female nor in male Senegalese sole breeders.
Similar results have been previously found in other fish
species using both in vitro and in vivo treatments with
DA antagonists (tilapia: [70]; striped bass [71]: and red
sea bream: [72]. The results from the present study

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S.M. Sharaf / Theriogenology 77 (2012) 1709 1716

indicate that the endogenous DA inhibition would not


be the cause for the absence of spontaneous egg release
in cultured Clarias gariepinus breeders.
In conclusions, this study demonstrates that a combination of GnRHa and pimozide at GP(30 kg1 8
mg) and GP(40 kg1 16 mg) is effective for induced spawning within a predictable time period
(10 11 h) in C. gariepinus.
The combined treatments (GnRHaPIM) have a
preference compared with Ovaprim which gave the
same results, they have a reliable response and low
cost. Ovaprim is more than 3 to 5-fold of the cost of
(GnRH PIM). Therefore, this method could be useful
tool for commercial catfish breeders to ensure spawning
success.

[9]

[10]

[11]

[12]

[13]

Acknowledgments
The author acknowledges all the members in Animal
Production and Fish Resources Department and Fish
Research Center, Suez Canal University, Ismailia,
Egypt for their assistance in fish husbandry and sampling. The author also thanks the central laboratory for
aquaculture research Abbassa, Abo-Hammad, Sharkia,
Egypt.
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