La reproduction induite du muge à grosses lèvres
(Chelon labrosus, Mugilidae, Osteichthyes)
Donatella Crosetti, Cristina A. Cordisco 1
Central Institute for Marine Research (ICRAM),
1. Corresponding author: [email protected]
Crosetti D., C.A. Cordisco, 2004 – [La reproduction induite
Crosetti D., C.A. Cordisco, 2004 – Induced spawning
du muge à grosses lèvres (Chelon labrosus, Mugilidae,
of the thick-lipped mullet (Chelon labrosus, Mugilidae,
Osteichthyes)]. Mar. Life, 14 (1-2) : 37-43.
Osteichthyes). Mar. Life, 14 (1-2) : 37-43.
Des essais sur la reproduction induite du muge
Trials to induce spawning of thick-lipped mullet (Chelon
, Mugilidae, Osteichthyes) ont été
, Mugilidae, Osteichthyes) to obtain highly viable
effectués dans le but d’obtenir des alevins de bonne
seed were carried out in a hatchery of central Italy.
qualité. Les expériences se sont déroulées dans une
Females received one of five different inhibiting and
ferme d’élevage de poissons située en Italie centrale
stimulating hormone treatments during the natural
pendant la saison de reproduction de l’espèce (mars-
spawning season of March - April. The treatments
avril). Seules les femelles ont reçu les traitements
induced spawning completely or partially in 9 of the 11
hormonaux pour stimuler ou inhiber. Ceux-ci ont induit la
females selected. Five females spawned a total of 6810 g
reproduction, complète ou partielle, de 9 femelles
eggs with high viability (59 - 88% fertilisation rate) and
sur 11 traitées. Cinq femelles ont pondu au total 6810 g
subsequent hatching rate. Of the four most
d’œufs ayant une haute vitalité (59-88 % de fertilisation)
physiologically effective treatments which were all similar
avec un bon pourcentage d’éclosion. Des quatre
in results, the least invasive protocol was the combination
traitements physiologiquement plus efficaces, le
of a first injection of a homogenate of 7 carp pituitaries
protocole le moins invasif a été obtenu à partir de la
followed 24 h later by a second injection of 50 µg LHRH-a
combinaison suivante : 7 hypophyses de carpe/kg de
poids frais et 24 h plus tard de 50 µg LHRH-a/kg de poids.
Mugilidae, Chelon labrosus
, induced spawning,
Mugilidae, Chelon labrosus, reproduction induite,
The large family of mullets (Mugilidae) plays an
important role in aquaculture in Italy, and production
contributes around 3000 mt annually to the domestic
Experimental trials were carried out during the
market. Two species of mullet are particularly appre-
natural spawning season of thick mullet in central Italy
ciated by producers in Italy. These are the thick-lipped
(March-April) in the hatchery of a fish farm located on
mullet (Chelon labrosus), which is noted for the quality
the Orbetello Lagoon about 150 km North of Rome.
of its flesh, and the leaping grey mullet (Mugil
Breeders selected for our trials came from the farm’s
cephalus), which has some processed products with
broodstock of 65 C. labrosus adults which had been
high added value, such as dry roe and smoked fillets.
captured over a number of years and held in an outdoor
Much is raised in the extensive systems of the North
pond (400 m3); moreover mature wild individuals of the
Adriatic ‘valli,’ in ways which have been a tradition for
same species were caught at the lagoon barriers during
centuries. Such traditional culture of mullets depends
on the harvest of wild fry which school in estuaries and
A few days before the beginning of the experimental
coastal waters in large numbers and the need to obtain
trials, the captive broodstock were harvested from the
a steady supply of fry, independent of conditions in the
outdoor pond and transferred to two circular PVC tanks
natural environment, have been stressed for years in
(10 m3), each shaded by tarpaulin, where they were held
order to increase the mullet output. Artificial propaga-
until the treatment. Wild females selected for hormonal
tion has never reached a commercial scale, and culture
treatment were given the first dose the same day they
still remains dependent on the collection of wild fry.
had been captured. The trials lasted from March 24th to
Upon time various hormonal approaches have been
tested on different mullet species in many regions of
Each fish was identified individually on site by an
the world, with particular emphasis on the genus
electronic passive integrated transponder (PIT), injected
Mugil, and especially on the cosmopolitan species M.
into the muscle. All fish subjected to handling were first
cephalus (Crosetti, Cataudella, 1995; Kuo, 1995).
anaesthetised with 2-phenoxy-ethanol (0.3 mL.L-1), and
The first experiences on C. labrosus were carried out
about one third of this dose during any transportation
by Cassifour, Chambolle (1975), who injected female fish
with 20 mg /kg /d progesterone for 6 days, and subse-
Observations on the ovaries of wild females sampled
quently obtained eggs both by natural spawning and
during the natural spawning season showed that
manual stripping. Later Cataudella et al. (1988a, 1988b)
females below 950 g in weight were immature. This
induced three females to spawn after injection with
criterion was thereafter taken to be the basic minimum
5000 IU hCG, fertilized the eggs, and successfully mass-
for female selection. All males, on the other hand, were
propagated larvae for the first time.
selected as broodstock, indipendent of their weight.
More recently, the effects of a range of inhibiting and
Other diagnostic criteria for female ripeness were
stimulating substances were investigated, in different
more subjective, such as ‘fullness of belly’ or redness of
combinations and dosages, on 50 female thick-lipped
genital opening (Kuo et al., 1974). However, the most
mullet over a period of seasons (Crosetti, 1998; Crosetti
reliable criterion for ovarian maturity which could be
et al., 1998). Spawning was induced, at various levels
obtained in the field by observation alone, as identified
of success, both with homoplasic and heteroplasic
by Shehadeh et al. (1973), was an extrusion of oocytes
pituitaries (mullet and carp), hCG, LHRH-a, and dompe-
sampled with an intraovarian catheter.
The present study concentrates on further refinement
of the spawn-inducing experimental protocol on the
Three small circular PVC tanks (6 m3) were located in
thick lipped mullet in order to identify the optimal effect
a greenhouse, partially shaded with dark tarpaulins, in
use of hormones to obtain the mass production of high
preparation for injection and follow-up spawning.
Natural ambient conditions maintained both watertemperatures and diurnal light variations. Water tempe-ratures ranged from 12 - 18°C in the tanks containingthe experimental pools of fish, and subsequently from14.5 - 18°C in the spawning tanks containing the
Chelon labrosus, induced spawning
Chelon labrosus, reproduction induite
treated fish. On the days the females spawned, water
out immediately before the first and second injection,
temperatures ranged from 16.5 - 17°C, and salinity 30 -
and subsequently 48 h and 192 h after the first
injection. Sampling was not performed when females
A total of 12 females (5 from the wild pool and 7 from
appeared to be overly stressed, in particular if injured or
the captive pool, and identified #1 - #12) and 16 males
were then selected from the two experimental pools
A sub-sample of the extracted oocytes were placed in
and divided among the spawning tanks. In each tank
9‰ physiological solution and examined in vivo, with
the males always out-numbered the females by at least
the naked eye or low-magnification binocular, to
one. The weights of the selected females ranged from
determine their quality and wether females were ready
950 - 3000 g and 400 - 2000 g for the males.
for the hormone treatment. Another sub-sample was
Hormone treatments were only given to females,
fixed in 70% ethanol and saline solution for
whereas males were selected for running milt and
measurement, and another was fixed in Bouin’s liquid
therefore not treated. Each experimental treatment was
for 4 h, rinsed in 90% ethanol, and then stored in 90%
alcohol for later histology; to prepared the slides, the
samples were inclused in paraffin making sections of 6
– LHRH-a (luteinising hormone-releasing hormone
?m in thickness; the sections were stained in Mayer’s
analogue, synthetic peptide realising gonadotropin
The exact stage of development of the oocytes was
– Domperidone (synthetic dopaminergic blocking
determined in the field. About 10 eggs were mounted
on a slide and observed under the microscope.
These particular hormones, in a range of doses and
Application of gentle pressure on the cover glass and
combinations, had proven to be the most promising for
viewing the number and size of oil droplets in each
induced spawning of thick-lipped mullet by Crosetti
oocyte enabled identification of the maturation level.
Oocyte development stage were recorded according to
As shown on table I
each hormonal treatment was
Kuo et al. (1974) and later confirmed by histology. Only
given to females either in 2 or 3 injections 24 hours
females bearing oocytes at least at the secondary yolk
intervals apart. The treatments with 3 injections were
globule stage were selected for hormone treatment.
performed for comparison with the results of Yashouvet al. (1969), who injected leaping grey mullet (M.
cephalus) with 3 doses of carp pituitaries and LHRH-a.
Females spawned naturally in the tanks, and the eggs
As control, one female was given three injections of
were fertilized naturally by the males. Eggs were
physiological solution 9 ppt (2 mL each). Due to the
collected with a framed hand net and transferred to a
small number of mature females available, treatments
container (25 L) of high-saline water (45 ppt salinity)
and without aeration. Fertilised and buoyant eggs were
The commercial dry carp pituitaries were ground in a
collected from the water surface, weighed, and
pestle and mortar, and homogenized in saline solution
transferred to one or more incubation tanks. Here, the
9 ppt (2 mL). The homogenate was left to deposit for 15
fertilised eggs were observed regularly to follow early
minutes at room temperature before the supernatant
cell divisions, and later embryonic development.
was decanted for use. Domperidone (DOM), commer-cially available in 10-mg tablets under the trademark ofMotilium® (Janssen-Cilag, Cologno Monzese, Italy) was
prepared according to Fermin (1991). LHRH-a DAla6 wasa commercial preparation (Sigma-Aldrich, Milan, Italy),
diluted in saline solution in the laboratory and stored in
Histological observations are shown on table II
first injection, most females had oocytes at thesecondary yolk globule stage. At the time of the second
injection, 24 h later, the oocytes of some fish showed
For identification of the stage of development, or to
no change, whereas others had oocytes developed to
follow maturation after hormone treatment, oocytes
the tertiary yolk globule stage. Two females (#3 and #8)
were sampled with a polyethylene cannula (3 mm i.d.)
had oocytes at the tertiary yolk globule stage at first
inserted into the genital opening. Sampling was carried
injection, which developed to the migratory nucleus
Chelon labrosus, induced spawning
Chelon labrosus, reproduction induite
stage by the time of the second one. Both spawned
out in previous years with different hormonal substan-
ces, such as mullet and carp pituitary homogenates,
Oocytes sampled from females receiving a third
hCG, LHRH-a, and domperidone, gave poor results in
injection, two days after the first, showed further
spawning and production of highly viable eggs of
progressive maturation and these fish were not
mullets (Cataudella et al., 1988b; Crosetti, 1998).
disturbed again as they prepared to spawn. The oocytes
However, they provided considerable information on
of the control female (#7), which was a recently
the reproductive biology of C. labrosus, and some of
captured fish at the secondary yolk globule stage, did
the basic protocols for induced breeding of the species.
not change throughout the experiment (Table II).
Prior to the natural spawning season, vitellogenesis ofboth wild and captive female thick-lipped mullet
proceeds to the secondary or tertiary yolk globule
Spawning success and egg yields are given in table I
stage, but is then blocked until final maturation leading
Between 50 and 91 h after receiving their first
to ovulation is triggered by the environmental stimuli of
injections, 9 of the 11 females induced with various
seaward migration and spawning grounds, or the
treatments completed maturation and ovulated, either
artificial stimuli of hormone treatment. Consequently,
completely or partially. These 9 females yielded 7925 g
female mullets migrating from the lagoon, and
of eggs. Yields from individual females were not always
captured at a fish barrier, have attained the same level
accurately recorded as some fish spawned in the same
of maturity. Therefore, to reduce handling, oocyte
tank at about the same time. Therefore, for example,
sampling of these fish is optional, and selection criteria
eggs from females #1, #2 and #3, and females #10 and
can be confined to females which have soft and full
The 5 females (#1, #2, #3, #8, #12), which spawned
During the natural spawning season of thick-lipped
out completely, yielded 6810 g of eggs within 2 h, or
mullet, all wild females selected for the trials at the fish
86% of the total quantity of eggs obtained during in
barrier and weighing over 950 g were presumably ripe,
this study. Fertilisation rate ranged from 42 – 88%. The
as shown by previous observations on 50 females
other 4 females spawned only partially, and the eggs
(Crosetti, 1998). This weight can be considered the
were judged to be of poor quality as they had a low
lower limit at which females reach maturity, and can be
hatching rate. These fish never completely emptied
used as broodstock. On the other hand, the smallest
their ovaries, and at 192 h after the first injection still
bore degenerated eggs which could be expelled
Successful spawning was obtained in 9 out of 11
manually with slight pressure. One female, #9, spawn
females receiving the experimental treatments. This
out but the eggs proved to be not viable and were
high percentage was obtained with females receiving
hormone treatments of (i) 7 carp pituitaries +50, +100
Two (#4 and #6) of the 11 treated females, together
and +200 µg LHRH-a per kg body weight, and (ii)
with the control (#7), showed no sign of oocyte
domperidone 10 mg +100 µg LHRH-a per kg body
maturation or hydration, and did not spawn. When
weight, given in two injections within a 24 h interval.
sampled 192 h after their first injections, all had oocytes
Eggs with high viability, determined by fertilisation and
hatching rates, were obtained from only 5 large females,
Eggs taken from all sources measured 1.2 - 1.4 mm in
whereas small batches of poor quality eggs were
diameter and contained between 2 - 12 oil droplets.
obtained from females which spawned incompletely.
These minimum dosages for the thick-lipped mullet
are only slightly less than those required for the Mugil
cephalus. Lee et al. (1987, 1988) found the combinationof 20 mg carp pituitary homogenate (equivalent to
As a rule, the advantage to use the carp pituitary
approximately 7 pituitaries glands) and 200 µg LHRH-a
homogenates is that it contains, besides hCG, also
was the minimum dose required to induce spawning
others hormones with synergic effect on the
gametogenesis, so that it is much used in the induced
Benefits of applying hormone treatment in 3 injec-
spawning of fishes. Similary, combined treatment with
tions instead of 2 were not evident, and only subjected
LHRA-a and dopamine antagonist, domperidone,
the fish to additioned handling and stress, and possibly
induces ovulation and spawning. Several trials carried
unsuccessful spawning (Zohar, Mylonas, 2001).
There is an on-going debate on whether induced
oocytes with more than one oil globule produce themost viable larvae. Stand here the successful results
The dosages of hormones to induce spawning of
with thick-lipped mullet in this study, and also by
thick lipped mullet in captivity are typical for the
Cataudella et al. (1988b) indicate that eggs containing
family Mugilidae. Current practices with a variety of
multiple (2-13) oil droplets produce viable larvae.
hormones, such as carp pituitary homogenate,
Similarly, some other authors, which studied other
luteinizing hormone releasing hormone analogue, and
Mugilidae species, assert this opinion (Cassifour,
domperidone and are all physiologically effective to
Chambolle, 1975; Van der Horst, Lasiak, 1989). On the
some degree. The least intrusive protocol, in terms of
other hand, these results disagree with the statement
the quantity of hormone used and the number of
of Kuo et al. (1973) for M. cephalus, which suggested
injections, is the combination of (i) a first injection of a
multiple oil droplets to be a sign of premature
homogenate of 7 carp pituitaries followed 24 h later by
inducement by stripping manually, and Nash et al.
a second injection of 50 µg LHRH-a per kg female
(1974), which asserted that spontaneous release by the
bodyweight. Anyway all tested protocols seem to be
female produced eggs with a single oil droplet.
reliable for the production of highly viable eggs from
There was no clear difference in spawning success
carefully selected females, with the best chance of high
between wild broodstock females which had been in
captivity for a year or more, or wild females capturedshortly before induced breeding. Captive broodstockwere invariably fatter and easier to handle than wild
Our results confirmed that the thick-lipped mullet
The research was part of a project financed by the
spawns a large volume of eggs. Spawned eggs can
General Directorate for Fisheries and Aquaculture,
amount to 57 - 65% of the female’s body weight, and
Ministry of Agriculture, within the third Plan for
Fisheries and Aquaculture. The authors wish to
There are no records of mullets spawning sponta-
thank Prof. S. Cataudella, who provided ideas and
neously in captivity, and this continues to be confirmed
encouragement for the project, V. Brasola and technical
by the records from this paper. There have, however,
staff of the La Rosa Hatchery who provided the facilities
been reports of ‘spontaneous’ spawning in previous
trials. Crosetti (1998) recorded one female whichspawned 8 days after being injected with 5000 IU hCGas a third injection, following failure with two previoustreatments 13 days earlier. The oocytes of the female inquestion were as perfect at day 13 as at day 0. The longtime-gap between the last injection and spawningsuggests these events to be spontaneous and notinduced. It is thought that the hormone preventsoocytes from undergoing atresia, brought on bycaptivity and handling stress, thus enabling the femaleto mature slowly and eventually spawn. On the otherhand, the female controls start undergoing atresiabetween 72 - 120 h after the first injection. Cataudella etal. (1988b) reported a similar reaction in three Chelonfemales, which spawned 9, 11, and 14 days after a singledose of 5000 IU hCG.
C. labrosus, induced spawning
C. labrosus, reproduction induite
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UNIVERSIDAD NACIONAL DE CÓRDOBA FACULTAD DE CIENCIAS QUÍMICAS DEPARTAMENTO DE FARMACIA C I M E CENTRO DE INFORMACIÓN SOBRE MEDICAMENTOS Ciudad Universitaria (5000) Córdoba - Tel: 351-4334127/ 63 int 110 Fax: 351-4334127 - E-mail: [email protected] DATOS DEL SOLICITANTE: FICHA N º: F116-07 NOMBRE: AURELIO F. GARCÍA ELORRIO FECHA: 5/09/07 Hora: DI