Marine_life_14_.pdf

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: c.cordisco@icram.org 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 (Chelon labrosus, Mugilidae, Osteichthyes) ont été labrosus, 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: at
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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