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Animal Reproduction Science 119 (2010) 115122

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

nduced spawning and embryonic development of Liza ramada rearedn freshwater ponds

ostafa A. Mousa

ational Institute of Oceanography and Fisheries, Alexandria, Egypt

r t i c l e i n f o

rticle history:eceived 7 November 2009eceived in revised form5 December 2009ccepted 21 December 2009vailable online 6 January 2010

eywords:iza ramadapawning inductionggmbryonic development

a b s t r a c t

The possibility of inducing and synchronizing spawning can be very useful to facilitatefish farming, particularly in species that achieve ovarian development in captivity withoutovulation occuring. The present study was undertaken to observe the morphological andnormal embryonic development of thin-lipped mullet, Liza ramada, after spawning induc-tion of fish reared in freshwater fish farms. The use of pregnyl (HCG) as a priming injectionat a dose of 20,000 IU/kg body weight followed by a second injection of 40,000 IU HCG/kgbody weight 24 h later, proved to be effective in inducing final oocyte maturation, ovulationand spawning in L. ramada at 5260 h after hormonal injection. The mean number of theovulated eggs for each female was 700 80.3 eggs g1 body weight. The mean rates of buoy-ancy, fertilization and hatching were 46 7.1, 55 8.4 and 60 6.6, respectively. Fertilizedeggs were kept under normal environmental conditions in seawater at 2021 C. The firstcleavage occurred at 40 min, epiboly began at 5 h, the embryonic body was formed at 24 hand hatching occurred at 48 h after spawning. Newly hatched larvae were approximately

2.5 mm (total length) and similar to those of the other mullet species in terms of exter-nal features except no pigment spots were present over the yolk. The mouth and foregutopened on the 5th day after hatching; at which the total length of larvae was 3.5 mm; thehindgut and anus had developed prior to hatching.

The induced ovulation technique using acute injections of hormones is an important stepin the development of the mullet culture.

. Introduction

The thin-lipped grey mullet, Liza ramada (Risso) is anmportant and attractive species for farming in sea, brack-sh and fresh water. Its notable characteristics include aesh that is very tasty, and, therefore, achieves higher mar-et prices with respect to other fish species. In Egypt, grey

ullet fingerlings are reared successfully in freshwater-

olyculture ponds together with common carp: Cyprinusarpio, grass carp; Ctenopharyngodon idella, silver carp;ypophthalmichthys molitrix and Nile tilapia; Oreochromis

Tel.: +20 101082930; fax: +20 34801174.E-mail address: mostafa mousa2002@yahoo.com.

378-4320/$ see front matter 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.anireprosci.2009.12.014

2010 Elsevier B.V. All rights reserved.

niloticus. However, the culture of mullet is closely depen-dent on the availability of their fry. The present frycollection stations from nature cannot satisfy the increas-ing demand for the juveniles. Consequently, it is desirableto develop and establish practical techniques for artifi-cial propagation. Spawning induction of cultured fish byhormonal treatments is one of the ways to produce fryfor stocking ponds. A supply of mature female brood-stock is important, particularly for mass propagation ofjuveniles. However, mature mullet females cannot be col-

lected from the natural habitat, since they migrate to thesea for spawning (Mousa, 1994). Like females of manycommercially important fishes, thin-lipped grey mullet donot undergo final oocyte maturation (FOM), ovulation orspawning in captivity (Mousa, 1994; Mousa and Mousa,http://www.sciencedirect.com/science/journal/03784320http://www.elsevier.com/locate/anireproscimailto:mostafa_mousa2002@yahoo.comdx.doi.org/10.1016/j.anireprosci.2009.12.014

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116 M.A. Mousa / Animal Reprod

1997; Mousa and El-Gamal, 1999). Without exogenous hor-mone stimulation, ova will not advance to final maturationand ovulation, but will undergo atresia and degenerate.Consequently, it is necessary to induce maturation and ovu-lation. The use of pregnyl (human chorionic gonadotropin;HCG) has been effective in inducing final oocyte matura-tion and ovulation in thin-lipped grey mullet (Mousa, 1999;Mousa and Mousa, 2006).

Therefore, in light of the recent increasing interest inthe culture of mullets in Egypt, the aim of the present studywas to acclimate the captive broodstock thin-lipped mulletfrom freshwater to saline water and induce spawning. Also,a detailed description of developmental stages is needed,because the previous description by Yashouv and Berner-Samsonov (1970) is not sufficiently detailed, and does notdocument yolk sac absorption and first feeding.

2. Materials and methods

2.1. Broodstock management

The fish used in the present study were stocked inoutdoor 400-m2 earthen ponds of El-Serw Fish ResearchStation (in April 2008) with running fresh water at anambient temperature of 1535 C. Dissolved oxygen con-tent was above 5.0 mg O2 l1 and pH-value ranged from 7.5to 8.2. The light regime was that of natural photoperiod(1214 l h). Fish were fed floating pelleted diet containing32% crude protein, 5% lipid, 0.2% vitamins and 0.2% mineralswith daily rate of 3% of the body weight. Sexes were identi-fied based on spermiation for males and slightly distendedabdominal condition for females. Mature breeders of thin-lipped grey mullet, at least 2 years old, with total weightsranged from 250 to 550 g and total lengths ranged from27 to 35 cm, were collected alive, by draining water com-pletely, during the spawning season (NovemberJanuary)from culture ponds. There were 10 females and 30 malesselected from the captured fish used in the inductionspawning trials. All procedures used in the present studycomplied with local and or national animal welfare laws,guidelines and policies. Minimum sample sizes for theexperimental work were statistically determined and nomore fish than necessary used. Fish densities were keptwell below allowable levels. Furthermore, all fish wererestocked in fish farm after spawning.

2.2. Seawater acclimation and induction of spawning

The present experiments were carried out, during thenatural spawning season of thin-lipped grey mullet, atEl-Matareyya Research Station in December 2008. Thefish obtained from El-Serw Fish Research Station (fresh-water habitat) were anesthetized in a solution (40 mg/l)of clove oil (Sigma) before handling according to Mousa(2004). Mature females of thin-lipped grey mullet wereselected on the basis of the presence of a soft, swollen

abdomen and protruding genital papillae. The maturityand the oocyte diameters of the females were staged byobtaining in vivo biopsy of the ovary using a polyethylenecannula (Shehadeh et al., 1973). The females that wereused possessed oocytes whose diameters were greater

cience 119 (2010) 115122

than 600 m. Ripe males in which milt could be easilyextruded, by gentle pressure on their bellies, were used.Selected breeders were acclimated in 2000-l circular fiber-glass tanks (10 fish/tank). In brief, fish were transferredto water with 10 salinity (for 12 h) which graduallyincreased to 35 (for another 12 h). Acclimated breed-ers were transferred to 500-l fiberglass tanks equippedwith constant running ozonated seawater (35) and aer-ation (female + 3 males/tank) for induction of spawningwith human chorionic gonadotropin (HCG) pregnyl (NileCo. for Pharmaceuticals, Cairo, ARE). Water temperatureranged from 20 to 21 C. Pregnyl was injected into thedorsal musculature of fish adjacent to the dorsal fin as apriming injection at a dose of 20,000 IU/kg body weight fol-lowed 24 h later by a resolving injection of 40,000 IU HCGfor females. All males received a single injection of HCG ata dosage of 1000 IU/kg body weight at the time of femaleresolving injection. The used doses were calculated empir-ically depending on a series of preliminary experimentsdetermining the optimal dose.

Hormone treatments were evaluated using the fol-lowing criteria: (i) egg production, (ii) buoyancy, (iii)fertilization rate, and (iv) hatching rate as well as thepercentage of normal hatched larvae. The eggs were col-lected from each spawning tank (10 tanks) approximately2 h after the estimated time of spawning. Eggs were col-lected with PVC pipes and siphoned into a plastic circularegg-collection basket (10 l) with nylon bottom (mesh size200 m). The total eggs volume was estimated by cylin-der. Number of ovulated eggs produced by each femalewas estimated by eggs ml1 egg volume multiplying bytotal eggs volume, and then the number of eggs g1 bodyweight was observed for each female. Buoyancy, fertiliza-tion and hatching rates were determined for each female(three replicates of 500 eggs from each fish were randomlytaken and used to estimate fertilization rate and hatch rate).These percentages were calculated using the following for-mulae:

Buoyancy (%) = 100 no. of buoyant eggsno. of ovulated eggs

.

Fertilization (%) = 100 no. of fertilized eggsno. of buoyant eggs

.

Hatching (%) = 100 no. of hatched larvaeno. of fertilized eggs

.

Fertilization rates were determined under a dissectingmicroscope approximately 1 h after spawning, when eggswere at the cleavage stage.

2.3. Eggs incubation and embryonic development

Eggs incubation took place at 21 C, in natural photope-riod. At the beginning of the experiment 30 of fertilizedeggs were randomly taken from each female and used to

measure the mean egg diameter. Buoyant viable fertilizedeggs, from the different females, were stocked in nylonincubators with a volume of 10 l (mesh size, 200 m) as500 eggs l1. The eggs incubators were placed on watersurface in 500-l circular fiberglass tanks (spawning tanks).

M.A. Mousa / Animal Reproduction Science 119 (2010) 115122 117

Table 1Body weight, egg production, buoyancy, fertilization rate, hatching rate (three replicates of 500 eggs from each fish were randomly taken and used toestimate fertilization rate and hatch rate) and normal hatched larvae, during the induction spawning of the thin-lipped mullet Liza ramada, using thepregnyl (HCG) as a priming injection at a dose of 20,000 IU/kg body weight followed, 24 h later, by a resolving injection of 40,000 IU HCG/kg body weight.

Spawnedfemale number

Bodyweight (g)

Number ofovulated eggs(g1 fish)

Buoyancya

(%)Fertilizationrateb (%)

Hatchingratec (%)

Normalhatchedlarvae (%)

1 295 600 50 45 55 952 294 725 57 40 50 903 336 650 51 61 70 944 550 750 47 59 60 955 392 675 40 55 58 966 350 700 48 50 60 957 540 600 45 57 57 928 382 740 40 58 65 969 250 760 50 55 55 9710 320 850 32 75 70 100

Mean 370 101.3 700 80.3 46 7.1 55 8.4 60 6.6 95 2.7(250550) (600850) (3257) (4075) (5070) (90100)

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ll mean values are presented S.D. Values in brackets represent the rana Buoyancy rate (%) = 100 no. of buoyant eggs/no. of ovulated eggs.b Fertilization rate (%) = 100 no. of fertilized eggs/no. of buoyant eggs.c Hatching rate (%) = 100 no. of hatched larvae/no. of fertilized eggs.

uring the experiment, the flow rate of seawater into theanks was 25% from tank water per hour. Before the use,eawater was treated by ozone, UV light and filtering.oth penicillium (10 IU/ml) and streptomycin (0.01 mg/ml)ere added once every day to ensure the reduction of

acterial growth based on previous studies (Nash andhehadeh, 1980). The flow of water was stopped for 1 h dur-ng the addition of antibiotics to ensure their effectivenessn the bacteria may be found on eggs. In order to deter-ine the common embryonic developments, 30 eggs were

aken from each incubator in every 30 min until morulatage and then hourly intervals. Whenever there was anvident difference during the embryonic development, thehotographs were taken before or after the due time.

. Results

.1. Induced spawning

In freshwater ponds, thin-lipped grey mullet femalesttained to prespawning stage, whereas the males attainedo ripe stage. Prespawning females contained vitellogenicocytes varying in diameter from 600 to 650 m. Vitel-ogenic (tertiary yolk) oocytes had a centrally locatederminal vesicle (GV). The breeders were successfully accli-ated to seawater (35) prior to hormonal injection. High

ate (100%) of final oocyte maturation (FOM), ovulation andpawning was achieved in thin-lipped grey mullet femalestilizing the pregnyl (HCG) as a priming injection at aose of 20,000 IU/kg body weight followed, 24 h later, byresolving injection of 40,000 IU HCG/kg body weight. All

njected females (10) were spawned at a time of 5260 hfter the priming injection. The number of the ovulatedggs, rate of buoyancy, fertilization, and hatching for the

ifferent females are shown in Table 1. The number ofhe ovulated eggs ranged between 600 and 850 eggs g1

ody weight with a mean value of 700 80.3. The rates ofhe buoyancy, fertilization and hatching ranged between257%, 4075% and 5070% with mean values of 46 7.1,

mum and maximum) of data.

55 8.4 and 60 6.6, respectively. High percentages of nor-mal hatched larvae were obtained as 90100, with a meanvalue of 95 2.7.

3.2. Embryonic development

The developmental stages of the embryos are shown inTable 2 and Figs. 1 and 2. The results are organized as astaging guide. The present method for naming the stages;used anatomical features to give each stage a name.

3.2.1. Fertilized eggThe eggs were pelagic and transparent. The ovulated

eggs varied in diameter between 0.9 and 1.0 mm. Afterfertilization, several changes occurred in the appearanceof the egg. The perivitelline space formed 40 min afterspawning. The fertilized eggs had between 5 and 8 oil glob-ules but they subsequently developed and hatched. Duringdevelopment it was observed that the droplets merged. Onhatching the larvae had one oil globule (rarely two) locatedin the yolk sac. Between 5 and 8 oil droplets gathered in theupper space of the egg. The blastodisc was situated on thebottom side of the floating egg (Fig. 1A).

3.2.2. CleavageFirst cleavage begins at approximately 40 min and the

next six cleavage cycles occur at regular intervals ofapproximately 35 min. It is characteristic of these earlycleavages that they occur synchronously, all cells cleavingat almost the same time (Fig. 1BH). First cleavage occurredat the animal pole 40 min after spawning. The egg showed atypical discoidal cleavage and two blastomeres of equal sizewere formed (Fig. 1B). The egg attained the 128-cell stage4:10 h after spawning (Fig. 1H). The oil droplets gradually

began to fuse to form larger droplets.

3.2.3. MorulaAfter 128-cell stage (4:10 h) it was difficult to observe

the other symmetrical divisions, but they continued to

118 M.A. Mousa / Animal Reproduction Science 119 (2010) 115122

Table 2Summary of development of the thin-lipped mullet L. ramada under normal environmental conditions in seawater at2021 C.

Stage of development Time after spawning Description

Fertilized egg 0 Single cell at animal pole.2-cell stage 40 min First cleavage.4-cell stage 1:15 h Second cleavage.8-cell stage 1:50 h Third cleavage.16-cell stage 2:25 h Fourth cleavage.32-cell stage 3:00 h Fifth cleavage.64-cell stage 3:35 h Sixth cleavage.128-cell stage 4:10 h Seventh cleavage.Early morula 5:00 h The blastodisc consisted of many blastomeres

and the oil droplets decreased in number to410.

Mid morula 5:30 hLate morula 6:00 hEarly blastula 7:00 h Cell division becomes less synchronous and it

becomes difficult to count cells.Mid blastula 10:00 hLate blastula 12:00 h Blastocoel formation.Gastrula 19:00 h Embryonic area well marked.24-h embryo stage 24:00 h Optic vesicles appeared.26-h embryo stage 26:00 h The brain vesicles can be identified as well as the

eye rudiment.31-h embryo stage 31:00 h The appearance of first pigmentation.32-h embryo stage 32:00 h Optic vesicles well developed.36-h embryo stage 36:00 h Heart formed.44-h embryo stage 44:00 h Lens fully formed; pigmentation gets dark.Hatching 48:00 h Hatching completed; 50 h.Pro-larvae 3:00 h after hatching The body remained curved for several hours after

hatching.1-day-old larvae 1 days after hatching Its body straightened; gut was straight and

pigmentation was more pronounced throughoutthe head and body.

3-day-old larvae 3 days after hatching Its mouth was under development and the eyebecomes pigmented.

5-day-old larvae 5 days after hatching Opening of mouth. The differentiation of theanterior alimentary canal is nearly complete.

A total of 30 eggs were examined.

divide. During the incubation, morula stage was observedat 5:00 h after spawning (Fig. 1I). The blastodisc consisted ofmany blastomeres and the oil droplets decreased in num-ber to 410 (Fig. 1I and K).

3.2.4. BlastulaThe blastula describes the period when the blastodisc

becomes multilayered. During this period, cell divisionbecomes less synchronous and it becomes difficult to countcells (Fig. 1LN). A blastocoel was formed inside the blas-todisc 12:00 h after spawning (Fig. 1N). The oil droplets hadfused to a single droplet.

3.2.5. GastrulaDuring gastrula stage 19:00 h after spawning, the

blastoderm shows true expansion as the sheet of cells pro-liferate and spread toward the vegetal pole (Fig. 1O). Theblastoderm covered one-quarter of the egg and a thick-ened part of the blastoderm became an embryonic shield(Fig. 1O).

3.2.6. Embryonic body formationAn embryonic body was formed from the embryonic

shield 24:00 h after spawning (Fig. 2A). Six to eight somiteswere discernible. 26 h after the spawning, the brain vesi-cles and the eye rudiment could be identified; in addition,

810 somites were observed (Fig. 2B). The appearance offirst pigmentation was determined at 31 h after spawn-ing (Fig. 2C). The pigment cells appeared below the oilglobule and along the embryonic body; no pigment spotspresent over the yolk. 32 h later, the embryo was raisedabove the yolk surface; optic vesicles were developed andthe tail is separated from the yolk (Fig. 2D). Then, the for-mation of optic lens was observed at 36 h (Fig. 2E and F).During this stage, the heart formed; blood circulation com-menced, embryos showed slight movements; the numberof somites increased to 20 and pigment cells appearedalong the embryonic body (Fig. 2E and F). 44 h after thespawning, the lens fully formed; pigmentation got dark andvarious colour combinations were noticed (Fig. 2G and H).Also, otic capsules were observed (Fig. 2FH).

3.2.7. Hatching (newly hatched larvae)Forty-four hours after spawning, the body reached

three-quarters of the circumference of the egg as the tailelongated (Fig. 2G). Hatching occurred at 48 h after spawn-ing (Fig. 2I), at a water temperature ranging from 20 to

21 C. The body of a newly hatched larva remained curvedfor several hours after hatching (Fig. 2J and K). The larva hada large oval-shaped yolk sac with an oil droplet at its pos-terior end. The anus is formed, but the mouth and foregutare not. No pigmentation was recognized over the yolk.

M.A. Mousa / Animal Reproduction Science 119 (2010) 115122 119

Fig. 1. Cleavage and embryonic development of Liza ramada. (A) Unsegmented fertilized egg with animal pole marked off. (B) Two-cell stage (first cleavage)40 min after spawning. (C) Four-cell stage (second cleavage) surface view, 1.15 min after spawning. (D) Eight-cell stage (third cleavage), 1.50 h afterspawning. (E) 16-cell stage (fourth cleavage), 2:25 h after spawning. (F) 32-cell stage (fifth cleavage), 3:00 h, (G) 64-cell stage, 3:35 h, (H) 128-cell stage,4.10 h. (IK) Multicell morula stages: (I) early morula stage 5:00 h, (J) mid morula stage 5:30 h and (K) late morula stage, 6:00 h. (LN) Blastula stages (topv stula staa

3

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iews): (L) early blastula 7:00 h, (M) mid blastula 10:00 h and (N) late blarea well marked and embryonic streak visible. Scale bar = 1 mm.

.3. Early larva

.3.1. Larva 24 h after hatchingLarvae, 24 h after hatching had an average total length

f 3 mm (n = 30) and 25 somites. Its body straightened;

ge 12:00 h (blastocoel formation). (O) Gastrula stage 19:00 h; embryonic

gut was straight and pigmentation was more pronouncedthroughout the head and body. A membranous fin was vis-ible on the posterior body (Fig. 2L). The newly hatchedlarvae remained suspended in the water and quivered occa-sionally.

120 M.A. Mousa / Animal Reproduction Science 119 (2010) 115122

Fig. 2. Embryonic and early larval development of L. ramada. (A) 24-h embryo stage; embryonic rudiment became raised and distinct; optic vesiclesappeared; six or eight somites marked off. (B) 26-h embryo stage; the brain vesicles can be identified as well as the eye rudiment; the number of myotomesincreased to 8 to 9. (C) 31-h embryo stage; the appearance of first pigmentation was determined. (D) 32-h embryo stage; optic vesicles well developed;the tail is rising from the yolk. (E and F) 36-h embryo stage (side view); heart formed; the number of somites increased to 20; pigment cells appearedalong the embryonic body; otic capsule was observed. (G and H) 44-h embryo stage; lens fully formed; pigmentation gets dark. (I) Hatching started; 48 h.

e, 24 hy. (M) 3r and lo

(J) Hatching completed; 50 h. (K) Pro-larvae, 3 h after hatching. (L) Larvaanus; pigmentation was more pronounced throughout the head and bod5-day-old larvae; 3.5 mm; opening of mouth; good development of uppe(H) = 0.25 mm and scale bar of (LN) = 1 mm.

3.3.2. Three-day-old larvaThe average TL of 3-day-old larvae was 3.5 mm (n = 30);

the yolk sac was reduced in size and its mouth wasunder development and is invaginated but not yet opens.

after hatching; 3 mm; 25 somites; uncoiled hind gut between yolk and-day-old larvae; 3.5 mm; the mouth is invaginated but not yet open. (N)wer jaws. Scale bar of (AE), (G) and (IK) = 0.5 mm; scale bar of (F) and

Although, the yolk sac was not completely absorbed, thedifferent parts of the alimentary canal (esophagus, stom-ach and intestine) began to differentiate. The eye becamepigmented (Fig. 2M).

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M.A. Mousa / Animal Reprod

.3.3. Five-day-old larvaThe average TL was 3.5 mm (n = 30). The mouth was

pened and good development of upper and lower jaws,ble to take food (Fig. 2N). Yolk was diminished. Oil glob-le was also reduced. The differentiation of the alimentaryanal was nearly complete. The pigmentation extends fromhe head along the trunk, except for the final section of theail (Fig. 2N).

. Discussion

In the present study, high rate (100%) of final oocyteaturation (FOM), ovulation and spawning was achieved

n thin-lipped grey mullet females reared in freshwatersh farms utilizing the pregnyl (HCG) as a priming injec-ion at a dose of 20,000 IU/kg body weight followed, and4 h later, as a resolving injection of 40,000 IU HCG/kgody weight. All injected females (10) were spawned260 h after the priming injection. In our previous stud-

es (Mousa, 1999), we also successfully used pregnyl (HCG)s a priming injection at a dose of 15,000 IU/kg bodyeight followed 24 h later by a resolving injection of

0,000 IU HCG in combination with 200 g LHRH-a/kgody weight, to induce final oocyte maturation and ovu-

ation in thin-lipped grey mullet. There are a severaltudies which applied a variety of hormonal treatments fornducing final maturation and spawning of Mugil cephalusemale (with vitelline oocytes; tertiary yolk stage) with

uch higher doses: 50,00080,000 IU HCG/kg body weightKuo et al., 1973b), 2848 mg of fresh mullet pituitarieslus 10,00080,000 IU chorionic gonadotropin/kg of bodyeight (Kulikova and Gnatchenko, 1987) and 20 mg carpituitary homogenate/kg body weight of fish, followed by00 g LHRH-a/kg of fish (Lee et al., 1987; Suzuki et al.,991).

The induced spawning of thin-lipped grey mulletllowed us to integrate the data on the development ofhis species. Although, the previous studies by Yashouvnd Berner-Samsonov (1970) for thin-lipped grey mullet,resented some of developmental stages; however, theirescriptions are incomplete, because the developmentaletails and/or the sequence of stages are not included.herefore, our studies present a detailed description ofmbryonic and early larval development of thin-lippedrey mullet.

All fertilized eggs of thin-lipped grey mullet observedrom 10 females were pelagic and similar to those of other

ugiliformes fishes (Nash and Shehadeh, 1980). The nar-ow perivitelline space of fertilized eggs of thin-lippedrey mullet may be a distinguishing feature from that ofther fish species. Furthermore, fertilized eggs of thin-ipped grey mullet have more than one oil globule andhese subsequently developed and hatched. During devel-pment the droplets were merged and on hatching thearvae had one oil globule (rarely two) located in the yolkac. Similar observations were reported by Yashouv and

erner-Samsonov (1970).

The present description of embryonic and larval devel-pment of thin-lipped grey mullet, presented in Table 2nd Figs. 1 and 2, was similar to other Mugiliformes fishesNash and Shehadeh, 1980; Boglione et al., 1992), European

cience 119 (2010) 115122 121

Sea Bass; Dicentrarchus labrax (Saka et al., 2001), giltheadsea bream; Sparus aurata (Kamaci et al., 2005) except dif-ferences in the time course of the different stages andthis may be due to the difference in the incubation tem-perature. It is well established that the egg developmentand hatching time are both temperature dependent. More-over, the time required by fertilized fish eggs to hatchdecreases with increasing temperature. In the presentinvestigation, the propagation of thin-lipped grey mul-let is conducted within the most desirable temperaturerange and mostly at the optimum level. The fertilized eggsdeveloped and hatched within 48 h at 2021 C in normalseawater (35 g/l). Yashouv and Berner-Samsonov (1970)noted that under laboratory conditions the eggs of M.cephalus and Mugil capito developed and hatched within3644 h at 2232 C. Kuo et al. (1973a) stated that hatchingof M. cephalus eggs was evident 3638 h after fertilizationat 24 C, and 4850 h at 22 C. However, development andhatching of the thick-lipped mullet (Chelon labrosus) occurs3 days after spawning, at a water temperature ranging from15.7 to 18.7 C (Boglione et al., 1992).

During the embryonic development of thin-lipped greymullet, the pigment spots appeared on the oil globule andalong the embryonic body but no pigment spots presentover the yolk. Yashouv and Berner-Samsonov (1970) notedthat in early larval stages of M. capito light pigmentationpresent on forehead and on oil globule, absent on yolk sac.We found that newly hatched larvae of thin-lipped greymullet had 25 somites and one oil globule situated in theposterior part of the yolk. Moreover, feeding began 5 daysafter hatching. Tung (1973) recorded 24 myotomes in thenewly hatched larvae of M. cephalus. The author added thatfeeding began 35 days after hatching. However, in C. labro-sus the mouth opening formed 6 days 8 h after spawning(Boglione et al., 1992).

5. Conclusion

In the present study, we succeeded in the artificialspawning and present a detailed description of embryonicand early larval development of L. ramada, an economicallyimportant and valuable fish species in fisheries. This inves-tigation adds new information to the early life history offish in the order Mugiliformes.

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Induced spawning and embryonic development of Liza ramada reared in freshwater pondsIntroductionMaterials and methodsBroodstock managementSeawater acclimation and induction of spawningEggs incubation and embryonic developmentResultsInduced spawningEmbryonic developmentFertilized eggCleavageMorulaBlastulaGastrulaEmbryonic body formationHatching (newly hatched larvae)Early larvaLarva 24h after hatchingThree-day-old larvaFive-day-old larvaDiscussionConclusionReferences