eggs and pre larvae development for humpback grouper

Aquacultura Indonesiana (2005) 6 (3) : 115–121ISSN 0216–0749

Hak cipta oleh Masyarakat Akuakultur Indonesia 2005 115

Introduction

Humpback grouper, Cromileptes altivelis, isbegining to be developed for commercial business,and has been spawned under controlledenvironment. Though, it could be spawned undercontrolled environment, the spawning must be donecollectively (several broodstock in one tank).Usman (1999) reported that, in a tank in the ratio of1: 1 or 2 : 1 male to female.

Humpback grouper fishes do their spawningduring the night, around 22.oo h till 04.oo h. Spawningactivity usually occurred during the dark moon, thatis 3 days before the appearance of a new moonand 5 days after appearance of the moon at the

beginning of the month (Sudaryanto et al.,1999;Usman, 1999). The eggs that have been fertilizedare transparent, floating on the surface, while theunfertilized eggs are white in color and would sinkto the bottom. (Lies in Polovina and Ralnston, 1987;Antoro et al., 1999 and Usman, 1999). The fertilizedeggs would developed into embryos and finallywould hatched out to become larvae.

During the development of embryos tolarvae, they would passed through some stages.Woynarovich and Horvath (1980), reported that thedevelopment of embryo occurred immediately afterfertilization of the egg cells by sperm. The fertilizingprocess begins with the cleavage of blastula,gastrula, organogenesis and finally to hatching. It

Eggs and Pre Larvae Development for Humpback Grouper,Cromileptes altivelis, (Valencee) Larvae

U. Bulanin1, C. R. Saad2, M. S. Kamarudin2, R. Affandi3 and A. Sudaryono4

1) Department of Aquaculture, Faculty of Fisheries, Bung Hatta University, Indonesia,2) Department of Agrotechnology, Faculty of Agriculture, UPM, Malaysia

3) Department Aquatic Resources Management, Faculty of Fisheries and Marine Science,Bogor Agiculture University, Indonesia.

4) Department of Animal Science, Faculty of Agriculture, UNDIP, Semarang

Abstract

U. Bulanin, C.R. Saad, M.S. Kamarudin, R. Affandi and A. Sudaryono. 2005. Eggs and pre larvae developmentfor humpback grouper, Cromileptes altivelis, (Valencee) larvae, Aquacultura Indonesiana, 6 (3): 115–121. Thisexperiment were conducted to determine the embryonic and pre-larvae development for humpback grouper,Cromileptes altivelis, under controlled condition. The above study was carried out from spawning of broodstocksto obtain the fertilized eggs till fully yolk sac and oil droplet absorption. The results showed that the mean diameterof humpback grouper egg was 828.69 45.91 m. The fertilized eggs hatched in 20 hour 10 minutes at the temperatureof 27 to 28o C. The hatched larvae had a mean length of 1.86 mm, and a mean height of 0.460 mm. The yolk sacs andoil droplets were totally utilized after 63 and 65 hour after hatching (HAH), respectively. One day larvae had anaverage length and height of 2.253 mm, and 0.615 mm, respectively.

Keywords: Humpback Grouper (Cromileptes altivelis); Larvae; Hatching; Egg

Abstrak

Penelitian ini telah dilakukan untuk menentukan perkembangan embrio dan pre larva pada ikan kerapu bebekCromileptes altivelis, pada kondisi terkontrol. Penelitian diatas dicari dari pemijahan induk hingga menghasilkanpembuahan telur sampai kuning telur penuh dan tetesan minyak terserap. Hasil menunjukkan bahwa diameter rata-rata telur ikan kerapu bebek adalah 828,69 45.91 m. Pembuahan telur ditetaskan dalam 20 jam 10 menit padatemperatur 27–28oC. Larva yang menetas mempunyai panjang rata-rata 1,86 mm dan tinggi 0,460 mm. Jumlah Kuningtelur dan tetes minyak yang digunakan setelah 63 dan 65 jam setelah menetas (HAH) secara berturut-turut. Larvaberumur satu hari mempunyai panjang dan tinggi secara berturut-turut 2,253 mm, dan 0,615 mm.

Kata kunci: Ikan Kerapu Bebek (Cromileptes altivelis); Larva; Penetasan; Telur

Aquacultura Indonesiana, Vol. 6, No. 3, Desember 2005 : 115–121

Hak cipta oleh Masyarakat Akuakultur Indonesia 2005116

was also explained that the series of stages in thedeveloping embryo were (1) swollen of fertilizedegg, (2) first cleavage or 2 cell stage, (3) 4 cellstage, (4) morula (early stage), (5) morula (laterstage), (6) blastula, (7) gastrula, (8) closing of theblastopore (9) development of tail and head buds,and (10) egg ready for hatching. Meanwhile Balon(1985) stated that during the embryonicdevelopment it is divided into three stadia, they arecleavage of the egg, embryonic and free embryonic(eleutheroembryo) stages.

The duration of the embryo development tolarva varies, depending on the size of yolk sac(Nontji, 1984), environment and species (Effendie,1978). For example brown marbled grouper,Epinephelus fuscoguttatus, hatching took placewithin 19 hours after fertilization (Anindiastuti etal. , 1999), and spotted coral-trout fish,Plectopomus maculatus, is about 16 to 18 hoursafter fertilization (Diani et al., 1991).

Effendi (1997) reported that the developmentof larvae is divided into two stages of pro-larvaeand post larvae. Pro-larvae stage is when the larvaestill has it yolk sac and transparent body. Post larvaestage is when the larvae had fully used the yolksac and formation of organs until larva looked likethe adult fish. Therefore, the morphologicaldevelopment of each species of the grouper fishesis different to each other. The objective of this studywas to observe the egg till yolk sacs and oil dropletfinished.

Materials and Methods

The eggs were obtained form the result ofthe natural spawning by manipulation of theenvironment. Water in spawning tank was loweredup to a height of 60 cm in the morning at 8.oo h andmaintained till 15.oo h, when the water level wasagain raised to 275 cm level. The broodstockspawned collectively, with the ratio of 10 male and19 female in a round tank of 80 tons volume. Theeggs, after fertilization were taken and incubatedin an aquarium with the size of 60 x 40 x 40 cm or96 litre volume at the temperature of 27 to 28 oC.The eggs were observed under a “Nikon”microscope for development of embryo until theyhatched. For each changing stages pictures weretaken serially.

The observation of the volume of yolk sacand oil droplets were done every three hours until

the yolk sac and oil droplets were used. For everyobservation 10 larvae were used. The volume ofyolk sac and oil droplets were calculated based ona formula from Kohno, et al. (1986). V = / 6 x Lx H2 for yolk sac and V = 4/3 x x r3 for oil dropletabsorption, which V is Volume, = 3.14, L is length,H is Height, and r is radius.

Results

Embryonic Development

The fertilized eggs of humpback grouper arerounds, transparent, and pelagic. They do not stickto each other and aggregate together when there isno aeration. The eggs diameter were 707.84 to864,64 m with an average of 828.71 45.91 m.The fertilized eggs would develop to become embryo,then hatch after reaching their perfection. Theduration for eggs to embryo development are asshown in Table 1 and Figure 1.

Ten minutes after fertilization, the perivitellinespace was formed and the chorion filaments wereobvious. Blastomer will be visible in about 15 minutesafter fertilization (AF). In about 25 minutes AF, 2blastomers undergoes cleavage and woulddeveloped into 4 cell stage in 45 minutes. Theblastula stage would occur in 4 hours 40 minutesand would developed into gastrula in 7 hours 25minutes AF. At this stage, the egg would have been

Table1. The embryo development of humpback grouper, Cromileptes altivelis, at 27–28 oC.

The embryo development Time

Hour Minute

Fertilization 00 002 cell satge 00 254 cell stage 00 458 cell stage 01 0516 cell stage 01 1532 cell stage 01 3064 cell stage 02 08Morula 02 45Blastula 04 40Gastrula 07 25Formation of embryo 10 25Development of head 12 30and tail budsComplete embryonic 18 10developmentTail coming out of shell 19 30

Eggs and pre larvae development for humpback grouper (Usman Bulanin et al.)


covered by blastoderm for 1/4 of its body.Gastrula stage was formed when the blastodermhas covered about ¾ of the egg.

After the gastrula stage, the eggdevelopment continues into the embryo stages ororganogenesis (organs formation). During theembryo development, head and tail formation wasin tandem. When the embryo developed into itsperfect form, it was very active, especially at theposterior side. About 19 hours 5 minutes AF, thetail appeared from its casing and generally the larvaewould hatched in about 20 hours 10 minutes AF.

Larvae Development and Absorption of Yolk Sacand Oil Droplet

The larvae morphology development startedfrom hatching until it reaches the yolk sacs and oildroplet were finished as shown in Figure 2. The

newly hatched larvae have a yolk sac and an oildroplet. The average volume of the yolk sac is0.15456 m3, and the oil droplet is 0.00352 m3.The rate of yolk sac and oil droplet absorption canbe seen in Table 2, Figures 3 and 4 .

The newly hatched larvae would depend onthe yolk sac for growing and developing. Threehours after hatching 8.57% of the volume of yolksac and 7.39% of oil droplet had been absorbed.The rate of yolk sac and oil droplet absorption werevery fast up to 33 hours after hatching (HAH) afterwhich the remaining volume of yolk sac was only8.34% and the oil droplet was 21.02% respectively(Appendix 1). The yolk sac was totally absorbedwhen the larva was at 60 to 64 HAH. Therelationship of the yolk sac absorption versus theage is linear with an equation of Y = -0.0001x +0.0064 ; r 2 = 0.96, while the oil droplet had a

Figure 1. Stage of embryonic development of humpback groupers, Cromileptes altivelis, A) fertilization, B) 1 cellstage, C) 2 cell, D) 4 cell, E) 8 cell, F) 32 cell, G) 64 cell, H) morula, I) blastula, J) gastrula, K) formation ofembryo, L) development of head and tail buds, M) complete embryonic development, N) tail coming outof shell and O) hatching



Figure 2. Larva development (A) Few minutes after hatching, (B) 1 days old and (C) 2 days old.A B C

0

20

40

60

80

100

120

0 6 12 18 24 30 36 42 48 54 60 63

Age of larvae (hour)

The

abs

orpt

ion

of y

olk

sac

and

oil

drop

let

(%)

YsOd

Figure 3. The absorption rate of yolk sac (Ys) and oil droplet (Od) of humpback grouper, Cromileptes altivelis, larvae.

Table 2. The rate of yolk sac and oil droplet absorption of humpback grouper, Cromileptes altivelis, larvae

Age (hrs) Yolk sac Oil Droplet

Length(mm)

Height(mm)

Volumeµm3 % Length

(mm) Volume µm3 %

0 0.8852 0.5523 0.15456 100 0.8880 0.00352 100

6 0.8326 0.4777 0.09942 64.32 0.1732 0.00272 77.27

12 0.6821 0.4185 0.06253 40.46 0.1636 0.00229 65.06

18 0.5941 0.3539 0.03894 25.19 0.1439 0.00156 44.32

24 0.4510 0.2725 0.02766 17.90 0.1349 0.00129 36.65

30 0.3579 0.3037 0.01727 11.17 0.1142 0.00078 22.16

36 0.3253 0.2561 0.01117 7.23 0.0883 0.00036 10.23

42 0.2847 0.2155 0.00692 4.48 0.0702 0.00018 5.11

48 0.1686 0.1356 0.00163 1.05 0.0533 0.00007 1.90

54 0.1429 0.0983 0.00080 0.52 0.0488 0.00006 1.70

60 0.0889 0.0794 0.00029 0.19 0.0291 0.00001 0.28

63 0.0334 0.0259 0.00001 0.01 0.0120 0 0

33



quadratic relation with age with an equationof Y = - 1.59E-07x2 + 7E-06x – 2E-05 ; r 2 = 0.97(Table 4; Figure 4).

The average total length of the humpbackgrouper larva that was just hatched was 1.865 mmand the average height was 0.460 mm (Figure 5).At the moment the yolk sac was totally absorbed,the larva’s length had increased to 2.54 mm, and itsheight had increased to 0.790 mm respectively. Therate of growth for larva from hatching to total yolksac absorption was 0.11 mm per day or 0.049 mmper hour. During this period the process ofdevelopment for digestive system, eyes, mouthopening and pigment also occurred. The mouth

would opened before the yolk sac was totally usedup while eyes pigment was not yet visible.

Discussion

The humpback grouper eggs were round,pelagic, transparent, non-sticking and would coalescewhen there was no aeration. The shape andcharacteristic of the humpback grouper eggs werethe same as other marine coral fishes (Leis inPolovina and Ralstons, 1987; Lim at al., 1990; Doiand Singhagraiwan, 1993) cod, Gadus morhua,(Huse in James, 1991), red sea bream fish (Fukusitoin James, 1991) and Sillaginodes punctata,(Burce, 1995). The diameter of the humpbackgrouper eggs vary from 707.84 to 864.64 m withan average size of 828 45.95 m. Tang (1979) inPolovina and Ralstons (1987), reported that the sizeof the eggs ranged from 0.80–0.83 mm. There weresimilar observations in other marine and freshwaterspecies such as, cod, red sea bream, red snappers,river catfish, Mystus nemurus, and etc (Leis inPolovina and Ralstons, 1987; Lim et al., 1990 Husein James 1991; Fukusito in James 1991; Doi andSinghagraiwan, 1993; and Tang, 2000).

The humpback grouper eggs, which hadbeen fertilized would developed into embryos andhatched at 20 hours 10 minute after fertilization atthe temperature of 27–28oC. Minjoyo et al. (1999)reported that eggs of humpback grouper wouldhatched after incubation for 17–19 hours AF at thetemperature of 27–29oC, 17 hours 45 minutes attemperature of 27–30.5oC (Tridjoko et al., 1996)and 22 hours at 28.4oC (Tang et al., 1979 inPolovina and Ralstons, 1987). The difference in theincubation period was due to the temperature.

Figure 4. Relationship of age (hour) with yolk sac (a) and oil droplet (b) for humpback grouper, Cromileptes altivelis, larvae.

y = -1E-04x + 0.0062R2 = 0.9524

00.00020.00040.00060.00080.001

0.00120.00140.00160.0018

45 48 51 54 57 60 63 66Age (hour)

Abs

orpt

ion

of y

olk

sac y = -1E-07x2 + 7E-06x - 2E-05

R2 = 0.9735

0.0000.0000.0000.0000.0000.0000.0000.0000.000

45 48 51 54 57 60 63 66Age (hour)

Abs

ortio

n of

oil

drop

let

A B

Table 3. The length and height (mm) of humpbackgrouper, Cromileptes altivelis, larvae duringthe yolk sac and oil droplet absorption

Age (day) Length (mm) Height (mm)

0 1.865 0.460

6 1.968 0.516

12 2.150 0.547

18 2.218 0.574

24 2.281 0.604

30 2.305 0.632

36 2.323 0.649

42 2.340 0.667

48 2.365 0.699

54 2.416 0.730

60 2.481 0.753

63 2.540 0.790



Lower temperature will slow down the embryodevelopment process, because the energy neededin metabolism process is relative less. Fukusito inJames, (1991), reported that the red sea bream eggsdid not develop at the temperature of 10oC. WhileDoi and Singhagraiwan, (1993) stated that hightemperature would accelerate the egg developmentfor Lutjanus argentimaculatus fish. Besidestemperature, incubation times, also was affectedby salinity and species. The red snapper, Lutjanusargentimaculatus, hatched at 17 hours 30 minutesafter fertilization at the temperature of 27.8–29.7oC (Doi and Singhagraiwan, 1993), Lutjanus sp.fishes at 24–45 hours and 20–45 hours afterfertilization for humpback groupers (Leis in Polovinaand Ralstons, 1987), Epinephelus fuscoguttatus,in 19 hours at 28–30oC (Anindiastuti et al., 1999).

The absorption of yolk sac and oil droplet ofthe humpback grouper larvae was faster at thebeginning until up to 33 hours after hatching (HAH),by which time then the rate of absorption wouldslow down till all the yolk sac was utilized. The rateof yolk sac absorption, depend on the rate of larvalgrowth. The rate of length increment was fast forthe larvae that were just hatched till 33 hours at0.014 mm per hour, and after which time the rateof length increment would slow down and similarlythe absorption rate followed closely with only 0.0018mm per hour. The fast rate of length increment till33 HAH was due to the availability of nutrientsource from the yolk sac, which was heavily usedfor maintenance and growth. During this time, thelarvae were still inactive and the other organs have

not yet developed. After 33 HAH the nutrientsource, mainly the yolk sac, were mostly used upfor maintenance and organs formation, the larvaebegan to start swimming actively. This observationwas similar to the one observed by Kohno et al.(1986) who reported that the rate of larvae’s lengthincrement at the first phase depends on the rate ofthe yolk sac absorption. It was also observed thatthe yolk sac absorption phase for sea bass, Latescalcarifer, was divided into: 1) fast phase, whichwas from 0 until 16 HAH, 2) the slow phase, whichwas from 16 to 60 or 70 HAH (until the yolk sacwas totally utilized). Swanson, (1996) also reportedthat the rate of yolk sac absorption increases withthe increased of larvae’s age, even at day-1, theyolk sac volume of the milkfish, Chanos chanos,larvae remained only 12–25%.

In this trial it was shown that the humpbackgrouper fish larvae started to utilize the yolk sac atday-3 (60 till 64 HAH), while Tridjoko et al. (1996)reported that the yolk sac was completely used upon the third day. The difference on the duration ofthe yolk sac utilization was due to the environmentalfactors, especially the temperature and the salinitydifferences, yolk sac size and dissolved oxygen.Complete utilization of yolk sac by day-3, was alsofound in other fishes such as E. fuscoguttatus(Anindiastuti et al., 1999), Lates calcarifer (Kohnoet al., 1986), Mystus nemurus (Tang, 2000).Higher temperature would increased the rate ofabsorption process (Kohno and Slamet 1990;Kamler, 1992 in Swanson, 1996 and Swanson,1996).

0

0.5

1

1.5

2

2.5

3

0 6 12 18 24 30 36 42 48 54 60 63

Age of larvae (hour)

The

leng

th a

nd h

eigh

t (m

m)

LengthHeight

n = 10 larvaeFigure 5. Length and height of humpback grouper, Cromileptes altivelis, larvae during yolk sac and oil droplet absorption.



The humpback grouper eggs hatched at 20hours 10 minutes AF at the temperature of 27–28oC. The yolk sac is completely absorbed at 63 HAHand oil droplet at 65 HAH. The larvae’s length thatwas just hatched out is 1.86 mm with an averageheight of 0.46 mm.

References

Anindiastuti, N., Rausin, Mustamin dan E. Sutrisno.1999. Paket Usaha Budidaya Kerapu Macan,Epinephelus fuscoguttatus . DepartemenPertanian, Dirjen Perikanan, Balai Budidaya LautLampung. 35 hlm.

Antoro, S., E. Widiastuti dan P. Hartono. 1999. BiologiKerapu Tikus, Cromileptes altivelis, dalamPembenihan ikan Kerapu Tikus. DepartemenPertanian, Dirjen Perikanan, Balai Budidaya LautLampung, 88 hlm.

Bruce, B.D. 1995. Larval development of king georgewhiting, Sillaginodes punctata, school whiting,Sillago bassensis, and yellow fin whiting, Sillagoschomburgkii, from south Australian waters.Fishery Bulletin, (93) : 27–43.

Doi, M. and T. Singhagraiwan. 1993. Biology and Cultureof the Red Snapper, Lutjanus argentimaculatus.The research Project of Fishery ResourceDevelopment in The Kingdom of Thailand, 51 p.

Diani S., B. Slamet, dan P.T. Imanto. 1991. Studipendahuluan pemijahan alami dan perkembanganawal larva ikan kerapu sunu, Plectropomusmaculatus. J. Penel. Budidaya Pantai, 7 (2): 10–19.

Effendie, M.I. 1978. Biologi Perikanan. Bagian I. Studynatural history. Fakultas Perikanan InstitutPertanian Bogor, Bogor.

Effendie, M.I. 1997. Metode Biologi Perikanan. YayasanDewi Sri, Bogor. 97 Hlm.

James, P.M. 1991. CRC handbook of mariculture. Vol. II.Finfish Aquaculture. CRC Press. Boca Raton, AnnArbor- Boston. 25 pp.

Kohno, H., Hara, S. and Y. Taki. 1986. Early larvaldevelopment of seabass, Lates calcarifer, withemphasis on the transition of energy sources.Bulletin of the Japanese of Scientific Fisheries,52 (10): 1719–1725 .

Kohno, H. and B. Slamet. 1990. Growth, survival andfeeding habits of early larval seabass, Latescalcarifer at different thermal conditions. Pressedition, Research For Coastal Aquaculture, 1: 37–44.

Leis, J.M. and D.S. Rennis. 1983. The larvae of Indo–Pasific coral reef fishes. New South WalesUniversity press, Sydney, Australia. pp. 79–83.

Lim, L.C., T.M. Chao and L.T. Khoo. 1990. Observationson the breeding of brown marble grouper, E.fuscoguttatus, Sing. J. Pri. Ind. 18 (2): 66–842.

Minjoyo, H., Sudaryanto dan E. Widiastuti. 1999.Pemeliharaan larva, dalam pembenihan ikankerapu tikus. Departement Pertanian Dirjen.Perikanan Balai Budidaya Laut, Lampung. 88 Hlm.

Nontji, A. 1984. Telur Ikan. Oseana, LIPI, IX(1) : 22–30.Polovina, J.J. and S. Ralston. 1987. Tropical snappers

and groupers. Biology and FisheriesManagemen, Westview Press/Boulder andLondon. 659 pp.

Subaidah, S., M. Murjani, Y.L. Nuraini dan M. Yoriksa.1997. Studi pendahuluan perkembangan awallarva ikan kerapu tikus, Cromileptes altivelis. J.Pen. Perikanan, Lokal Budidaya Air PayauSitubundo. 9 hlm.

Sudaryanto dan S.K. Yuhono. 1992. Studi awalpemeliharaan larva kakap merah, Lutjanus johniiBloch, Depertemen Pertanian, Direktorat JenderalPerikanan. Buletin Budidaya Laut, Lampung. (4):9–19.

Swanson, C. 1996. Early development of milkfish: effectof salinity on embryonic and larval metabolism,yolk absorption and growth. Journal of FishBiology, (48): 405–421.

Tang, U.M. 2000. Kajian biologi, pakan dan lingkunganpada awal daur hidup ikan baung, Mystusnemurus, (Cuvier and Valenciennes 1945).Disertasi, Program Pascasarjana, InstitutPertanian Bogor, Bogor. 115 Hlm.

Tridjoko, B. Slamet, D. Makatutu dan K. Sugama. 1996.Pengamatan pemijahan dan perkembangan telurikan kerapu bebek, Cromileptes altivelis, padabak secara terkontrol. J. Penelitian PerikananIndonesia, 2(2): 55–62.

Woynarovich, E. and L. Horvath. 1980. The artificialpropagration of warm water fin fish. A. manual forextention. FAO. Fisheries Technical Paper no.20/FIR/T.20.

eggs and pre larvae development for humpback grouper

Documents