Improved Artificial Breeding Techniques Of Tiger Groupe1

Improved Artificial Breeding Techniques of Tiger Grouper (Epinephelus fuscoguttatus) Hussin Mat Ali, Ahmad Daud Om, Shaharah Mohd Idris, Sufian Mustafa and Teoh Pik Neng Marine Finfish Production and Research Center, Tanjung Demong, 22200 Besut, Terengganu.

Abstract Artificial breeding of tiger grouper (Epinephelus fuscoguttatus) at Marine Finfish Production and Research Center, Besut, Terengganu (MAPREC) began in February 1995. The first breakthrough was achieved two months later in April 1995 using naturally spawned eggs. However, attempts to improve the breeding techniques and to commercialize the findings were not immediately carried out due to several technical and managerial constraints. In 2004 and 2005, artificial breeding of tiger grouper was resumed with reference developments in the field of system operation, larval nutrition and health management. Additional broodstock of various sizes ranging from 3.0 kg to 6.0 kg each were subjected to broodstock management methods such as natural and induce spawning in order to have a regular spawning and consistent egg quality. The eggs produced were hatched and reared under specific rearing methods and critical aspects of production in order to achieve the targeted survival rate of 3-5% for 35-45 day after hatching (DAH). The juveniles produced were then nursed in several types of nursery facilities to achieve 40 – 60% survival rate or 2% final survival rate when the fry attained 7.5 – 10.0 cm in total length (TL). Abstrak Pembiakan aruhan ikan kerapu harimau (Epinephelus fuscoguttatus) di Pusat Pengeluaran dan Penyelidikan Ikan Laut, Besut, Terengganu (PPPIL) bermula pada Februari 1995. Kejayaan pertama berlaku dua bulan selepas itu iaitu pada bulan April 1995 menggunakan telur semulajadi. Bagaimanapun, percubaan untuk meningkatkan teknik pembiakan dan untuk mengkomersial penemuan yang diperolehi telah tergendala akibat dari masalah teknikal dan pentadbiran. Pada tahun 2004 dan 2005, pembiakan kerapu harimau telah dimulakan semula dengan merujuk kepada perkembangan sistem operasi lapangan, pemakanan rega dan pengurusan kesihatan. Penambahan induk dari pelbagai saiz dari 3.0 kg hingga 6.0 kg telah dijalankan pengurusan induk seperti pembiakan aruhan dan semulajadi supaya peneluran akan berlaku dan kualiti telur yang konsisten akan didapati. Telur yang dihasilkan ditetaskan dan di ternak dengan kaedah yang spesifik dan aspek pengeluaran yang kritikal supaya kadar hidup dapat ditingkatkan dengan kadar 3-5% pada hari 35-45 selepas penetasan. Ikan juvenil yang dihasilkan akan diternak dengan beberapa kaedah asuhan untuk mencapai kadar hidup 40-60% ataupun 2% kadar hidup akhir apabila ikan telah mencapai saiz 7.5 – 10.0 cm panjang totalnya.

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INTRODUCTION Aquaculture of high value marine finfish species continues to develop rapidly in Southeast Asia.

Many grouper members of the family Serranidae, subfamily

Epinephelinae bring high prices up to US$70/kg wholesale in the live markets of Hong Kong and Southern China (McGilvray and Chan, 2001). Increasing market demand and the real perceived profitability of the live reef food fish trade has led to many Southeast Asian and Pacific countries focussing on suppling this apparently lucrative trade through aquaculture (Sadovy et al., 2003). Tiger grouper, Epinephelus fuscoguttatus or commonly known as blotchy grouper or flower cod is a popular marine food fish of high market value in Southeast Asia because of its excellent taste and scarcity value. It is widely distributed in the Western Pacific and Indian Oceans. In Indonesian waters, the species is a common target fish for fishermen and considered to be a desirable fish for culture (Kohno et al., 1991). It has been farmed commercially in marine cages and ponds in Taiwan, Thailand, Malaysia, Singapore and China (Rimmer, M. 1998). The culture of the tiger grouper has not been popularized in the world due to rare availability of seed. In Malaysia, the species is naturally available and being cultured in Sabah and Langkawi Island. The important of tiger grouper to aquaculture industry in Southeast Asia has been realized since late 1980’s. In 1995, MAFPREC started the tiger grouper fry production research using wild broodstock

imported from neighboring country. The first

breakthrough was achieved two months later in April 1995 using naturally spawned eggs (Hussin, M.A. et al., 1996). However, attempts to improve the breeding techniques and to commercialize the findings were not immediately carried out due to several technical and managerial constraints. In 2004 and 2005, artificial breeding of tiger grouper was resumed. Additional broodstock of various sizes ranging from 3.0 kg to 6.0 kg each were subjected to broodstock management methods such as natural and induce spawning in order to have a regular spawning and consistent egg quality.

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To date, a handfull amount of research on tiger grouper has been done on various aspects of captive breeding techniques including broodstock development and maintenance, spawning, larval rearing and fry nursery. The aim of this article is to provide an overview of the research findings and observation on captive breeding of tiger grouper at MAFPREC for the purpose of commercialization by target groups and to enhance local industry of tiger grouper fry production. BROODSTOCK DEVELOPMENT The purpose of broodstock development

is to supply good quality

spawners. A

succesful hatcery operation is based on healthy broodstock. Collection of broodstock is the first bottleneck because the mature fish are expensive

and less availability in

captivity. Broodstock can be obtained from the wild or from ponds, where young groupers are reared until sexual maturity. Matured broodstock is scarcely available in Malaysia except in Sabah, thus demanding higher prices ranging from RM 120.00 – 230.00/kg. Tiger grouper is a protangynous hermafrodite, meaning that the fish first sexually matured as female and change to male in the later part of its life. The smallest matured female appeared at MAFPREC at average body weight of 2.4 kg and later transformed into male at average body weight of 8 kg. Some females do not change the sex even they grow to 10 kg of body weight.

This may happen due to probably to captive

conditions (Ketut Sugama et al., 2001). In 2005, MAFPREC proved that functional male and female of tiger grouper can be raised in indoor tanks. This method of growing young fish in captive conditions enables to select the strong and the fast growing fish. Current research conducted on induced maturation of male and female tiger grouper at ealier age or smaller size is been carried out.

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Disease free broodstock especially of Viral Nervous Necrosis (VNN) is now a major concern to fish breeders and fish farmers which is possible if the broodstock is raised in bio-secure facilities using disease free juvenile. Success or failure of larval rearing depends mostly on the control of VNN. Once VNN breaks out during larval rearing, a high mortality occurs and in many cases, almost all larvae die within a few days. Up to now, however no treatment method for VNN has been developed. To control the disease, therefore it is important to make continuous effort to prevent contaminating VNN virus into rearing water, to decrease the virus density in rearing water and to reduce stress to larvae.

BROODSTOCK MAINTENANCE At MAFPREC, tiger grouper broodstock are kept in 50 to 150 ton tanks made of concrete, circular shape and at 1-2 m water depth. Broodstock tank are used not only for culturing but also for spawning.

Since broodstock

swim around the tank during

spawning activities, it is recommended to use circular tanks to minimize the risk of their colliding with the tank wall during the activities (Ketut Sugama et al., 2004). Tanks are located both outdoor and indoor under prevailing weather conditions. The outdoor tanks were unshaded while the indoor tanks were provided with 75% translucent roofing. Broodstock tank were continuously cleaned and supplied with new seawater at a daily or altenate day exchange rate of 100% using naturally sand –filtered seawater. During reproductive performance, the broodstock tanks should be covered

with parachute

netting. This is due to the cases of fish jumping out from the tank during spawning season especially the female. This behaviour occurred only during spawning season when the pairs became active, chasing each other and jumping near the surface of the water. These behaviour lasted till spawning occurred. The broodstock were fed daily at 1-2% total body weight with fresh fish. It is strongly recommended to feed broodstock on squid once a week in order to enhance the quality of their eggs. Vitamin E and C at a dosage of 200IU

and 2000mg per kg of fish

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were also given at least a week before spawning to enhance gonad development as well as to improve eggs and larval quality (Ahmad Daud, per.comm). Prophylactic treatment with 30 ppm formalin in 24 hours bath treatment for 3 consecutive days is carried out monthly. On the other hand, therapeutic treatment is applied in accordance to diagnostic results and recommendation by disease experts. In MAFPREC, despite some of the tiger grouper broodstock has been used for more than 10 years, due to proper maintenance and handling of broodstock will prolonged their life spent and reliability. SPAWNING Tiger grouper observed to spawn regularly either naturally or through hormone induction. However, natural spawning can be expected only from the newly introduced matured spawners obtained from the wild. Observations on spawners obtained from 1995 and 2005 indicated that natural spawning occurred only for about a year starting from the 2 nd month in captivity. Spawning program for the following years must be carried through hormone injection. A single injection of Human Chrionic Gonadotropin (HCG) at a dosage of 250-500IU/kg and 500-1000IU/kg of properly selected male and female, respectively is sufficiently induced fish to spawn. Other hormones such as GnRH (5 – 20 µg/kg of fish) is also applicable either solely or in combination with HCG. It was common for un-injected spawners within the same tank to also indirectly induced and spawned naturally 2 to 3 days later.

The regularly induced spawners performed

satisfactorily for the first 3 to 5 years and subsequently showed irregular response on the following years. It is suggested that the replacement of spawners after 3 – 5 years. The spawners may be kept until 5 to 10 years in captivity but some may changed to male after they attained 8-10 kg body weight. Sex of broodstock can be investigated only at new moon periods. If a fish matured as male, white milt is secreted from genital pore by gentle pressing abdomen part from head toward the tail direction by hand. If a fish matures as female, the fish has very swollen

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abdomen with eggs which can be sucked out through genital pore by cannulation (Liao, I.C. et al., 2001). LARVAL REARING Larval rearing of tiger grouper from hatchling to complete metarmorphosis may take about 35 to 45 days depending on prevailing environmental conditions and culture management (Chao, T.M. et al., 1993). Larval rearing period at feeding transition from endogenous to exogenous (5 days old), from smaller to bigger zooplankton (15 – 20 days old) and from live to inert food (30 – 40 days old) are the most critical parts. Supplementary of green water in larval rearing tank is crucial for tiger grouper larvae at least until 20 days old. Application of good quality micro encapsulated diet from ealier stage of larval rearing (20 days old onward) may eliminate ‘shock died syndrome’ when they entered juvenile stage. Suggested larval rearing protocol is shown in Table 1. Three kinds of feed items are used for larval rearing: rotifers (two strains, namely SStype rotifer that is 120 – 150 µm in lorica length and S-type rotifer that is 151 – 200 um in lorica length, artificial diets and Artemia. The following are the details of a feeding method. Chlorella or green water from tilapia fish culture is used as the green water at the density of 0.5 to 2 x 106 cells/mL. The water should be supplied to larval rearing tanks in order to serve as food for rotifers and maintain a color (for homogenous light intensity and water turbidity) of rearing water. Maintaining a color is effective in lowering larval floating to the water surface and aggregation. SS type rotifers should be supplied to the larval rearing tank in the morning of day 3 when larval mouths open. The density of the rotifers are 5 – 20 ind/mL starts on day 3 until day 25. As the rotifers uneaten by larvae propagate in the rearing tank, the density of rotifers in rearing water should be counted twice a day. It is recommended to provide rotifers at least until day 25. Prior to supplying rotifers to the rearing tank, the rotifers

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should be enriched with Algamac 2000 for highly unsaturated fatty acids (HUFA) for more than 6 hours. It is advisable to treat rotifers by washing with fresh water during the enrichment to prevent bacterial diseases. Harvesting Therapeutic treatments Prophylactic treatment Micro encapsulated diets Artemia (1-5 nauplii/mL) Rotifer (5-20 ind/mL) Green water top up

yes

(500,000 – 2 x 106 cells/mL) Shading (75%) Tank cleaning (bottom siphoning) Water change (%) Water level (m) Tank shape Tank size (m3) Age (Day After Hatched)

yes nil 0.5

nil 0.7

0-5

6-10

yes

yes

5-10 1.0

10-15 15-20 20-30 1.0 1.0 1.0 Circular or square 10-20 11-15 16-20 21-25 26-30

yes 30-40 1.0

40-50 1.0

31-35

26-40

Table 1 : Improved Larval Rearing Protocol Artemia is a feed item of easy handling and larval preference. However, the amount of Artemia used for larval rearing should be minimized due to its low nutritional quality and high price. Artemia should be enriched for HUFA before feeding to larvae. The feeding density of Artemia nauplii are 1 – 5 ind/mL. To minimize mortality from nutritional deficiency, feeding of artificial diets, which contain sufficient nutritional value for larvae, should be started as early as possible. However, artificial diets easily deteriorate rearing water. Considering these, it is recommended to start feeding at day 14 when larvae become larger and strong enough for water exchange such as flowthrough. Since larvae prefer live foods to artificial diets, it is very important to consider combination of feeding artificial diets and live foods for success in the early weaning on to artificial diets.

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Filtered seawater by using 0.45µm filter bag is use as a rearing water. At stocking of eggs, the tank is filled with 0.5% seawater. The water should not be changed except adding green water , until day 5. At day 6, the volume of rearing water is increased to 0.7% and until day 11, is increased to 100%. Between day 11 and 15, 5 – 10% of tank water is daily replaced by new seawater. It is recommended to carry out continuous water exchange to increase the exchange rate. Organic wastes of rotifers and larvae, excess foods, dead larvae and other dirt always accumulate on the bottom of rearing tanks. These should be removed by siphoning with a hose. The first bottom cleaning should be done at day 7. After the start feeding of artificial diets, it may be required every day. Tiger grouper larvae are very sensitive to environmental conditions and easy to die due to stress. Therefore, careful management is required for the larval rearing of this species. To improve survival rate in larval rearing, it is necessary to eliminate or minimize the mortality factors. Factors that contribute to larval mortality are : a) Surface tension Larval between day 0 and 5 are easily trapped at the water surface by the surface tension. Once the larvae trapped at the water surface, the larvae cannot escape from the trapping and eventually died. When larvae receive a stress like the trapping, they secrete sticky mucus. The mucus from the trapped larvae accelerates the trapping of other larvae. Result from that, a significant number of larvae die in a short time. Measures such as adjustment strength (0.1L/min for 0-14 day old larvae while 0.61L/min for 15 day old and above larvae). There should be 12 position of aeration points and maintaining colour of rearing water should be taken into account to prevent motality. b) Nutritional deficiency

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Larval mortality is observed also at day 25. This is suspected to be due to nutritional deficiency. To prevent this problem, early weaning larvae on artificial diets which have sufficient nutritional value is effective. For this purpose, the following measures should be taken, to introduce a commercial compound feed as artificial diet. In MAFPREC case, we used love larvae as inert feed and start feeding at the same time as to feeding artemia. Light is a very important factor for larval feeding. If either light intensity is too low or photoperiod is too short, larvae can not feed well. For larvae at the initial stage, light intensity of more than 1,000 lux (at the surface of the rearing water) should be provided. Light intensity in the tank should be controlled as even as possible using black plastic netting hung around the tank. By this operation, even if light intensity is little lowered, well developed larvae can still be feed. FRY NURSERY Tiger grouper fry are nursed in two stage. The first stage are rearing the fry from 2.0 – 4.0 in total length (TL). At this stage, the fry grows rapidly into fingerling, reaching 15-25 g at the end of the first nursery stage. Size grading is undertaken everyday. The fry are given adults artemia at a density of 1-2 ind/mL. If large size plankton example mysis is available, of course, it can be fed to the fry as supplementary food. In case where minced fish is fed to the fry, special attention should be paid to the deterioration of culture water. In this stage the fry should be totally weaned to pelleted feeds. Nursery culture tank should be provided with flowthrough seawater system as well as an aeration system. In practical experience at MAFPREC, it appears that using shallow tank or calvert are easy to manage for feeding and size grading purposes. This technique produce much better effect in decreasing cannibalism while it is applied with other manipulations such as routine grading, suitable feeding practise and optimal stocking density.

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Although the optimum stocking density of tiger grouper are not known, it appears that the density of 4 fry/L may be suitable. If stocking density is too high, due to the flowthrough system, if there is an electricity failure, then it might suffer from stress and dying. Almost all dead juveniles during the nursery culture at MAFPREC, are due to cannibalism. When exerting cannibalism, juvenile fish generally wholly consume their prey for a series of reasons.

To date attempts to mitigate cannibalism in

MAFPREC are mainly concerned with environmental manipulation such as reducing the salinity. The present study demonstrates for the first time the effect of salinity cannibalism.

This resulted in better survival (40 – 60%) for juvenile grouper at

salinity of 15 ppt. This method requires less labour and time and is more convenient and applicable to prevent cannibalism in the larviculture of tiger groupers. In the second stage, the tiger grouper fingerlings are reared from 4.0 to 5.0 in (day 70 to day 90) in larger tanks (5 – 10 ton tanks). Feeds are provided manually to satiatian 3 times daily. The size of the pelleted feed is increased gradually as the fish grows. Feeding rate on the other hand is reduced from 6% in stage 1 to 4% in stage 2. Size grading is undertaken only once during this stage due to cannibalism are not critical at this stage. Recently, In MAFPREC, a recirculation system was designed and still under research, for nursing the tiger grouper fry under intensive rearing and mass production of tiger grouper fingerlings.

CONCLUSION

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A significant amount of research on the aquaculture of tiger grouper has been conducted for the last ten years and commercial production has just begun. Most of the research and production to date has been oriented to the hachery phase and much research remains to be done on that phase. Although grow-out production has begun, based on techniques and facilities develop for the rearing of other fish species, the majority of tiger grouper research in the future should be aimed at optimizing the larviculture

and nursery fry

production by meeting their species-specific

requirements for the highest-density production at lowest cost.

REFERENCESS Chao, T.M., Lim, L.C. and Khoo, L.T. (1993). Studies on the breeding of brown – marbled grouper

Epinephelus fuscoguttatus (Forsskal) in Singapore. In.

Proceedings of finfish hatchery in Asia. Hussin, M. A., Ali, A., Nik-Daud, N-S., and Nik-Razali, N-L. (1996).

Natural

spawning and larval rearing of tiger grouper (Epinephelus fuscoguttatus (Forsskal) in tank: A preliminary result. In. Proceedings in National Fisheries Symposium 1996. Ketut, S., Tridjoko, Bejo, S., Suko, Ismi., Eri, S. and Shogo, K. (2001). Manual for the seed production of humpback grouper, Cromileptis altivelis. Kohno, H., Imanto, P.T., Diani, S., Slamet, B. and Sunyoto, P. (1991). Bull. Pen. Perikanan, Special Edition No.1: 27-35pp Liao, I.C., Su, H.M. and Chang, E.Y. (2001). Techniques in finfish larviculture in Taiwan. Aquaculture 200, 1-31.

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McGilvray, F. and Chan, T.T.C. (2001). The trade in live reef food fish: A Hong Kong Perspective, International Marine Life Alliance, Hong Kong. Rimmer, M. (1998). Grouper and snapper aquaculture in Taiwan.

Austasia

Aquaculture 12: 3-7. Sadovy, Y. (2000). Regional survey of fry/fingerling supply and current practises for grouper mariculture: evaluating current status and long term prospects for grouper mariculture in Southeast Asia, PP. 120. University of Hong Kong, Hong Kong SAR, China.

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