Download 401 Prosiding Forum Inovasi Teknologi Akuakultur 2015 DIGESTIVE

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Prosiding Forum Inovasi Teknologi Akuakultur 2015
DIGESTIVE ENZYME ACTIVITY OF PROTEASE, α-AMYLASE, AND LIPASE OF
RABBITFISH (Siganus guttatus) LARVAE
Kamaruddin, Usman, and Samuel Lante
Research Institute for Coastal Aquaculture
Jl. Makmur Dg. Sitakka No. 129, Maros 90512, Sulawesi Selatan
E-mail: [email protected]
ABSTRACT
One of the main constraints in seed production of rabbitfish is the high mortality during larval stage. Lack
of suitable size of <150 mikron of live feed become the main issue in hatchery because rabbitfish require
very small feed, also rabbitfish larvae have low capacity to digest the feed. The aim of this study was to
determine the activity of digestive enzyme including protease, α-amylase, and lipase in rabbitfish larvae, as
a basic for feed management. Rabbitfish larvae were reared in two fiber glass tank filled with 700 L of
sewater (30-31 ppt salinity) and equipped with aeration. Test fish were newly hatched rabbitfish larvae
which were stocked at density of 50 fish/L. Live feeds used were rotifers and Artemia nauplii. Measurement
of the digestive enzymes activity was conducted when the larvae age at 2, 5, 10, 15, 20, 25, 30, and 35 days
after heatching (DAH). The results showed that the highest enzyme activity during endogenous phase was
the protease of 0.0442 U/mL/minute and the lowest was the α-amylase of 0.021 U/mL/minute. During
exogenous phase, the peak enzyme activity of protease occurred at 25 DAH, for the amylase occured in 15
DAH and for lipase 25 DAH. The enzyme activity was associated with age of the rabbitfish larvae and related
to the phase where larvae need to obtain life feed.
KEYWORDS: digestive enzyme, rabbitfish larvae
INTRODUCTION
Rabbitfish is one of captured fish which has a great potential for aquaculture because of its high
value and local demand for local consument. This fish has a delicious taste and commonly served in
restaurants in areas of South Sulawesi (Rachman Syah et al., 2007). Lante et al. (2007) had successfully
bred this fish in the hatchery and produced juvenile, but survival rate was still very low. The main
constrain of the seed production is that the larvae still rely on live feed rotifers, (Branchionus rotifer
or B. phlycatilis) and Artemia/Artemia salina/nauplii. Price of artemia are too expensive for this larvae
which lead to the higher price of the seed. In addition, prolonged use of live feed is not practical and
its mass culture highly depends on weather (Kurokawa et al., 1998). Therefore, application of artificial
feed for larvae and juvenile become necessary. Utilizationof micro-diet is more efficient and can be
produced any time and stored for longer periode (Gatesoupe & Luquet, 1981).
More importantly, its nutritional composition can be formulated according to the fish requirement.
However, several studies reported that the use of artificial feed for larva and juvenile fish produced
lower growth and survival rate than fish fed live feed (Duray & Bagarinao, 1984; Haryati, 2002). The
lower growth is likely due to the inapproppriate feeding regime associated with the development of
digestive system of the larvae. Therefore, to improve the utilization of artificial feed in seed production
of rabbitfish, it is important to understand the feeding strategy by determining their digestive enzymes
activity. The aim of this study was to determine the activity of digestive enzyme (protease, á-amylase,
and lipase) of rabbitfish larvae.
MATERIALS AND METHODS
This experiment was conducted in the Hatchery Installation of Institute for Brackish water Aquaculture Research and Development, located in Lawallu, Barru Regency, South Sulawesi. The rabbitfish
larvae were reared in two fiber glass filled with 700 L of seawater with salinity 30-31ppt and equipped
with aeration. Newly hatched larvae were stocked with stocking density of 50 fish/L. During the
Digestive enzyme activity of protease, α-amylase, and lipase ..... (Kamaruddin)
402
rearing periode, larvae were fed with rotifers and artemia nauplii depending on the age asshown in
Table 1.
Table 1. Feeding scheme for rabbitfish (Siganus guttatus) larval rearing
Age of larvae (day) live feed
0
2
4
8
10 12 16 18 20 22 24 26 28 30 32 35
-----------------------------------------------------
15-20 ind/mL -------
300-500 sel/mL
30-35 ind/mL
---------------------------------- Nannoucculata
-----------------------------------------
----------------------------- 25-30 ind/mL ------------------
Rotifera
Naupli Artemia
Observation on the digestive enzymes activity was carried-out by taking1 g sample of larvae at
age of 2, 5, 10, 15, 20, 25, 30, and 35 days post hatching, respectively. Samples were washed with
distilled water then wiped out the water with filter paper.Each sample was collected intomicro-tube
and stored in coolbox to tranfer for enzymatic analysis. Collected samples were finely minced by
mortal and homogenized by adding 10mL of 4°C distilled water, then centrifuged at 15,000rpm for
20-minat 4°C. Supernatant was taken as a crude enzyme extract and measured for the enzymatic
activity in cluding protease, α-amylase, and lipase. Measurement of the enzyme activity were carriedout at Laboratory of Nutrition and Feed Technology of Institute for Brackish water Aquaculture Research
and Development Maros.
Measurement of protease activity based on Bergmeyer & Grassi(1983) using casein substrate and
tyrosine as a standard, by measuring the ability of the enzyme to hydrolyze proteins, thus resulting
tyrosine, measurements were performed by using as spectrophotometer with a wavelength of 550nm.
Protease activity was calculated according to the equation (Bergmeyer & Grassi,1983) :
 Act - Abl  P
U = 
 x
 Ast - Abl  T
where:
U
=
Protease enzyme activity (unit)
Act =
Value of sample absorbance
Abl =
Value of blank absorbance
Ast =
Value of standard absorbance
P
=
Dilution factor;
T
=
Time of incubation (minutes)
Similar to protease activity, measurement of α-amylase activity also based on the method of
Bergmeyer & Grassi (1983). Starch was used as a substrate with citrate buffer (pH 5.7). Its activity
was expressed as mg maltoseliberated from starchin 30 minutesat a temperature of 32oC. Maltose
was measured using a spectrophotometer at wavelength of 550nm. α-amylase activity was calculated
using the following formula (Bergmeyer & Grassi,1983):
α - amylase activity =
where:
ss
=
Ab l =
Ast =
Value of sampel absorbance
Value of blank absorbance
Value of standard absorbance
ss - Abl
P
x
Ast - Abl
T
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Prosiding Forum Inovasi Teknologi Akuakultur 2015
P
T
=
Dilution factor (mL)
=
Incubation time (minute)
Lipase activity was determined according to Tietz and Friedreckin Borlongan (1990) based on the
measurement of fatty acid sproduced by enzymatic hydrolysis of the triglyceride spresenting and
stable emulsion of olive oil. Buffer used was 0.1MTri-HCl (pH 8.0) and the substrates were olive oil.
Volume of NaOH standard solution used to titrate the hydrolized fatty acid was used as an index of
lipase activity of crude enzyme extract. One unit of lipase activity was defined as the volume 0,05NNaOH
required to neutralized the fatty acid produced 6 hours of incubation with the substrate and after
correction with the blank. Lipase activity was calculated using the following formula (Tietz &
Friedreckin Borlongan, 1990):
Lipase activity = ( A - B ) x N NaOH x
where:
A
=
B
=
N
=
P
=
T
=
1000 =
P
T
Volume of NaOH for titration of the sampel (mL)
Volume of NaOH for titration of the blank (mL)
Normality of NaOH for titration
Dilution factor (mL)
Time of incubation (menute)
Conversion from m molto μmol.
The Relative Changes of Enzyme Activity
The relative changes of each enzyme activity is calculated using the following equation:
 A - A t -1 
PR (%) =  t
 x 100
 At −1 
where:
PR
=
=
At
=
At-1
Relative change
Enzyme activity at time t
Enzyme activity at time t – 1 (enzyme active before t)
RESULTS AND DISCUSSIONS
Enzyme activities and relative changes of protease, á-amylase and lipase of rabbitfish larvae at
the age of 2 to 35 days are presented in Figures 1to 3.
The digestive enzyme activity observed on larvae at 2 days of age showed that the activity of the
protease, á-amylase, and lipase were 0.0442 U/ mL/min, 0.0210 U/ mL /min and 0.0350 U/mL/min,
respectively (Figure 1, 2, and 3). Although at this age larvae still do not have the ability to digest
exogenous feed, enzymes activities detected during this stage came from hydrolysis process of eggs
yolk which contained proteins, fats, and carbohydrates. Larvae obtained energy from this catabolism
process. As reported by Sarasquete et al. (1992), during egg development until the early stages of
larvae where secretions organs still undeveloped proteins, carbohydrates and lipids degradations
are requlated by endogenous enzymes produced in oocytes and yolk sac of newly hatched larvae
Kamler (1992) found that protein is a dominant element in the fish eggs and most of the protein is
transformed into embryonic tissue and partly converted into energy. In addition, Fyhn (1989) in
Kamler (1992) through the study of the development of embryos and larvae of halibut and cod found
that free amino acids and EFA are very important as the energy sources during embryonic development
Digestive enzyme activity of protease, α-amylase, and lipase ..... (Kamaruddin)
Figure 1.
Figure 2.
Figure 3.
404
The protease activity for 35 days rabbitfish larvae at the age of 2 to 35 DAH and its
relative change
The α-amylase activity for 35 days rabbitfish larvae at the age of 2 to 35 DAH and
its relative change
The lipase activity for 35 days rabbitfish larvae at the age of 2 to 35 DAH and its
relative change
in marine fishes. Free amino acids were importantly needed by the larvae as energy source when
energy from egg yolks are nearly finished. In contrast, previousstudy by Effendi (1996) found that
betutu larvae (Oxyeleotrismarmorata) at 2 days of age, produced protease enzyme activity much higher
around 0.216 m/mL/min) than found in the present study. Furthermore Mathavan et al. in Anggoro
(1992) stated that the yolk was the only source of energy for the development of the embryo. When
egg yolk finish, larvae need exogenous enzymes which obtained from live-feed.
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Prosiding Forum Inovasi Teknologi Akuakultur 2015
During the exogenous phase, protease activity found in this study was higher than the other
enzymes probably because of high proteins content in rotifer fed to the larvae. Haryati (2002) reported
that rotifer sconsisted of 54.32% protein; 11.86% fat; and1.01% ash,while the nutrent contents of
artemia nauplii are 55.27% protein; 16.02% fat and 7.20% ash (Watanabe, 1988). In the present study,
Table 2. Enzymes activity(U/mL/min) of rotifers and Artemia naupli
Kinds of life feed
Rotifer
Artemian nauplii
Enzymes activities (U/mL/minute)
α-amilase
Protease
Lipase
0.359
0.370
0.072
0.370
0.049
0.204
protease activity was alsodetected higher than activity of α-amylase and lipase life feed as shown in
Table 2.
Generally, the three digestive enzymes of protease, á-amylase and lipase increased with the
increas of the age and development of the digestive organs of the larvae. Kawai & Ikeda (1973)
reported that increase of enzyme activity was influenced by two factors: (1) fully developed enzyme
producing organs, (2) increased useof life feed at the end of yolk stage. Walfordetal (1991) also
stated that the life feed consumed by fish larvae contribution toan increase of the digestive enzymes
activity.
During the exogenous phase, increased lipase activity was lower than the increase of the protease
activity, but higher than the á-amylase activity at each larval group. This was probably due to the
lower lipase activities in both live feeds used during larval rearing (Table 2). In addition, lipid
content of thoselife feeds was also lower than their protein contents. Haryati (2002) reported that
the lipid content of rotifer was only 11.86% and for Artemia nauplii was 16.02% (Watanabe, 1988).
Similar to protease, lipase activity also increased with the increase of larval age and development of
the digestive organs. Kapoor et al. (1975) reported that development of the digestive organs
couldaffected the production of enzymes produced by a gland found in the digestive organs such as
intestines, pancreas, stomach, and intestinal wall.
The time where high digestive enzymes activity occured could be used as a basic references to
applyartificial feed. Accordingto Gawlickka et al. (2000), digestive enzyme activity was a good indicator
to determine the capacity of digestion, when high activityoccured, it might indicate that physiologically
larvae are ready to processexogenous feed. Based on the activity of digestive enzymes found in this
study, artificial feed could be applied afterthe age of 20-days of the rabbitfish larvae. This assessment
revealed that there was a clear relationship between the digestive enzyme activity and the development
of the digestive organs. When anatomical and histological structure of the digestive tracts have not
yet well developed, only small amount of endogenous enzymes are secreted and then activity increased
with increasing the larval ageuntil digestive organs have permanently developed.
CONCLUSIONS AND RECOMMENDATIONS
The increased activity of digestive enzymes related to the larvae age and the role of live feeds as
exogenous feed. Maximum relative increase of the protease activity in larvae occurred at the age of
20 DAH and the α-amylase enzyme occurred when the larvae age at 15 DAH, where as lipase occurred
when larvae age of 25 DAH.
Based on the activity of digestive enzymes found in this study, artificial feed could be applied
afterthe age of 20 DAH of the rabbitfish larvae.
REFERENCES
Anggoro, S. (1992). Efek osmotik berbagai tingkat salinitas media terhadap daya tetas telur dan
vitalitas larva udang windu, Penaeus monodon Fabricius. Disertasi, Program Pascasarjana. IPB. Bogor.
230h.
Digestive enzyme activity of protease, α-amylase, and lipase ..... (Kamaruddin)
406
Bergmeyer, H.U., & Grassi, M. (1983). Methods of Enzymatic Analysis. Volume ke-2 Weinheim: Verlag
Chemie.
Borlongan. T.G. (1990). Studies on the lipases of milkfish Chanoschanos. Aquaculture 89:315-325.
Duray, M., & Bagarinao, T. (1984). Weaning of hatchery-bread milkfish larvae from live food to artificial diets. Aquaculture, 41: 325-332.
Effendi, I. (1996). Perkembangan enzim pencernaan larva ikan betutu, (Oxyeleotrismarmorata belkr).
Tesis Magister Sains, Program Pascasarjana, InstitPertanianBogor, Bogor. 89 hal.
Gawlicka, A., Parent, B., Horn, M.H., Ross, N., Opsta, I., & Torrissen, O.J. 2000. Activity of digestive
enzymes in yolk-sac larvae of atlantic halibut Hippoglossus hippoglossus: indication of readiness for
first feeding. Aquaculture 184: 303-314.
Hara, S., Cono., H., & Taki, Y. (1986a). Spawning behavior and early life history of the rabbitfish,
Siganusguttatus in the laboratory. Aquaculture, 59: 273-285.
Haryati. (2002). Respon larva ikan bandeng (Chanoschanos Forskal) terhadap pakanbuatan dalam sistem
perbenihan. Disertasi. Program Pascasarjana Institut Pertanian Bogor, 165 hal.
Kamler, E. (1992). Early life history of fish: an energetics approach. Fish and Fisheries Series 4. Chapman
and Hall. London-New York-Melbourne-Madras. 267 p.
Kapoor, B.G., Smith, H., & Verighina, E.A. (1975). The alimentary canal and digestion in Teleost. In:
Russel, F.S., & Young, M. (Eds.). Mar. Biol. 13. Acad. Press. London, New york. San Francisco. P. 109211.
Kawai, S., & Ikeda, S. (1973). Studies on digestive enzymes of fishes. IV. Development of the digestive
enzymes of carp and black sea bream after hatching. Bull. Jpn. Soc. Sci. fish. 38:877-881.
Kurokawa, T., Shiraishi, M., & Suzuki, T. (1998). Quantification of exogenous protease derived from
zooplankton in the intestine of Japanese sardine (Sardinopsmelanoticus) larvae. Aquaculture, 161:491499.
Lante, S., Usman, Palinggi, N.N., & Rachmansyah. (2007). Uji coba pemijahan dan pemeliharaan larva
ikan beronang Siganusguttatus. Laporan Hasil Penelitian, Balai Riset Perikanan Budidaya Air Payau,
7 hal.
Rachmansyah, Usman, Lante, S., & Ahmad, T. 2007. Rabbitfihs (Siganus guttatus) Breeding and larvae
rearing Trial. Aquaculture Asia XII (3): 39-40.
Sarasquete, M.C., Polo, A., & Yufera, M. (1995). Histology and histochemistry of the development of
digestive system of larva gilthead sea bream. Sparusaurata L. aquaculture, 130:79-92.
Walford, J., Lim, T.M., & Lam, T.J. (1991). Replacing life food with microencapsulated diets in the
rearing of sea bass Lates calcarifer larvae: do the larva ingest and digest protein membrane
microcapsules. Aquaculture, 92: 225-235.
Watanabe, W.O. (1988). Larvae and larval culture. Pages: 117-152 in C.S., Lee., M.S., Gordon, and
Watanabe, W.O. (Editors). Aquaculture of milkfish (Chanos-chanos): State of the art. Oceanic Institute
Hawai.
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DISKUSI
Nama Penanya:
Bedjo Slamet
Pertanyaaan:
Penelitian ini perlu diperdalam, sejak umur berapa mulai diberikan pakan buatan, kalau bisa
dimasukan seawal mungkin
Tanggapan:
Menerima saran dari Pak Bedjo Slamet