Molekul, Vol. 13. No. 1, Mei 2018 : 80 – 91 Doi: 10.20884/1.Jm.2018.13.1.418

Molekul, Vol. 13. No. 1, Mei 2018 : 80 – 91 doi: 10.20884/1.jm.2018.13.1.418

The Age Dependent Activities of Digestive Enzymes in Rasbora, Rasbora lateristriata Blkr., (Pisces: Cyprinidae)

Untung Susilo1*, Purnama Sukardi2, Ridwan Affandi3

1Faculty of Biology, Jenderal Soedirman University, Purwokerto, Indonesia. 2Faculty of Fisheries and Marine Science, Jenderal Soedirman University, Purwokerto, Indonesia. 3Faculty of Fisheries and Marine Science, Bogor Agricultural University, Bogor, Indonesia. *email: [email protected]; [email protected] Received February 8, 2018; Accepted March 29, 2018; Available online May 31, 2018

ABSTRACT This study was to evaluate the digestive enzyme activity included a protease (trypsin & chymotrypsin), lipase, carbohydrase (amylase & cellulose) and alkaline phosphatase in Rasbora. This research was carried out using three different ages (2, 4 and 6 months) and in each age consisted of six groups (replicates). In this study 150 fish (±2 months age), 120 fish (± 4 months age) and 90 fish (± 6 months age) were used. All digestion enzyme activity was measured by the spectrophotometric method, except the lipase activity was by the titration method. The results showed that the distinctness of age resulted in a significant difference on total protease, trypsin, lipase, cellulase and alkaline phosphatase (P <0.05), but no significant difference in amylase activity (P> 0.05). Total protease and trypsin activities were higher in fish of age two months than fish age four and six months, but the activity of lipase, cellulase and alkaline phosphatase were higher in fish age of four months compared to two months age fish. Fish, with distinct age has the different nutrient digestion capacity as expressed by differences in the activity of the enzyme digestion, except amylase. These results contribute to the future development of digestive physiology, especially in Rasbora. Keywords: alkaline phosphatase, carbohydrase, lipase, protease, Rasbora

INTRODUCTION Lipase and carbohydrase activities are varied among fish with different feeding guilds The ability of fish to digest the feed is highly in which omnivorous fish show a lower lipase dependent on the activity of digestive activity than carnivores (Langeland et al., enzymes, such as protease, lipase, 2013). Amylase activity is generally higher in carbohydrase, and alkaline phosphatase. Acid herbivorous fish than in omnivores and protease or pepsin is particularly active in the carnivores (Day et al., 2011b). The ability of fish with stomach, as observed in Mormyrus fish to digest cellulose is highly dependent on Rume (Odedeyi & Fagbenro, 2010), Etroplus the presence of microbial symbionts in their suratensis and Oreochromis mossambicus digestive tracts (Maity, Kundu, Pramanik, & (Sankar et al., 2014), but not in fish without Patra, 2011; Ganguly & Prasad, 2012). stomach (Day, German & Tibbetts, 2011a) and Past studies have shown cellulase the Mediterranean fish (Caruso, Denaro, & activity in Oreochromis niloticus (omnivore), Genovese, 2009). but not in Horabagrus brachysoma (omnivore) The activities of alkaline protease such and carnivorous Parachanna obscura and as trypsin and chymotrypsin, have also been Gymnarchus niloticus (Fagbenro et al., 2005; studied in a variety of fish (Kamarudin, Otoi, Prasad & Suneesha, 2013). Alkaline & Saad, 2011; Savona, Tramati, & Mazzola, phosphatase activity was also affected by 2011). These enzymes showed different changes in the feed quantity and quality (Liu, activities in the fish digestive tract, according Zhang & Wang, 2010), but studies on Labeo to feed quality (Debnath et al., 2007; rohita indicated otherwise (Debnath et al., Chaudhuri, Mukherjee, & Homechaudhuri, 2007). The effect of age or size of the fish on 2012; Abdel-Warith, Younis, & Abdualla, the digestive enzyme activity has also been 2013), feeding guilds (Falcon-Hidalgo, documented in previous studies (Klahan, Forrellat-Barrios, Farnes, & Hernandez, 2011; Areechon, Yoonpundh, & Engkagul, 2009; Langeland, Lindberg, & Lund, 2013), Thongprajukaew, Kovitvadhi, Engkagul, & phylogeny (German, Horn, & Gawlicka, 2004; Rungruangsak-Torrissen, 2010; Susilo, Kumar et al., 2007), and sex (Thongprajukaew Yuwono, Rachmawati, Priyanto, & Hana, & Kovitvadhi, 2013). 2015). However, the effect of age on enzyme

80 The Age Dependent Activities of Digestive Enzymes in Rasbora Untung Susilo, at al. activity changes when diet and feeding guilds EXPERIMENTAL SECTION differ (Day et al., 2011a; Savona et al., 2011; Materials and Instruments Pena et al., 2015). In spite of elaborate study Nα-p-tosyl-L-arginine hydrochoride on digestive enzyme on various fish species, methyl ester (Sigma-Aldrich, AG), N-benzoyl- no published data are available for Rasbora L-tyrosine ethyl ester (Sigma-Aldrich, AG), digestive enzyme activities related to its age. Sodium acetate (Merck, AG), Folin & Feed consumed by fish will be used for Ciocalteu’s phenol reagent (Sigma-Aldrich), growth if it has been digested in the Starch (Bio Basic Canada, High Purity), gastrointestinal tract of fish. The digestive carboxymethylcellulose (Merck, Technical process of feed involving digestive enzymes Grade), Tris (hydroxymethyl) aminomethane such as proteases, lipases and carbohydrases, (Tris) (Sigma-Aldrich, ACS reagent, >99.8%), requires the compatibility between the feed hydrochloric acid (Merck, 36.5-38.0%), 3,5- consumed and the capacity of fish digestion, in dinitrosalicylic acid (DNS) (Sigma-Aldrich, order to achieve feed efficiency (Caruso et al., >98%), p-nitrophenyl phosphate (pNPP; 2009). The digestion product by the enzyme is Sigma-Aldrich, AG), p-nitrophenol (Sigma- a simple compound and readily absorbed by Aldrich, AG), NaOH (Sigma-Aldrich, AG), the fish intestine to support growth. Among the single centrifuge (Eppendorf, 5415 R), digestion products is a phosphate-bonded spectrophotometry (Hitachi, U-2900), channel compound which is a substrate for alkaline pipette (Serana), waterbath (JEIO-TECH, WB- phosphatase, so the hydrolysis product 20E). becomes easily absorbed (Ducasse-Cabanot et al., 2007; Silva et al., 2010). Therefore, a good Experimental Design feed quality supported by its enzyme capacity The fish sample (F1) was obtained from will be an important factor to support fish Wet Laboratory of Biology Faculty, Unsoed, growth, otherwise when low feed quality will Purwokerto. Fish with three different age have an effect on the poor of nutrient intake for maturity (age 2, 4 and 6 months) were rearing fish growth (Thongprajukaew et al., 2011; in three difference concrete tanks (125 x Santigosa et al., 2011). 250x50cm) and feed twice days at 08:00-09:00 Rasbora (Rasbora lateristriata Blkr.) is pm and 15:00-16:00 pm. Fish were fed with a wild fish that has the potential to be artificial diet containing crude protein 33.71 %, developed, because it has good taste and high crude lipid 5.67 % and NFE 38.96 % as a nutritional value. Biological studies of the previous study on Rasbora (Susilo, Sukardi & genus Rasbora have been conducted, but Affandi, 2016) during two weeks rearing. In mostly focused on the taxonomy aspects of the this study 150 fish (age ±2 months, mean fish (Kottelat, Whitten, Kartikasari, & weight 0.28±0.04 g), 120 fish (age ±4 months, Wirjoatmodjo, 1993), its length to weight mean weight 0.44±0.08 g) and 90 fish (aged ±6 relationships (Muchlisin, Musman, & Azizah, months, mean weight 0.74± 0.12g) were used. 2010), fecundity and spawning frequency Fish was fasted for 24 h before the sampling. (Muchlisin, Musman, Fadli, & Azizah, 2011), At the end rearing fish from each concrete natural feed (Sulistiyarto, 2012) and its tanks divided into six pools sample. distribution (Rosadi, Yuli, Setyohadi, & Crude enzyme preparation. Bintoro, 2014). Using a tissue Homogenizer (Heidolph A scientific study related to the digestive Dixie 900), the digestive organs were isolated physiology of fish will be necessary to support and homogenized (1: 8 w/v) in a cold solution the domestication efforts of Rasbora in the of 50 mM Tris-HCl buffer (pH 7.5) containing future. Therefore, a research focusing on the 10 mM NaCl. Homogenates were centrifuged digestive capacity associated with digestive at 12.000 RPM (Eppendorf, 5415 R) for 15 physiology as expressed by digestive enzyme minutes at 4 °C and the supernatant was stored activity provides new insight , especially for in the refrigerator at -80 °C (Thongprajukaew Rasbora. The objective of this study was to et al., 2010) until used for enzyme activity evaluate the digestive enzyme activities of total measurements. Soluble protein contents in the protease, trypsin, chymotrypsin, lipase, supernatant were determined using albumin as amylase, cellulase and alkaline phosphatase a measurement standard. with different age in Rasbora.

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Determination of Digestive Enzymes Titration method with olive oil emulsion Activity as substrate was used to measure lipase activity A modification of casein hydrolysis was (Borlongan, 1990). Reagent mixture consisted used to measure the activity of total protease of a substrate (2000 µL), 0.1 M Tris-HCl (Thongprajukaew et al., 2010). The buffers buffer at pH 8.0 (3000 µL) and extract enzyme included glycine-HCl 0.1 M (pH 2.0), citrate (500 mL) was incubated for 2 h at 37 °C, then 0.1 M (pH 5.0), phosphate 0.1 M (pH 6.9), stopped by the addition of 3000 µL ethyl Tris-HCl 0.1 M (pH 8.1) and Glycine-NaOH alcohol 95 %(v/v). The reaction mixture was 0.1 M (pH 10.0) and KCl-NaOH (pH 12.5) 0.1 titrated with 0.01 N NaOH using 0.9 % (w/v) M. The reaction mixture consisting of casein phenopthalien in ethanol as indicator. The 1% (w/v) in buffer (450 µL), buffer (450 µL) same procedure was conducted to blank tube and crude enzyme extract (100 µL) were except enzyme extract was added after ethyl incubated for 60 min at 37 °C. This reaction of alcohol and immediately before titration. the mixture was stopped by the addition of Lipase activity expressed as units of activity 1000 µL TCA 8% (w/v) reagent. per mg protein. Lipase activity unit (U) was The same procedure was done to blank defined as the volume of 0.01 N NaOH tube except that the enzyme extract was added required to neutralize the fatty acid released after the TCA 8%(w/v). After storing for 1 during the 2 hours of incubation of the hour in 4 oC, the sample was centrifuged at substrate and after correction by the blank 6000 rpm for 10 minutes. The absorbance of sample. supernatant was measured at a wavelength of A standard protocol of 3,5-dinitro- 280 nm. Protease activity was calculated using salicylic acid (Sigma-Aldrich, AG) using a standard curve of tyrosine. The amount of starch as substrate was used to measure enzyme required to catalyze the formation of 1 amylase activity (Klahan et al., 2009), whereas µg tyrosine min-1 was defined as one unit of phosphate 0.1 M (pH 6.9) and Tris-HCl 0.1 M enzyme activity. (pH 8.1) reagents were used as the buffer. A The trypsin activity was measured using Reagent mixture which consisted of a substrate Nα-p-tosyl-L-arginine hydrochloride methyl (750 µL), buffer (700 µL) and extract the ester (TAME; Sigma-Aldrich, AG) as substrate enzyme (50 µL) was incubated for 20 min at (Khishimura, 2008). Reagent mixture consisted 37 °C and the reaction was stopped by the of 100 µL of enzyme extract and 300 µL of 10 addition of 1500 µL DNS 1 %(w/v). The mM TAME in 2600 µL of 46.0 mM Tris-HCl reaction mixture was placed in boiling water buffer at pH 8.1 with 11.5 mM CaCl2. for 5 minutes. Reactions were started by adding the enzyme The same procedure was conducted to extract into a mixture of TAME and buffer blank tube, except enzyme extract was added solution. TAME hydrolysis were measured as after the DNS 1 %(w/v). The absorbance of the change in absorbance at 247 nm for 3 the reaction mixture was measured at 540 nm, minutes at 30 oC. Trypsin activity was after previously added 3000 µL of doubly expressed as the change in absorbance min-1 distilled water. The amount of maltose mg-1 protein in enzyme extract. removed from substrate was determined from a N-benzoyl-L-tyrosine ethyl ester standard curve of maltose. Amylase activity (BTEE;Sigma-Aldrich, AG) was applied as a was calculated as the amount of maltose substrate to measure the activity of released (µmol) min-1 mg-1 protein in enzyme chymotrypsin (Lazzari et al., 2010). Reagent extract. mixture consisted of 100 µL of enzyme extract Measurement cellulose activity used 3,5- and 1400 µL of 2.0 mM BTEE in 1500 µL of Dinitrosalicylic acid (Sigma-Aldrich, AG) 80.0 mM Tris-HCl buffer at pH 7.8 with 0.1 M method with carboxymethylcellulose (CMC) CaCl2. Reactions were started by addition of as substrate (Savona et al., 2011). Reagent enzyme extract to the mixture BTEE and mixture consisted of a substrate (300 µL), 0.1 buffer solution. BTEE hydrolysis was M sodium acetate buffer (300 µL) and enzyme measured as change absorbance at 256 nm for extract (60 µL) were incubated for 10 minutes 3 minutes at 30 oC. The activity of at 40 °C then the reaction was stopped by the chymotrypsin was expressed as the change in addition of 900 µL of DNS 1% (w/v) reagent. absorbance min-1 mg-1 protein in enzyme The reaction mixture was placed in boiling extract. water for 15 minutes. The same procedure was

82 The Age Dependent Activities of Digestive Enzymes in Rasbora Untung Susilo, at al. done at blank tube, except enzyme extract was was expressed in U (µg p-nitrophenol added after the DNS 1 % reagent. The liberated) mg-1 protein in enzyme extract. absorbance of the reaction mixture was Statistical Analysis measured at 640 nm, after previously added Enzyme activity values were tested by 3000 µL of doubly distilled water. The amount analysis of variance (ANOVA), followed by of glucose released from substrate was Tukey’s multiple comparison tests. Statistical determined from the glucose standard curve. analysis was performed using SPSS 18.0 Cellulose activity was calculated as the amount package version of Windows software. of glucose released (µmol) min-1 mg-1 protein in enzyme extract. RESULTS AND DISCUSSION A modified method using p-nitrophenyl phosphate (pNPP; Sigma-Aldrich, AG) as the There were significant differences of total substrate was used to measure alkaline protease activity between the age of the fish (p phosphatase activity (Debnath et al., 2007). <.05) and the high protease activity was found The reaction mixture consisted of 1000 µL of at pH 6.9 to pH 10, as well as the fish ages of substrate buffer (0.1 M Glycine, pH 10.4; 3 two months have the highest protease activity mM p-nitrophenyl phosphate in 1 mM MgCl2) (Table 1). and the enzyme extract (50 µL) were incubated In this study, the total protease activity for 30 minutes at 30 oC, then the reaction was was not found at pH 1.9 and it showed a low at stopped by the addition of 1500 µL of 3.0 N pH 5.1. This phenomenon indicated that there NaOH. The same procedure was conducted to was no pepsin-like activity that was active at blank tube, except enzyme extract was added acidic pH. These results were different from after of 3.0 N NaOH. The product of previous studies on Betta splendens hydrolysis of p-nitrophenyl phosphate (pNPP) (Tongprajukaew et al., 2010) , Tilapia rendali was measured at 405 nm. Alkaline phosphatase (Hlophe & Moyo, 2013) and Horabagrus activity was determined from a standard curve brachysoma (Prasad & Suneesha 2013) and of p-nitrophenol. Alkaline phosphatase activity Etropus suratensis and Oreochromis mossambicus (Sankar et al., 2014). Tabel 1. The total protease activity of Rasbora at different ages and pH activity Activity on various pH Age of -1 fish (U mg protein) (months) 1.9 5.1 6.9 8.1 10 12.5 2 (n=6) - 9.15±1.52a 127.56±26.11a 140.48±39.29a 131.81±42.00a - 4 (n=6) - 2.94±1.59b 91.92±24.02b 100.33±35.10ab 90.06±26.14ab - 6 (n=6) - 3.52±2.98b 80.21±13.86b 75.41±10.53b 69.26±26.02b -

Figure 1. Trypsin and chymotrypsin activities in Rasbora at different age. Means (+sd) with different superscript letter are significantly different (P<.05; n=5).

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The existence of the activity of pepsin or niloticus (Namulawa et al., 2013), Horabagrus pepsin-like seems to be related to the presence brachysoma (Prasad & Suneesha, 2013) and of a functional stomach. Rasbora on this Labeo rohita (Umalatha, Kushwaha & research may be included in the category of Gangadhar, 2016), but it is contrary to the fish with non functional stomach since there is results on Limia vittata & Gambusia no pepsin-like activity. This funding has also punctate (Falcon-Hidalgo et al., 2011) and confirmed with Zenarchopterus buffonis Hyporhamphus regularis ardelio (Day et al., (Abidin et al., 2016). At all the fish ages the 2011a). total protease activity measured at pH 6.9 to So it seems that the Rasbora changes in 10.0 showed a high activity indicating that protein digestive capacity, as indicated by protease digestion of the fish is active at a the increased activity of total protease, neutral to alkaline condition which occurred in dominated by the activity of trypsin and not by the segment of intestine. the activity of chymotrypsin. Trypsin activity The results of this study do not differ is higher than chymotrypsin, reflecting the from previous research on Betta spledens absence of growth inhibition in the fish, (Thongprajukaew et al., 2010), Odontobutis and the high activity of trypsin can be an obscures (Ye, Chen & Zhu, 2013) and indicator of increased growth and efficiency Lutjanus guttatus (Pena et al., 2015). The total of feed on fish ( Chan et al., 2008; protease activity was also found higher in two Rungruangsak-Torrissen et al., 2009). months old fish than in four and six months Lipase activity was found significantly old fish, which indicates that young fish have a different between the age of the fish (P <.05), higher protein digestive capacity than the and the lowest activity was found in the fish older. This is in contrast to results of previous with two months of age, but the activity of studies on fish omnivore Oreochromis lipase with four and six months of age did not niloticus (Klahan et al., 2009) and herbivore differ between the two (Figure 2). The results Hyporhamphus regularis ardello (Day et al., of this study were lower than previous studies 2011a) and Eurasian perch, Perca fluviatilis on Glyptosternum maculatum (Xiong, Xie, (Langeland et al., 2013). The higher activity Zhang & Liu, 2010). The incubation time of protease in two months old fish than in differences in this study were shorter than four and six months old fish may be previous research indicating one cause of the associated with the higher protein differences in the results of this research. requirements for growth, as was observed in However, the result is consistent with previous Oreochromis niloticus (Abdel-Tawwab, research on Oreochromis niloticus (Klahan et Ahmad, Kattab & Halaby, 2010). al., 2009). Results of previous studies had also Trypsin activity was significantly shown that the activity of lipase was in the different among the age or weight of the fish (p range of neutral to alkaline pH, which <.05) with the highest trypsin activity indicated that the lipase working on the found in fish age of two months (Figure 1), digestive tract of fish was a lipase secreted by but the activity of chymotrypsin was not the pancreas or hepatopancreas (Zambonino- significantly different among age or weight Infante & Cahu, 2007) with the intestine as the of the fish (p>.05). The results of this study did main segment of fat digestion (Wu, Hong & not differ from previous research on Lates Zhang, 2010).

Figure 2. Lypase activity in Rasbora at different age. Means (+sd) with different superscript letter are significantly different (P<.05; n=6)

84 The Age Dependent Activities of Digestive Enzymes in Rasbora Untung Susilo, at al.

Figure 3. Amylase activity in Rasbora at different age. Means (+sd) with different superscript letter are significantly different (p<.05;n=6). The higher activity of lipase in a four which reflects that the starch hydrolysis month old fish than in two months old fish in process occurs at a neutral to alkaline this study, was in line with the decrease in condition. protease activity. The phenomenon of a Amylase, that is active in such condition decrease in protease and increased lipase generally secreted by the pancreas or activity has been documented in fish that hepatopancreas and works in the area of obtained intake of protein and high intestine. Activities of amylase were dominant carbohydrate as occurred in Dentex dentex in the intestine with the alkaline condition has (Perez-Jimenez et al., 2009), Labeo brick been documented in Odontobutis obscures (Ye (Mondal, Kaviraj, & Mukhopadhyay, 2012) et al., 2013), Lates niloticus (Namulawa et al., and Megalobrama amblycephala (Habte-Tsion 2013), Etroplus suratensis and Oreochromis et al., 2013). mossambicus (Sankar et al., 2014) and Tilapia There was no significant differences rendali (Hlophe & Moyo 2013). Differences in amylase activity between age of the fish age of the fish in this study seem to also (P>.05), but amylase activity was significantly produce no significant difference of amylase influenced by the pH of incubation (P <.05) activity, which indicates no change in the with the lowest activity at pH 10 (Figure 3). In capacity of fish to digest complex this study, the highest amylase activity was carbohydrates, especially starch. A similar found at pH 6.9 and 8.1, but it decreased at pH phenomenon occurred in fish that has no 10.0. The results of this study are lower than in stomach as Hyporhamphus regularis ardelio previous studies on fish Betta splendens (Day et al., 2011a), but different results were (Thongprajukaew et al., 2010) and Diplodus seen in Horabagrus brachysoma (Prasad & puntazzo (Savona et al., 2011). High amylase Suneesha, 2013). activity at neutral to slightly alkaline pH,

Figure 4. Cellulose activity in Rasbora at different age. Means (+sd) with different superscript letter are significantly different (P<.05;n=6).

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Figure 5. Alkaline phosphatase activity in Rasbora at different age. Means (+sd) with different superscript letter are significantly different (P<.05;n=6) In this study the cellulose activity was phosphatase activity with the increase in lipase significant differences between the ages of the activity, was documented in fingerling fish (P <.05) and high activity in fish ages of Mesopotamichthys sharpeyi (Rahimi, Sayed four and six months (Figure 4). Cellulose Mohammad & Mohammad, 2015). Alkaline activity changed with the increase age of the phosphatase is an enzyme involved in the Rasbora. Six months old fish had higher breakdown of phosphate bonds so that it is cellulose activity than two months old fish, but easily absorbed in the substrate of proteins, did not differ from four months old fish. The lipids and carbohydrates, and the decrease in results of this study indicated that Rasbora at the intake of nutrients such as lipids can reduce six months age had higher ability to digest its activity (Ducasse-Cabanot et al., 2007), and crude fiber than fish of two months age. generally alkaline phosphatase works on the Increased capacity to digest crude fiber of the surface of intestine enterocytes microvillus fish indicated that in fish intestine, it is (Minjoyo, Tan-Fermin, & Macaranas 2003), suspected that symbiotic microorganisms so, the highest activity of alkaline phosphatase synthesize cellulose, because cellulose in the in larvae Rutilusfrisiikutum occur after fish intestine derives from the secretions of the microvillus is formed (Hassanatabar, Ouraji, gut of microbes (Krogdahl, Hemre & Esmaelli & Babaei, 2013). Mommsen, 2005; Kar & Ghosh, 2008). The activities of cellulose in the CONCLUSIONS intestine of fish were also shown in previous Fish with two months of age had higher protein studies on fish omnivore (Fagbenro et al., digestion than the older one, but the ability to 2005; Tongsiri, Mang-Amphan & digest carbohydrates, especially starch was not Peerapornpisal, 2010; Savona et al., 2011). influenced by differences in age. The ability of However, previous studies showed that Rasbora to digest fat and fiber increased with cellulose activity was related to the the increase age of the fish as well as feed composition of the feed in the digestive tract. absorption efficiency, improved with the Carnivorous fish that consumed macro algae increase of age reflected by the activity of had higher cellulose activity compare to fish alkaline phosphatase. that did not consume the macro algae

(Chaudhuri, Mukherjee, & Homechaudhuri, ACKNOWLEDGEMENTS 2012). Alkaline phosphatase activity showed a Thanks to the Indonesian Ministry of Research significant difference between the age of the and Higher Education that has provided fish (P <.05), and high alkaline phosphatase research funding through the Doctoral activity in fish age of four months (Figure 5), Dissertation Grant from the Ministry of but it was not different with six months old Research, Technology and Higher Education in fish. Increased activity of alkaline phosphatase 2016 so that this research can be accomplished. in four month old fish in this study reflected the increase of nutrient absorption ability with REFERENCES decreasing the activity of protease and Abdel-Tawwab, M., Ahmad, M.H., Kattab, increasing lipase activity in Rasbora. The Y.A.E. & Shalaby, A.M.E., 2010. phenomenon of the increase of alkaline

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