Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agricultural University, Dramaga Campus, Bogor 16680, Indonesia

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1 HAYATI Journl of Biosciences Jnury 2015 Vol. 22 No. 1, p EISSN: Avilble online t: DOI: /hjb Growth Response nd Feed Utiliztion of Gint Gourmi (Osphronemus gormy) Juvenile Feeding Different Protein Levels of the Diets Supplemented with Recombinnt Growth Hormone DARMAWAN SETIA BUDI 1*, ALIMUDDIN 2, MUHAMMAD AGUS SUPRAYUDI 2 1 Study Progrm of Aquculture Science, Deprtment of Aquculture, Fculty of Fisheries nd Mrine Science, Bogor Agriculturl University, Drmg Cmpus, Bogor 16680, Indonesi 2 Deprtment of Aquculture, Fculty of Fisheries nd Mrine Science, Bogor Agriculturl University, Drmg Cmpus, Bogor 16680, Indonesi Received June 12, 2014/Accepted December 11, 2014 The purpose of this study ws to exmine the effect of dietry supplementtion with recombinnt growth hormone () on the growth nd dietry utility of juvenile gint gourmi. The ws mixed with chicken egg yolk nd spryed on to rtificil feed with different protein levels (34, 28, nd 21%; isoenergy). Ech tretment group of gourmi ws pired with control group tht received feed of the sme protein level, but without supplementtion. Juvenile of gint gourmi (weight g) were fed diets contining, to pprent stition, 2 times week. Fish were rered from less thn 2 months old for 42 dys in 100 L glss quri t n initil density of 10 fish per qurium. At the end of this period, the biomss nd dily growth rte (SGR) of the fish were mesured nd the feed conversion rtio clculted nd compred. Our dt showed tht fish fed -supplemented diets experienced higher growth thn fish in the control groups nd showed tht fish with higher protein diets experienced higher growth thn the groups with less protein diets. The group with the highest biomss gin, SGR, nd lowest feed conversion rtio (FCR) ws the group fed 34% protein diet supplemented with. Furthermore, biomss gin, SGR, nd FCR in the tretment group with 28% protein diet mtched the mesurements of the non- control group receiving 34% protein diet (P > 0.05). We conclude tht gint juvenile gourmi given feed supplemented with recombinnt growth hormone will experience incresed growth nd dietry utility compred with gourmi given the sme feed without supplementtion. Key words: feed protein levels, growth, gint gourmi, INTRODUCTION Gint gourmi (Osphronemus gormy) is n importnt quculture commodity in Indonesi. Gint gourmi grows t reltively slow rte, which cn hinder efforts to increse gint gourmi production to meet high mrket demnd. However, if incresing the production of gint gourmi is difficult, it my be possible to increse the growth of ech fish. There re severl known strtegies for incresing fish growth rte. The use of recombinnt growth hormone () in fish diet is n esy method tht cn yield quick results in fish growth. Mny studies hve exmined the effect of supplementtion on fish species such s Nile tilpi (Acost et l. 2007), rinbow trout (Llorente et l. 2004), blone (Moriym et l. 2008), nd yellow til (Pedroso et l. 2009). Regrding gint gourmi Corresponding uthor. Phone: Fx: , E-mil: drm.jhders@gmil.com specificlly, prior reserch hs shown tht ppliction vi immersion method significntly increses the growth rte of gint gourmi (Irmwti et l. 2012). The ppliction of with 3 mg/kg dose in gourmi feed lso resulted in higher growth thn control groups without (Sfir 2012). This study ims to exmine the combined effect of ppliction nd vrition of dietry protein levels, which hs not yet been explored. The cn increse metbolic cpcity, especilly lipid metbolism nd optimiztion of protein synthesis, on gint gourmi. Fish with fster reltive growth rte exhibited the protein sprring effect tht occurs with incresed growth hormone levels (Rsmussen et l. 2001; Irmwti et l. 2012). Growth nd development of body tissue in fish re influenced by the blnce of protein nd energy in their feed. Feed with high protein content will not necessrily ccelerte growth if the totl energy content is low. The energy content of feed is

2 Vol. 22, 2015 Growth of Gint Gourmi Feeding with Recombinnt Growth Hormone 13 minly used for primry metbolic ctivities, such s respirtion, ion trnsport/metbolites, regultion of body temperture nd other physiologicl functions. Energy for these ctivities generlly derives from non-protein nutrients (fts nd crbohydrtes). If the energy contribution from non-protein mteril is reltively low, protein must be tpped to produce energy. This reduces the mount of protein vilble for building body tissues. In other words, the ddition of non-protein nutrients for energy production cn minimize the use of protein s n energy source nd mximize its use for growth nd body mss (protein sprring effect). This improves the function of protein in supporting the growth of fish (Furuichi 1988; Hsn & Khn 2013). The supplementtion is expected to increse the efficiency of protein utiliztion in the diet. The purpose of this study ws to exmine the effect of different dietry protein levels supplemented with recombinnt growth hormone () on the growth nd dietry utility of juvenile gint gourmi. MATERIALS AND METHODS Experimentl Diets nd Production of. We used 3 types of experimentl feed, ech with different proportion of protein; i.e. 21, 28, nd 34%. Gross energy (GE) for ech type of feed ws the sme, pproximtely kcl GE/kg of feed. Feed formultion is shown in Tble 1. production ws performed using Escherichi coli BL21 contining pcold-1/relgh vector expression. Bcteril culture nd collection methods were performed s described by Alimuddin et l. (2010). Tble 1. Formultion nd chemicl composition of the experimentl diets Ingredients (%) Fish mel Soyben mel Pollrd Fish Oil Tpioc Vitmin nd minerl mix Protein diets (%) Proximte (% dry mtter) nd gross energy (GE) Crude protein Lipid Minerl mtter Crude fibre Crbohydrte* GE (kcl/kg)** , , , *Crbohydrte = Dry mtter (Crude protein + Lipid + Crude fibre + Minerl mtter); **GE = Gross energy protein 5.6 kcl/g, ft 9.4 kcl/g, crbohydrte 4.1 kcl/g (Wtnbe 1988). The ws coted with 20 mg chicken egg yolk nd then pplied to feed pellets t dose of 3 mg/kg (Sfir 2012). The 3 tretment groups tht received feedt ech protein level, coted with, were pired with control groups tht received the feed t the sme protein level but without, for totl of 6 experimentl groups. We conducted 3 repetitions of the experiment for ll 6 groups. Fish Rering. Juvenile of gint gourmi (BW g) were rered for 42 dys in 100 L glss quri t initil density of 10 fish per qurium. Wter ws chnged 50% per dy ech fternoon. Oxygen levels were mintined using ertion. Fish were fed three times dy (morning, fternoon, nd evening) until pprent stition. Tretment groups were given feed contining 2 times week, t n intervl of 3 dys. Dt Collection nd Sttisticl Anlysis. Fish body weight ws mesured every 14 dys until t the end of the experiment. For ech fish, we mesured heptosomtic index (HSI), proximte composition, excreted totl mmoni nitrogen (TAN), blood glucose nd triglycerides levels, liver nd muscle glycogen, nd enzyme ctivity, t the end of experiment. All dt were nlyzed by twowy ANOVA using SPSS sttisticl softwre with P > Dily growth rte (SGR) ws clculted by the eqution: t Wt 1 W0 SGR = x 100% SGR = dily growth rte; Wt = Averge weight of n individul t the end of the rering period (g); Wo = verge weight of n individul t the beginning of the rering period (g); t = length of time mintennce (dys). Feed conversion rtio (FCR) ws clculted using the eqution: FCR = [P/((Wt + Wm)-Wo)] X100; [FCR = feed conversion rtio; P = mount of feed given during rering (g); Wt = biomss of fish t the end of the rering period (g); Wo = biomss of fish t the beginning of rering (g); Wm = Weight of fish tht died during rering (g)]. Protein nd lipid retention were clculted bsed on Tkeuchi (1988). The liver ws removed from ech fish nd weighed for clcultion of the heptosomtic index (HSI = 100 x liver weight/body weight). Mesurement of Prmeters. Anlysis of totl mmoni excretion ws determined by mesuring totl mmonium nitrogen (TAN) in the culture medi using test kit NH 3 /NH 4+ (API Mrs Fishcre North Americ Inc., USA). Initil mesurement ws crried out fter the fish hd fsted for 24 h nd the quri

3 14 BUDI ET AL. HAYATI J Biosci were filled with new wter ( mg TAN/L). This ws performed 24 h fter the end of the fish were fed to stition. TAN ws clculted for ech unit of feed consumed per fish biomss, s modified from Supryudi et l. (2014). Blood glucose levels were mesured by enzymtic colorimetric method using liquicolor GLUCOSE test (Humn mbh, Germny). A complete proximte nlysis of ech fish ws crried out on the first nd lst dy of the experiment ccording to the methods of Tkeuchi (1988). Blood triglycerides were mesured vi enzymtic colorimetric test with lipid clering fctor (LCF) using liquicolor mono TRIGLYCERIDES (Humn mbh, Germny). Liver nd musle glycogen ws mesured using the procedure in Wedemeyer nd Ysutke (1977). Enzyme ctivity, such s protese, milse, nd lipse, were mesured ccording to Bergmeeyer nd Grssi (1983), Worthington (1993), nd Borlongn (1990). RESULTS Our study results showed tht supplementtion of fish diets resulted in higher growth thn diets without supplementtion, t ech protein level tested (Tble 2). The highest biomss gin, SGR, nd feed consumption, nd the lowest FCR were found in the group given 34% protein diet supplemented with. The lowest biomss gin, SGR, nd feed consumption, with the highest FCR were found in the group given 21% protein diet without supplementtion. Furthermore, biomss gin, SGR, feed consumption, nd FCR in the group given 28% protein diet supplemented with ws comprble to tht of the control group receiving 34% protein diet without supplementtion. In ddition, biomss, SGR, feed consumption nd FCR responses showed no interction between dietry protein levels nd supplementtion; but ech of them independently to ffect re prmeters (Tble 2). We found significnt correltion between dietry protein levels nd supplementtion, nd protein nd lipid retention, nd lso protein nd lipid body content. There ws n interction between dietry protein levels nd supplementtion. Protein nd lipid body content nd retention in tretment groups t ll dietry protein levels were higher thn tht of the groups t corresponding dietry protein levels who did not receive supplementtion, except for the groups receiving 28% protein diet (Tble 2). We found tht HSI ws inversely correlted to dietry protein levels, but tht supplementtion Tble 2. Biomss gin, specific growth rte (SGR), feed consumption, feed convertion rtio (FCR), wet protein body content, protein retention, wet lipid body content, nd lipid retention of the gint gourmi (Osphronemus gormy) fter feeding with different protein levels nd supplemented with recombinnt fish growth hormone () Prmeters Lipid retention (%) Wet lipid body content (%) Protein retention (%) Wet protein body content (%) Biomss gin (g) SGR (%) Feed consumption (g) FCR Dietry protein levels c b b c b b c bc e d b c b bc b c b c c c d c bc bc b b e d c b b e ** d c b b Non * Non Non 21% 28% 34% P<0.000 P<0.000 P< P < P < P < P < P < P < P < P < P < Two-wy ANOVA Protei diets (P) (R) PxR (Interction) Stndrd Error *Non is the dietry without supplementtion; **Different superscript letters in the sme column indicte significnt different t P < 0.05 bsed on Duncn s multiple rnge test.

4 Vol. 22, 2015 Growth of Gint Gourmi Feeding with Recombinnt Growth Hormone 15 significntly incresed the HSI vlue of groups t ech dietry protein levels (Tble 3). Furthermore, the HSI of the group receiving the supplemented 28% protein diet ws comprble with the HSI of the group receiving the 34% protein diet without supplementtion. In this study we did not find tht dietry protein levels nd supplementtion hd ny influence on ech other, nor on the effect of either fctor in regrds HSI. We found significnt increse in TAN excretion with the increse of dietry protein levels (Tble 3). The suplementtion decresed TAN excretion, except in the group receiving n supplemented 28% test diet compred to the control group without supplementtion. As with HSI, we found no evidence tht dietry protein levels nd supplementtion worked in ny wy to lter the effect tht ech fctor would hve independently on TAN excretion. TAN excretion in the group given the -supplemented 28% protein diet ws comprble to the TAN excretion mesured in the group given the 34% protein diet without the supplementtion, while TAN excretion in the group tht received the -supplemented 21% protein diet ws comprble to tht mesured in the group tht received the 28% protein diet without supplementtion. The lowest TAN excretion ws observed in the group receiving the supplemented 21% protein diet supplemented with, nd the highest TAN excretion ws found in the group tht received the 34% protein diet without supplementtion. The supplementtion significntly incresed the levels of blood glucose in tretment groups t ll protein levels, but we did not find tht dietry protein levels nd supplementtion hd ny influence on ech other, nor on the effect of either fctor in regrds to blood glucose levels (Figure 1). Blood glucose levels in groups receiving supplemented feed ( mg/dl) were on verge, cross ll protien levels, 20.67% higher thn those for groups without supplementtion tretments ( mg/dl). However, triglyceride levels in ll groups, both treted nd control, were comprble, with the exception of the group receiving 21% protein diet without supplementtion, whose triglycerides levels were significntly lower thn ll tretments (Figure 2). Both dietry protein levels nd supplementtion significntly ffected liver nd muscle glycogen levels (Tble 3). Unlike the other mesurements in this study, we did find tht dietry protein level nd supplementtion hd n interctive effect on ech other, incresing the impct ech fctor would ordinrily hve independently. Liver nd muscle Tble 3. Heptosomtic index (HSI), totl mmoni nitrogen (TAN) excretion, liver glycogen, muscle glycogen, milse ctivity, lipse ctivity, nd protese ctivity of the gint gourmi (Osphronemus gormy) fter feeding with different protein levels nd supplemented with recombinnt fish growth hormone () Prmeters Protese (U/mg substrte) Lipse (U/mg substrte) Amilse (U/mg substrte) Muscle glycogen (mg/g smple) Liver glycogen (mg/g smple) TAN excretion (mg/g feed/g fish) HSI Dietry protein levels b b b b b bc bc c bc b b c c b b b c b c bc b bc bc c c c d b bc b b ** d c e d Non * Non Non 21% 28% 34% P < P < P < P < P < P < P < P < P < Two-wy ANOVA Protein diets (P) (R) PxR (Interction) Stndrd Error *Non refers to the diet without supplementtion; **Different superscript letters in the sme column indicte significnt differences t P < 0.05 bsed on Duncn s multiple rnge test.

5 16 BUDI ET AL. HAYATI J Biosci Glucose level (mg/dl) b 21% 28% 34% Dietry protein levels Figure 1. Glucose level in blood (mg/dl) of gint gourmi (Osphronemus gormy) juvenile fter feeding with different protein levels nd enriched fish recombinnt growth hormone (). The different letters bove the br indicte significnt difference (P < 0.05) bsed on Duncn s multiple rnge tests. without suplementtion, with suplementtion. Triglycerides level (mg/dl) b 21% 28% 34% Dietry protein levels Figure 2. Triglycerides level in blood (mg/dl) of gint gourmi (Osphronemus gormy) juvenile fter feeding with different protein levels nd enriched fish recombinnt growth hormone (). The different letters bove the br indicte significnt difference (P < 0.05) bsed on Duncn s multiple rnge tests. without suplementtion, with suplementtion. glycogen levels of fish fed supplemented 21% protein diets were higher thn those in fish fed 21% protein diets without supplementtion tretments. The supplementtion in 21% protein diets tretment significntly incresed the ctivity of milse, lipse, nd protese; however supplementtion did not significntly ffect this ctivity for the 28 nd 34% protein tretments. There ws n interction between dietry protein levels nd supplementtion (Tble 3). b b DISCUSSION Administrtion of Incresed Fish Appetite. Appetite is thought to be influenced by incresed growth hormone stimultion induced by the hormone ghrelin (Volkoff et l. 2005; Debnnth 2010). The effect of supplementtion in incresing ppetite ws pprent in the incresed feed consumption of fish rered in this reserch (Tble 2). In others, feed consumption lso ws ffected by feed pltbility relted to dietry protein levels (Tble 2). Pltbility of feed reltes directly to its ttctiveness to fish, determining whether fish will seek, cpture, nd ingest (cceptbility) of feed. Pltbility of feed relting to nutrient content, especilly free mino cids such s glycine, lnine, nd betine; s well s energy of feed (Shmushki et l. 2007). In this reserch, the increse in pltbility of feed occurs in the feed with higher protein content. Food intke will ffect the ctivity of digestive enzymes. Digestive ctivity is influenced by the substrte directly nd by growth hormone (GH) indirectly (Debhnnt 2010; Mtrug et l. 2012; Irmwti et l. 2012). This ws supported by our results: dietry protein levels nd dministrtion triggered digestive enzyme ctivity, with trend tht enzyme ctivity (protese, mylse, nd lipse) of lower dietry protein levels tretments were the sme compred to higher dietry protein levels tretments; giving effect n increse in enzyme ctivity t 21% protein feed (Tble 3). The increse in blood glucose in groups receiving supplementtion ws comprble t ll dietry protein tretment levels (Figure 1); protein composition of feed did not seem to ffect blood glucose levels. Glucose functions s source of energy (Hemre et l. 2002). Blood glucose levels were higher in the groups receiving tretments thn in those without, regrdless of protein levels, nd we mesured higher energy vilbility in the blood fter the fish in these groups fter they fsted for 24 h. The high blood glucose levels were followed by n increse in liver nd muscle glycogen (Tble 3). This is consistent with the findings of Kersten (2001), tht unused glucose will be stored s glycogen nd the excess will be converted into triglycerides. We confirmed this process in our finding of n increse in triglyceride levels in groups given supplementtion, especilly in the group receiving the 21% protein diet (Figure 2). Incresed triglycerides were mesured in the treted fish, fter 24 h fsting, due to endogenous synthesis of triglycerides derived from glucose (lipogenesis) s

6 Vol. 22, 2015 Growth of Gint Gourmi Feeding with Recombinnt Growth Hormone 17 the result of liver glycogen mobiliztion nd the trnsport of free ftty cids from dipose tissue to the liver (Groff & Gropper 2000). Glycogen levels rise initilly becuse of n incrse in liver volumes (Tble 3). Yng et l. (2002) similrly sw increses in heptosomtic index (HSI) due to the ccumultion of glycogen in the liver of silverperch (Bidynus bidynus). The rise in HSI is lso relted to incresed body ft content (Tble 2 & 3), with HSI nd body ft content highest in the tretment group receiving 21% protein feed. This is in line with the Cheng et l. (2006) study of grouper (Epinephelus coioides) in which increses in body ft content, followed by n increse in liver ft content, resulted in n increse in the HSI. Incresed protein nd lipid retention due to supplementtion hs been reported in other studies (Promdonkoy et l. 2004; Hghighi et l. 2011). The presence of non-protein energy (crbohydrtes nd lipids) cn reduce the use of protein for energy (protein sprring effect) resulting in incresed protein retention nd decresed mmoni excretion (Supryudi et l. 2014). In our study, supplementtion llowed for incresed protein retention (Tble 2) nd decresed TAN excretion (Tble 3). This indictes tht higher protein retention decreses metbolite excretion of nitrogen (in the TAN form) due to the protein sprring effect during the production of energy in fish. This is consistent with Perez-Snchez (2000), who found higher nitrogen (protein) retention in fish receiving tretment, indicting tht functions to increse the utiliztion of non-protein nutrients s source of energy (protein sprring effect). The role of in reducing TAN excretion hs lso been reported in studies of trnsgenic tilpi (Kobyshi et l. 2007). On the other hnd, n increse in dietry protein cuses TAN excretion to increse (Tble 3). Previous reserch on Austrlin short-finned eel juveniles (Guo et l. 2012) nd blue fin trevlly (Supryudi et l. 2014) showed positive reltionship between protein intke nd mmoni excretion. This suggests tht mino cids from protein, deminted nd excreted s mmoni, re used to produce energy rther thn stored for growth, when fish re given protein supplemented diets (Mohnt et l. 2008). These results lso indicte tht low levels of dietry protein nd supplementtion cn reduce mmoni pollution from quculture. The increses in protein nd lipid retention we found due to supplementtion, were followed by improved FCR mesurements (Tble 2 nd 3). Incresed dietry protein long with supplementtion, resulted in decrese in FCR, suggesting more efficient feed utiliztion. This indictes tht supplementtion cn improve the efficiency of protein nd ft utiliztion to increse fish growth. The role of growth hormone in improving feed efficiency hs been reported in slmon (Devlin et l. 2004), muchloch (Nm et l. 2004), nd tilpi (Kobyshi et l. 2007). Furthermore, ll the prmeters tht hve been described re direct effects of the growth of juvenile gint gourmi. The results of this study showed tht the growth mesures of juvenile gint gourmi incresed in correspondence to increses in dietry protein nd to supplementtion. Yng et l. (2002) suggests tht low dietry protein content results in low growth in mny species of fish. Menwhile, the success of orl dministrtion of in boosting fish growth hs lso been reported in severl previous studies (Promdonkoy et l. 2004; Hghighi et l. 2011). In our study, we found the highest biomss gin nd best SGR in the tretment group receiving the 34% protein diet supplemented with. Biomss gin nd SGR in the tretment group receiving the 28% protein diet supplemented with ws comprble to tht mesured in the group receiving the 34% protein diets without supplementtion. Overll, incresed dietry protein, s well s supplementtion ech produced significnt improvements in the growth of juvenile gint gourmi, but we found no interction between the effects of these two tretment vribles. The interction between dietry protein levels nd supplementtion insted ws evident in prmeters tht supporting growth, such s protein nd lipid retention, blood triglyceride levels, liver nd muscle glycogen, nd the ctivity of digestive enzymes. In conclusion, the growth nd feed utiliztion of juvenile gint gourmi cn be incresed by recombinnt growth hormone supplementtion in different protein levels of the diets. ACKNOWLEDGEMENT This report is prt of thesis of the first uthor, which funded by BPPDN scholrship wrd from DIKTI. Thnks to Denny Whyudi, Rngg Grnm, nd Fjr Muln for ssisting in preprtion nd supporting in technicl implementtion of this reserch.

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8 Vol. 22, 2015 Growth of Gint Gourmi Feeding with Recombinnt Growth Hormone 19 Wtnbe T Fish nutrition nd mriculture. Deprtment of qutic Bioscience. Tokyo University of Fisheries. JICA. Wedemeyer GA, Ysutke WT Clinicl method for the ssessment of the effect of environmentl stress on fish helth. Tech Pp US Fish WildlServ. Wshington DC. p 89. Worthington V Worthington enzyme mnul: enzymes nd relted biochemicls worthingthon chemicl. New Jersey. p 399. Yng SD, Liou CH, Liu FG Effects of dietry protein level on growth performnce, crcss composition nd mmoni excretion in juvenile silver perch (Bidynus bidynus). Aqucult 213: