SUBSTITUTION OF FISHMEAL WITH SOYBEAN MEAL IN HUMPBACK GROUPER

Feeding experimEnts wer€ conduct€d to evaluat€ ths sff€cis of replacing fishmoal with soybran me6l in di6t on grolvth of humpback groupgr. Fifteen cagss of 1xlxl.2m3 each stocked with 16 humiback group€r juveniles (61..3t0,4 g/pc) wers set up randomly in seawat€r, Fish were fed to satiation twlce deily for 1 12 days, The control diet contained 61.9% fbhmeal (63.34% c|ud6 protein). Four isonitrogenous (480/6'cruds protein) and isocalorio (4,7 kcaug teed) diets supplemented with commercial phytase 'Rhonozym6,p" et o.07s% were formulated to contain different levels (8, 16, 24, and 32o/o) soybean meal (43.65% crud€ protsin) as a partial replacement for tishmeal. These diets contained total phosphorus levels between 3.64.s (10.4)% and 0.7-1.5 (r0.04)0/6 available phosphorus. Replacement of fishmeal with soybean meal (8 to 32016 replacament) was not significantly differ€nt (p>0.05) to the control diet on daily groMh rate (DGR), food conwFion ratio (FcR), protein effcierry ratio (PER), and daily food consumpton (DFc). However, the dietary levels of soybean meai significantly affec'ted (p<0.05) whole body protein and phosphorus retenlion. These data suggesl that addfion 9f pfrytase in diets could improve protein and phosphorus availability and reduce nirogen and phosphorus loading in the environment. Phytase can lherefore play an important role in formulating eco-triendly feed for humpback grouper. Based on P loading, supplementation of phytase is able to reduce fishmeal and replace up to 24olo with sovbean meal.


INTRODUCTION
The expansion of global aquaculture production increases the demand for aquaculture teeds.For carnivorous species, such as barramundi, protein can form up to 60% of the diet (William et a/., 2003) and fishmeal is the main source of dietary protein.
Fishmeal is readily recognized as the best source of dietary protein and n-3 fatty acid eicosapentaenoic acid (EPA) and decosahexaenois acid (DHA), which are essential dietary requirement of tropical marine species (King, 2004).Fishmeal is also one of the most expensive and demanded ingredients and has become the main and most critical ingredient in aquafeed production.The increasing cost and demand of fishmeal has encouraged feed manufaclure to search for cheaper alternative protein sources such as plant proteins.Fish nutritionists have tried to use less expensive plant protein sources to partially or totally replace fishmeal.Substitution with other ingredients, especially from plant origin, is likely to compromise nutrient balance and sometime fails to match the energy concentration achieved using fishmeal Plant protein sources contain high lipid and fiber and lack essential amino acids.Considering the increasing cost of fishmeal and doubt concerning its long-term availability, much research has been carried out to find alternative protein sources as fishmeal substitutes Williams et al., (2OO3) reported that for aquaculture to make a net contribution to human supplies, the present use of fishmeal in aquaculture daets must be substantiallv reduced.

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Research Inslitute for Coastal Aquaculture, Maros (South Sulawesi) ' Central Research Instituie for Aquaculture, Jakarta   From economics point of view, market availability and nutritional value, a prime candidate for replacing fishmeal (FM) in aquaculture diets is soybean meal (SBM).Although the protein content of SBM is less than that of FM, the essential amino acid profiles of processed SBM products @mpares well with that of FM when considered on a percentage of protein basis (NRC, 1993).Replacement of FM with SBM has tended to reduce fish groMh because of the numerous antinutritional factors.However, Nyirenda et al., (2000) reported that animal protein sources could totally be replaced by soybean meal in order to get similar groMh .ales in Oreochromis karongae.
SBM that originates from seed contains phosphorus primarily as the calcium-magnesium salt of phytic acid known as phytin.Phytin phosphorus is unavailable to animals with simple stomachs because of their lack of phytase (the enzyme required to liberate phosphorus from phytat) in the gastrointestinal tract Phytic acid content in diets can degrade groMh rate, FCR and protejn retention, influencing thyroid function, increasing mortalities, triggering cataract formation at Zn low level and causing abnormalities of pyloric cecal structure (Richardson et al., '1985 !! Halver, '1989).Although diets based on soybean meal generally contain less phosphorus than diets containing animal feedstuffs, increasing the availability of phosphorus from soybean meal products is desirable to restrict the amount of supplemental phosphorus required in diets and also to limit the amount excreted by fish into the environment.
The availability of phosphorus in SBM is between 29 and 54% (Lovell, 1989).
Phosphorus (P) is an essential element in fish diet for skeletal groMh, cellular structure, protein, lipid, and energy metabolism.Soybean meal contain more than 60% P in the form of phytat, a molecule of inositol phosphate.Only 10-30% P in soybean meal is available as inorganic P such as dicalcium phosphate (CaHPO4), which must be enhanced in diet to fulfill requarements.The combinataon of inorganic P addition in the diet and phytat-P content in feed ingredients (in an unavailable form) will resuh in the released high conCentrations of free P to the environment and cause eutrophication, algal blooms, fish death, smell, scum, increased turbidity, and sedimentation.For that reason, phytat-P in feed ingredients have to be converted to inorganic P by the eEyme phytase before becoming available to fish.Felix & Selvaraj (2004) reported that phytase is able to release the phosphorus bound in phytat and this permits feed manufacturers to reduce the fishmeal and lower the cost of feed oroduction.lmproved phosphorus utilization can also help reduce the discharge of nutrients into the environment.
Phytase can therefore play an important role in formulating eco-friendly aquafeeds.The use of the enzyme is able to reduce fishmeal inclusion by around 5% in most aquafeeds with potential for more as techniques are refined.This may help to reduce the demand for fishmeal from the aquaculture an coming years.The purpose of this study was to evaluate the feasibiljty of replacing some fishmeal with soybean meal supplemented with phytase in diets of humpback grouper, C. a/tlyells.

Experimental Cages
Feeding experiments were conducted at floating net cages facilities of the Research lnstitute for Coastal Aquaculture (RICA) in Awarange Bay, Barru Regency, South Sulawesi for 16 weeks from June to September 2003.Fifteen sea cages of 1x1x1.2m3were hung at one wooden raft and randomlv allocated to the drfferent treatments in such a way that each treatment was three replicated.

Experimental Design and Diets
A complete random design with three replicates (15 experimental units) was used to investigate the feasibility of replacing some fishmeal with sovbean meal supplemented wth phytase In drets of numpbact grolper, C. a/tiyelis during 16 weeks groMh assay.
Before the diets were formulated, all ingredients weie anallzed lor approximate composition (Table 1), using standard methods for crude protein, crude lipid crude frber and ash content (AOAC 1995).The percentage of crude protein in the diets was determined by semimicro-Kjeldahl anlysis, the percentage lipid was determined by the acid hydrolysis method, and crude fiber and moisture determined by drying 100'C until constant weight.
Five experimental diels were formulated to contain various percentages of soybean meal (SBM) as a partial replacement for fishmeal.The formulation of the diets used in this experiment is given in Table 2.The nutritional composition ot experimental diets is presented in Table 3.All diets were formulated to be isonitrogenous (48% crude protein) and isocaloric (4.7 kcal/g feed).The control diet contained 61.9% fishmeal (63.34% crude protein) and 0% SBM.The experimental diets were formulated to contain soybean meal (43.65% crude protein) at different levels (8, '16, 24, and 32o/ot as partial replacements for fishmeal and supplemented with commercial phytase "Rhonozyme-P" at O.O75Vo.These diets contained total phosphorus levels between 3.6-4.5 (10.4)% and 0.7-1.5 (t0.04)% available phosphorus.

Fish and Culture Condition
Juvenile humpback grouper (Cromileptes altivelis) were obtained from a commercial baclqard hatchery in Gondol, Bali and had an average weight of 7t0,2 g.
After arflval the fish were placed in sea cages of 2Px2 2m'wrth stocking densittes of 500 fish per cage for acclimation.The duration of acclimation was 2 months.During acclimation the flsh were fed a commercial feed twice daily at 07.00 and 16.00 hours to satiation.After acclimation, fish were selected to the uniform in weight with an initial mean (lsd) start weight of 6'1t0.4g.Sixteen fish were randomly distributed to floating net cages with 3 replication per treatment Sea cages were suspended from a floatang raft in seawater approximately 10 m depth and 1 m daily tidal fluctuation.Before the treatment diets.10 fish were sacrificed for carcass analysis to determine nutntronal composrtion of the initral fish.During the experiment, fish were fed by hand as much related feed as they would consume in 30 min at 07.00 and 16.00.Feeding was carefully monitored to minimize any food wastage The feeding trial lasted for 16 weeks.Fish were weighed individually at 4-weekly intervals.The individual fish in each cage were weighed using a top loading balance with a precision of 1 g At the end of the experiment.four representative fish from each cage were randomly sampled and frozen for determination of the whole body composition according to standard methods (AOAC 1995).

Chemical analysis
Representative samples of each feed were randomly taken from the feed and ground.lVaterial that passed through a 1 mm sieve used for analysis.At ,.{ S/ kS ,"|r"il"" brel pr;vid€d in 1 kg offinal diet: retinol, 432 mg: cholecalciferol, 7.125 mg: a-tocopherol, 1@ 92 mg; monadion€.3OO mgt thiaman,24O mgi riboflavin, 600;g; pyruoxin,240 mg; cyanocobalamin, 2.8 mg; ascorbic acad,3600 mg; folic acid' 120 mg; nicotinic acid.14zlo mo: DDartothenic acid.12m mE; biotin, 3 mg; and C|/L-methionin€. 1200mg nicotinic acid, 14zlo mg; Dpartothenic acid, 12m m9; biotin, 3 mg; and C|/L-methionin€. 1200mg b)Mineral mixture (vatu-e are in mg/'loo g diet): NaHPOa, 616; KH2PO.415: caco3, 2E2; F€ch 4HrO, 166; ZnSo.' 10; MnSO. 675: Cuso!, 2i Kl, 0.15; cosot.THro,0.1For determination of the body composition of the initial die plate fitted to a meat grinder.A uniform sample^of fish, 10 fish from the fish itock were combined for the whole fish body wai dried in an.oven at 60oC chemical analysis.For determination of the body overnight and then ground and sieved through a 1 mm composition of the fish at the end of the exPeriment, mesh size.A representative sample of feed or whole two fish from each sea cages were combined for fish body was analyzed for approxamate composition chemical analysis.Fish samiled for pooled chemical using standard methods for crude protein, total lipid' analyses were frozen and then chopped into small crude fiber and ash content (AOAC 1995) The pieces that could be easily minced thiough a 3 mm percentage of crude protein was determined by micro- SBM32 Kjeldahl analysis with distillation into 4% boric acid and titration with sulphuric acid using methyl red indicator for end point determination.Percentage lipid was determined gravimetrically following a chloroform;methanol extraction of the sample, and moisture was determined by drying at 105"C until constant weight.

Statistical analysis
Response data were analyzed tot statistical significance (P<0.05) by ANOVA in a complete randomized design Significant differences among m€ans were rdentrfied usjng the Least Significant Ditferent (LSD) piocedure.All percentage and ratio data were transformed to arcsine value prior to analysrs Homogeneity of vanances between samples was tested usrng the Banlet test normaltty data was tested using the Lillifors test and additivlty was tested using the Tukey test The signifjcance level of the tests was taken as 0 05.Statistical analvsis was perforrnaed us,ng l\,rlNrTAB ver 6 O

RESULTS
All fish accepted the experimental diets and the survrvat rate during 16 weeks feeding trial was between 86 and 94o/o Fish mortality during the 76 experiment was caused by parasite, Benedenia sp infection even though the fish were immersed in freshwater for 5-10 minutes every 4 weeks.
Humpback grouper juveniles fed diets with different soybean meal levels showed 10Q-129o/o weight gain during 16 weeks rearing period (Table 5).The weight gained was statistically not significantly difierent (P>0.05) for all test diets.The same response also happened in other biological variables such as daily groMh rate, survival rate, daily feed consumption, food conversion ratio, protein efficiency ratio, and fat retention.The DGR was lower for fish fed the control diet than tor the other four diets, but not significanfly different (p>0.05)Weight gain, DGR, DFC, PER, protein retention, phosphorus retention tend to Increase as the proportion of soybean meal in the diets increase, while lipid retention in the whole body of the fish decrease as dietary soybean meal increase, but there was no significant ditference (p>0.05)(Table 5).The proximate anatysis of crude protein and total lipld for fish carcasses (whote body) sampled at termination of the feeding trial correlated well with the protein retention and lipid retention (Table 6).These data suggest that humpback grouper juvenile can be fed diets containing up to 32% SBM without adverse effecls on groMh performance parameters.
Protein retention and phosphorous retention in the SBM 18 and SB|M24 treatments were signjficanfly higher than for other treatments (p<0 05) There was no significant difference in N loading, but replacement of fishmeal with soybean meal caused reduction of N load.Partial replacement of dietary fishmeal by soybean meal also srgniftcantly atfected the p loadrng (Table 6).The lowest P load was observed in the diets contajn'ng SBl,48 and SBM24 The highest p loading was found for the controt diet and for SBM32i 5.53 gP/kg fish produced and 5.44 gp/kg fish produced, respectively.The quadratic model analysis based on P load and P retention showed that the minimum of p load occured at 16.27Vo and 16.31Vo replacement of fishmeal by soybean meal respectively.These data indicated that supplementation of phytase in test diets coud increase phosphorus availability and reduce p nutflent release in the environment
The lack of significant differences in growth performance variables was possibly exdaned by the supplementation of phytase in four the experimental diets playing an essential roie in increasing the availability ol nutrient trom soybean meal.Phosphorus availability at SBM16 and SBlvl24 were higher than the other test diets and the control diet and showed a 2'l4.7Vo and 157yo increase respectively over the control diet (Table 7).Supplementation of phytase an test diets was also able to improve utilization of both N and P nutrients so that nutrient loading to environment could be minimized and even lower than in the fish fed the control diet.Phytase enhancement in test diets has certain capacities in improving the availability of nutrient in diets according to the amount of phytase added and the proportion of soybean meal in the feed formulation.

Table l .
I n dqt esi an Fi sh eie s Re se arch J oum d V ol.I 1 -2005 Compo6ition (%) of the local feed ingredients used lngledienb Crude protein Total lipld Crude tiber Ash

Table 3 .
Nutritional composition of the diets 1 1 -2005Biological response of humpback grouper fed with differoncB soybean levets in the diet Means in the same row followed by similar superscdpt are not significantly different(P>0.05) b)

Table 6 .
Whole body crude protean, crude lipid, and phosphorus of humPback grouper fed diets (Carter & Hauler 1998)with phytase-incorporated soybean based diets have been reported to sf,ow a ZZo/o improvement over control fish, as phosDhorus availabrlrty rncreased from 460/o to over 70% (iorster et a/, 1999) N.4tcrobiat phltase added to diets containing a higher proportion of plant protein have qgen :l.9wn lo improve phosphorus and protein drgestrbility in Aflantic salmon(Carter & Hauler 1998)Suglura el al .(2OO|J found that supptementatron of ph!.tase in flsh diet can improve the absorption of c,ude prote,n aqd va ous m nerals from dlets of soybean meal with low ash content, but absorotion was lower In dtets Content both Soybean meal and ftshmeat with high ash.phosphor.usdigestibitity ;ill rncrease as phytase level in diet jncreases.