Feed Peas in diets for shrimp tilapia and milkfish - Northern Pulse ...

Final Project ReportTitle:Utilization of feed peas (Pisum sativum) as alternative protein sources in diets forshrimp, tilapia, and milkfish.Implementing Personnel and Agencies:Southeast Asian Fisheries Development Center, Aquaculture DepartmentClarissa L. Marte, PhD, Head, SEAFDEC Research Division (clmarte@aqd.seafdec.org.ph)Myrna B. Teruel, Study leader, Shrimp (mbt@aqd.seafdec.org.ph)Corazon B. Santiago, PhD, Study leader, Tilapia (csantiago@aqd.seafdec.org.ph)Ilda G. Borlongan, Study leader, Milkfish (igb@aqd.seafdec.org.ph)Perla S. Eusebio, Study leader, Digestibility experiments (pse@aqd.seafdec.org.ph)USA Dry Pea and Lentil CouncilTimothy Welsh, Southeast Asia Representative (agsource@loxinfo.co.th)Funding:US Department of Agriculture, Emerging Markets ProgramUSA Dry Pea and Lentil CouncilTrial Period: July, 2001 through March, 2002

Table of ContentsSUMMARY ............................................................................................................................................................1-2STUDY 1. SHRIMP (PENAEUS MONODON, FABRICIUS)............................................................................................... 1STUDY 2. TILAPIA (OREOCHROMIS NILOTICUS L.)..................................................................................................... 1STUDY 3. MILKFISH (CHANOS CHANOS, FORSSKAL)................................................................................................. 2GENERAL CONCLUSIONS ............................................................................................................................................ 2Utilization Of Feed Pea, Pisum Sativum Meal, As An Alternative Protein SourceIn Practical Diets For Juvenile Tiger Shrimp, Penaeus Monodon Fabricius......................................................3-10ABSTRACT................................................................................................................................................................... 3INTRODUCTION............................................................................................................................................................ 3MATERIALS AND METHODS........................................................................................................................................ 4RESULTS...................................................................................................................................................................... 5DISCUSSION................................................................................................................................................................. 5ACKNOWLEDGEMENT ................................................................................................................................................. 9REFERENCES: .............................................................................................................................................................. 9Feed Pea (Pisum Sativum) As An Alternative Protein SourceIn The Diets Of Nile Tilapia, Oreochromis Niloticus (L.) ................................................................................11-19ABSTRACT................................................................................................................................................................. 11INTRODUCTION.......................................................................................................................................................... 11MATERIALS AND METHODS...................................................................................................................................... 11RESULTS.................................................................................................................................................................... 13DISCUSSION............................................................................................................................................................... 14ACKNOWLEDGEMENTS ............................................................................................................................................. 18REFERENCES.............................................................................................................................................................. 18Potential Of Feed Pea (Pisum Sativum) As An Alternative Protein SourceIn Practical Diets For Milkfish (Chanos Chanos Forsskal)...............................................................................20-26ABSTRACT................................................................................................................................................................. 20INTRODUCTION.......................................................................................................................................................... 20MATERIALS AND METHODS...................................................................................................................................... 20RESULTS.................................................................................................................................................................... 22DISCUSSION............................................................................................................................................................... 22ACKNOWLEDGEMENT ............................................................................................................................................... 26REFERENCES.............................................................................................................................................................. 26

SUMMARYThis report summarizes the results of experiments conducted to test the efficacy of feed pea meal (Pisumsativum) as an alternative protein source in the diets for tilapia, milkfish, and shrimp. All formulations werebased on the known nutrient requirements (e.g., protein, fat, carbohydrate, protein:energy ratio,indispensable amino acids) of the species specified.Study 1. Shrimp (Penaeus monodon, Fabricius)The potential of feed pea meal as an alternative protein source in practical diets for juvenile tiger shrimpwas assessed in a 12-week feeding trial. Formulated diets were made isonitrogenous (40% crude protein)and isocaloric (14.5 kJ g –1 ). The control diet contained fish meal, soybean meal, squid meal, shrimp meal,shrimp head meal as protein sources. Protein from feed pea meal replaced 0, 20, 40, 60, 80, and 100% ofthe protein from defatted soybean meal in the diets. These values were equivalent to 0, 5, 10, 15, 20, 25%,respectively, of the total protein in the diet. A negative control with no protein sources was added to thetreatment. Twelve shrimp post-larvae with an average weight of 0.02±0.01g were randomly assigned in 35,60-l oval tanks equipped with a flow-through seawater system. The shrimp were fed the formulated diets ata daily feeding rate of 20-25% body weight in 5 replicate samples.Results showed no significant differences in % weight gain (WG)(5162 to 5839); specific growth rate (SGR)(4.4-4.6); feed conversion ratio (FCR) (1.2-1.9); protein efficiency ratio (PER) (1.2-1.7); survival (SURV)(75-100) of shrimp fed diets with 0 up to the highest level of replacement. Weight gain of shrimp fed thenegative control (364) was significantly lower (P>0.05) compared to the rest of the treatments.The apparent dry matter (ADMD) and protein (APD) digestibilities of the dry feed pea in P. monodon werehigh at 73.38±4.98% and 92.74±2.62%, respectively. Digestibility coefficients for dry matter (71-77%) andprotein (83-88%) for the feed pea meal-based diets increased with increasing level of feed peareplacement.The results indicate that whole feed pea meal can be an alternative protein source for shrimp. It cansubstitute even up to 100% of the protein from defatted soybean meal, which is equivalent to 25% of thetotal protein in the diet. An inclusion level of up to 42% in the juvenile shrimp practical diet did not manifestany adverse effects on growth, feed efficiency, survival, body composition, and digestibility coefficients fordry matter and protein of the shrimp.Study 2. Tilapia (Oreochromis niloticus L.)The tilapia experiments made use of fish meal-based (experiment 1) and plant-based (experiment 2) dietsto allow maximum level of feed pea as a protein source. In both experiments, all test diets were madeisonitrogenous (30% crude protein) and isocaloric (14 kJ g –1 ). Water temperature during the feeding trialsranged from 22-27°CExperiment 1. Fish meal supplied about 28% protein in the diets. Feed peas (12.7-63.3% of the diet)substituted up to 50% of fish meal protein. Manually sexed male Nile tilapia, each weighing 32-39g atstocking, were used. The fish were reared in polyethylene tanks (58x37x27 cm) with aeration undercontrolled conditions. Water was static and was partially replaced once a day. Fish were allowed to feed tosatiation twice daily.The various inclusion levels of feed peas did not affect body weight, feeding activity, and feed efficiency ofthe tilapia. Weight gain after 9 weeks (range: 21.7±5.6 to 34.0±9.3g) did not differ significantly amongtreatments (P>0.05). Survival rates were highly variable (40-75%) and not significantly different. Mortalitywas not related to treatment. FCR (3.2 to 4.2) and PER (0.8 to 1.0) were not significantly different amongtreatments.Experiment 2. Feed peas in the diets ranged from 5.9-41%. Feed peas substituted up to 35% of the plantprotein (or up to about 30% of total dietary protein). The control diet contained fish meal, soybean meal,and copra meal as principal protein sources. Separate feeding trials were conducted on two strains of allmaletilapia (CLSU and BFAR strains) under controlled conditions using the same test diets and biggertanks (90x78x43 cm).There were no significant differences in weight gain among treatments after 12 weeks of feeding (45.5-±7.8to 57.4±12.4g for the CLSU strain; 52.5±4.4 to 74.1±9.2g for the BFAR strain). Moreover, FCR (3.2 to 4.0)or PER (0.9 to 1.0) were not significantly different among treatments. Survival was 100% in all tanks inboth trials.1

Digestibility Experiments. As ingredient in tilapia diet, feed pea has apparent dry matter digestibility(ADMD) of 69.3+1.8% and apparent protein digestibility (APD) of 87.2±2.0%. The digestibility of some ofthe diets used in experiments 1 and 2 was also determined in separate feeding trials. The ADMD of thediets increased as feed pea inclusion level increased. The APD of the fishmeal-based diets or the plantbaseddiets were all high, exceeding 90%.Overall, feed pea meal may substitute up to 50% of fish meal protein (equivalent to 63.3% feed pea) and35% of the plant proteins (41% feed pea) in the diets of Nile tilapia without any adverse effect on fishgrowth and survival.Study 3. Milkfish (Chanos chanos, Forsskal)The efficacy of feed pea meal as an alternative protein source for milkfish was tested in a 12-week feedingtrial. All test diets were made isonitrogenous (30% crude protein) and isocaloric (15.7 kJ g –1 ). The controldiet contained fish meal, soybean meal, meat and bone meal, and copra meal as protein sources. Feedpea meal was substituted at 0, 5, 10, 15, 20, 25, and 30% of total protein. A leading commercial milkfishfeed was also tested as an additional control. The experimental diets were fed to triplicate groups ofmilkfish fingerlings (mean initial weight =0.42±0.01g) at 10% body weight /day.Results showed that % weight gain (WG) of milkfish juveniles fed diets with 5% (WG= 833.1) and 10%(WG=835.3) feed pea inclusion did not significantly differ from that of the SEAFDEC control diet (0% feedpea inclusion; WG=834.8). However, % weight gain obtained for milkfish juveniles fed with 15%(WG=691.0) and 20% (WG=680.0) feed pea inclusion in the diet were significantly higher than thecommercial milkfish feed formulation (no feed pea inclusion, WG=581.4). Survival ranged from 70-90%.FCR was between 2.1 to 2.60 while PER ranged from 1.15 to 1.44.Dietary crude protein digestibility values of the experimental diets were 84.8 to 89.2% suggesting that feedpea protein is well digested by milkfish.Over-all results indicate that feed peas could be used as an alternate dietary protein source for milkfishjuveniles. Optimum level of inclusion without amino acid supplementation is 10% of the dietary protein(131g feed pea/kg dry diet). Dietary protein inclusion levels up to 20% feed peas (260g/kg diet) are stillacceptable.General ConclusionsThese feeding trials clearly demonstrate that whole feed pea can be incorporated as an alternative proteinsource in diets for tilapia, milkfish, and shrimp. Since feed peas have a moderate protein content (23-25%)compared to fish meal (65-70%), their incorporation in fish diets needs to be balanced with other proteiningredients, especially for fish that require high protein levels in their diet. In this case, feed pea cantherefore be used as one of the ingredients that would collectively replace fish meal in practical diets.The replacement of other plant protein sources with feed pea is possible as evidenced by the studiesconducted. Feed peas contain high levels of lysine, which could complement the amino acids of otheringredient substitutes that are deficient in such amino acids. The relatively low level of sulfur amino acids(i.e., methionine and cystine) in feed pea meal may limit the use of this plant protein source dependingupon the requirement of the species and the diet formulation.The pea variety used in these studies contains low levels of anti-nutritional factors and is similar to mostfeed pea varieties now supplied in global markets. Thus, positive results were obtained for shrimp andtilapia, even at high levels of incorporation in the diets. The relatively lower performance of milkfishcompared to shrimp and tilapia, even with feed pea comprising 15% of the diet, may be attributed to aminoacid deficiencies or possibly to effects from certain anti-nutritional factors. However, it is notable that thegrowth response of milkfish fed the 15% feed pea diet was still better than from the commercial milkfishdiet, which contained no feed pea, so further study is needed.The availability of feed pea meal as an alternative protein source for tilapia, milkfish, and shrimp may openavenues for commercial producers to reduce feed costs. Feed peas have a higher carbohydrate content(65-67%) compared to soybean meal (44-45%), which may provide a lower cost and more digestible energysource for the animals.2

Utilization Of Feed Pea, Pisum Sativum Meal, As An Alternative Protein SourceIn Practical Diets For Juvenile Tiger Shrimp, Penaeus Monodon FabriciusMyrna N. Bautista-Teruel, Perla S. Eusebio, and Timothy P. WelshAbstractThe potential of feed pea, Pisum sativum, meal as an alternative protein source in practical diets for thejuvenile tiger shrimp, Penaeus monodon, was assessed in several experiments. Six isonitrogenous dietswere formulated to contain 40% protein. Protein from feed pea meal replaced 0, 20, 40, 60, 80, and 100%of the protein from defatted soybean meal in the diets. These values were equivalent to 0, 5, 10, 15, 20,and 25% respectively of the total protein in the diet. A negative control with no protein sources was addedto the treatments. Twelve shrimp post-larvae with an average weight of 0.02±0.01g were randomlyassigned in 35, 60-l oval tanks equipped with a flow-through seawater system. The shrimps were fed theformulated diets at a daily feeding rate of 20-25% body weight for 90 days in 5 replicate samples. Nosignificant differences (P0.05) compared to the rest of thetreatments. Specific growth rates (SGR) of shrimps for the various treatments showed the same trend asthe percent weight gain. Survival of shrimp for all treatments, including the negative control, was generallyhigh at 75-100%. The apparent dry matter (ADMD) and protein (APD) digestibilities of the dry feed pea in P.monodon were high at 73.38±4.98 and 92.74±2.62, respectively. Digestibility coefficients for dry matter andprotein for the feed pea meal-based diets increased with increasing level of feed pea replacement. Therewere no significant differences (P

Materials and MethodsExperimental dietsThe feed ingredients used in this study were obtained from commercial suppliers through the SEAFDECpilot feed mill plant. Feed pea of US origin was supplied through a local distributor in Manila, Philippines.Prior to use, all dry feed ingredients were ground and sieved through a 60µm mesh screen sieve.Proximate analyses for each feed ingredient were conducted and the resulting data were used in dietformulation (Table 1). Six isonitrogenous diets were formulated to contain 40% protein (Table 2). Proteinfrom feed pea replaced 0, 20, 40, 60, 80, and 100% of the protein from defatted soybean meal in the diets.These values were equivalent to 0, 5, 10, 15, 20, 25%, respectively, of the total protein in the diet.Replacement of protein was based on the essential amino acid requirements of tiger shrimp (Kanazawaand Teshima, 1981; NRC, 1993; FDS Manual, 1994; Millamena et al., 1996-1999). A negative control withno protein sources was added to the treatments. Protein content of 40% in the shrimp diet has beenreported to be optimal for juvenile Penaeus monodon (Alava and Lim 1983; Bautista, 1986; Shiau et al.1991). The diet containing no feed pea meal served as the control diet. Other animal protein sources, suchas fish meal, shrimp meal, shrimp head meal, and squid meal, were included in the dietary formulations atthe same levels. The levels of wheat flour and rice bran were adjusted to maintain similar dietary proteincontents. Diets were prepared according to standard diet preparation in the laboratory as described in FDSManual, 1994. To prevent lipid oxidation during storage, all diets were packed in sealed plastic containersand stored inside a cold room at –20ºC until used in the feeding trials.Proximate analyses were conducted of the various diets prepared following the methods of Association ofOfficial Analytical Chemists (AOAC 1990). The amino acid composition of both soybean meal and feed peameal and experimental diets was determined using the method of Simpson et al., 1976.Feeding trialsGood quality and disease-free post-larval Penaeus monodon were sourced from a private hatchery inTigbauan, Iloilo, Philippines. They were placed in 2-tonne fiberglass tanks and fed with Artemia sp andcontrol diet for a week before they were transferred to experimental tanks. Twelve shrimp post-larvae withan average weight of 0.02 ± 0.01g were randomly assigned in 35, 60 L oval tanks equipped with a flowthroughseawater system. Prior to start of the feeding trial, shrimps were acclimatized to experimental diets.The shrimp were fed the formulated diets at a daily feeding rate of 20-25% of total body weight for 90 daysin 5 replicate samples. Shrimp were fed three times daily at 0800, 1300, and 1700h. The wet weights ofthe shrimp were recorded every 15-d interval for 90 days and the amount of feed given was adjusted everysampling day. All experimental tanks were cleaned before every feeding. Water temperature, salinity,dissolved oxygen, and pH were measured daily in all tanks. Total values for ammonia-nitrogen and nitritenitrogenwere measured once a week. Shrimp were collected at the beginning of the feeding trial forproximate analyses. At the conclusion of the feeding trial, all shrimp samples from each tank were pooled,freeze-dried and ground for proximate analyses of whole body composition following the standard methodsof AOAC (1990).Biological analysisDiet performance was evaluated by calculation of percent weight gain=final weight-initial weight/initialweight x 100; specific growth rate=100 (ln ave. final wt-ln ave. initial wt)/no. of culture days; feed conversionratio (FCR)= total dry feed intake (g)/wet weight gain (g); percent survival =final number of shrimp/initialnumber of shrimp x 100.In vivo digestibility experimentApparent digestibility coefficients for dry matter (ADMD) and crude protein (APD) of feed peas as ingredientand feed-peas based diets were measured using 1% Cr 2 O 3 as external indicator. The method by Cho. etal. (1982) was adapted in a ratio of 70:30 (reference diet to test ingredient) in each test diet. The controldiet used for growth experiment was used as reference diet. Composition of the diet is shown in Table 3.In the digestibility experiment for the test ingredient and feed pea-based diets, 10 shrimp with mean bodyweight of 14.53± 0.57g and 8 shrimp with mean body weight of 13.56±0.56g were stocked in 250 L conicalfiberglass tanks for the test ingredient and feed pea-based diets, respectively. The shrimp were acclimatedto experimental diets and laboratory conditions for 7 days before the experiment. All shrimp were fed at 8-10% of the total body weight 3x daily. A flow-through filtered seawater system with continuous aeration andflow rate of 600-700 ml/min was provided for each tank. Water temperature and salinity were maintained at24-48ºC and at 30-34 ppt, respectively. Culture period was 80 days. Fecal collection was done manually.The feces were allowed to float into a plastic scoop and then pipetted and gently transferred into acollecting vial. Care was taken to prevent the breaking-up of fecal strands to facilitate collection and toavoid leaching of nutrients. Thereafter, fecal samples were carefully rinsed with distilled water andimmediately stored in a freezer to retard bacterial decomposition. At the end of fecal collection, samples4

were thawed and pooled prior to freeze-drying. Dried fecal samples were thoroughly mixed and preparedfor chromic (Cr 2 O 3 ) (Carter et al., 1960) and crude protein analysis (AOAC, 1990). Apparent dry matterdigestibility (ADMD) and apparent protein digestibility (APD) were determined using the equation:% Digestibility = 100-100 X [{C d /C f } X {N f /N d }] where: C d = % chromic oxide in diet; C f = % chromic oxide infeces; N f = % nutrient in feces; N d = % nutrient in dietWater stability test for dietsWater stability of the diets was determined at 4, 8, 12, 16, and 24 h following the method described in FDSManual, 1994. Percent water stability was computed as final dry weight of feed / initial dry weight of feed X100.Statistical analysisAll data were analyzed by a one-way ANOVA using a Statistical Analysis Software Program of SPSS. TheDuncan’s Multiple Comparisons T was used to determine the differences between the treatment means(Duncan, 1955). Results were considered statistically significant at the level of P

Smith et al. (1999) reported that digestibility coefficients of feed peas in tiger shrimp were 80% for ADMDand 91% for APD. The ADMD value and APD value is almost the same as those obtained in this presentstudy (73% and 89-92%).In this study, no adverse effect on shrimp was noted on the use of whole unprocessed feed pea. Perhaps,processing of feed pea may even improve its present nutritional value. Cruz-Suarez et al., 2001 however,did not obtain any particular benefit with the use of processed feed pea (dehulling). In her study, raw feedpeas and peas processed by dehulling have similar nutritional value in shrimp diets. Results, however, maybe dependent on the type or variety of feed pea used. Studies with other species such as trout (Kaushik etal., 1993) and European sea bass (Giuveia and Davies, 2000) have emphasized the requirement fordehulling and extrusion cooking. Eusebio (1991) also found that processing of cowpea and rice bean hadno significant effect on growth and survival of P. monodon, although apparent protein digestibility of ricebean was improved. Shrimp are more tolerant than other species of certain anti-nutritional factors in theseed coat components (Cruz-Suarez et al., 2001).This study has demonstrated the acceptable nutritional value of whole feed pea as an ingredient in shrimpdiets, since this product can replace most commonly used shrimp feed ingredients, such as soybean meal.Inclusion of feed peas in shrimp diets may therefore be a function of diet formulation. An inclusion level upto 42% in the juvenile shrimp, P. monodon, practical diet does not give any adverse effect on growth,survival, and body composition of the animal.Table 1Proximate composition (% dry weight) of feed ingredients included in test diets*IngredientsMoistureCrudeProtein(N x 6.25)Crude fatCrudefiberNFE cFeed pea a 11.58 25.24 1.32 3.68 65.96 3.80Soybean meal b 10.78 42.67 1.37 4.03 44.87 7.06Seaweed 9.1 12.13 0.44 5.58 30.42 51.43Wheat flour 14.19 17.21 1.26 0.03 80.76 0.74Squid meal 15.34 78.84 5.19 0.55 5.38 10.04Shrimp meal 13.66 70.25 2.76 2.08 5.35 19.56Peruvian fish meal 9.3 74.64 7.32 1.02 0.76 16.26Shrimp head meal 10.08 47.44 3.00 12.04 9.74 27.78Rice bran 10.12 14.22 18.40 7.20 50.17 10.01*Means of 2 replicate samplesa Whole feed peab Defatted soybean mealcNFE-Nitrogen Free ExtractAshTable 2Percentage composition of experimental diets on a dry matter basis (g/100g dry diet)Diet No.Ingredients 1 2 3 4 5 6 7% Soybean replacement 0 20 40 60 80 100Neg.control% Total protein replacement 0 5 10 15 20 25 -Feed pea - 8.45 16.90 25.36 33.82 42.30 -Soybean meal 25.00 20.00 15.00 10.00 5.00 - -Wheat flour 17.00 13.00 9.00 7.00 5.00 5.00 17.00Rice bran 8.95 9.5 10.05 8.59 7.13 3.65 22.95Dextrin - - - - - - 49.00Common ingredients 49.05 49.05 49.05 49.05 49.05 49.05 11.05*Peruvian fish meal, 23; Ascetes, 3; squid meal, 2; shrimp head meal, 10; seaweed, 2.5; cod liver oil, 2;soybean oil, 1; soybean lecithin, 1; vitamin mix, 1.99; vitamin C, 0.01; mineral mix,1; dicalciumphosphate,1;carboxymethylcellulose, 0.5; ethoxyquin,0.05.**Vitamin mix. _-carotene ,3.0M.I.U.kg -1 ;cholecalceferol, 0.6M.I.U.kg -1 ; thiamine, 3.60g kg -1 ; riboflavin, 7.20gkg -1 ; pyridoxine, 6.60g kg -1 ; cyanocobalamine, 0.02gm kg -1 ; _-tocopherol, 16.50 gm kg -1 ; menadione, 2.40gm kg -1 ; niacin, 14.40gm kg -1 ; pantothenic acid, 4.00 gm kg -1 ; biotin, 0.02 gm kg -1 ; folic acid, 1.20gm kg -1 ;inositol, 30.00gm kg -1 ; stay C, 100.00gm kg -1 ; Mineral mix: P, 12.0%; Ca, 12.0%; Mg, 1.5%; Fe, 0.15%; Zn0.42%; Cu, 0.21%; K, 7.50%; Co, 0.011%; Mn, 0.160%; Se, 0.001%; Mo, 0.0005%; Al ,0.0025%; I, 0.04%.6

Table 3Composition of reference and test diets for in vivo digestibility experiment in shrimp,P. monodon juveniles (g/100 g feed)Feed IngredientReference Test DietDiet (70:30)Peruvian fish meal 23.00 16.10Squid meal 2.00 1.40Ascetes sp. 3.00 2.10Shrimp head meal 10.00 7.00Soybean meal, defatted 25.00 17.50Wheat flour 17.00 11.90Rice bran 8.90 5.50Cod liver oil 2.00 1.40Soy bean oil 1.00 0.70Vitamin mix 1.99 1.39Phosphated ascorbic acid* 0.01 0.01Mineral mix 1.00 0.70Dicalcium phosphate 1.00 1.00Seaweed 2.50 1.75Celufil 0.50 0.50Erthoxyquin 0.05 0.05Cr 2 O 3 1.00 1.00Feed peas - 30.00* Phosphitan C, feed grade, Showa Denko K.K. JapanTable 4Diet No.Proximate composition (%) of experimental diets*MoistureCrudeProtein Crude Fat CrudeFiberNitrogenFreeExtractAsh1 (0) 8.05 40.02 7.34 5.26 30.73 16.652 (20) 6.44 39.12 7.06 5.80 31.42 16.603 (40) 6.76 39.45 7.69 5.16 31.41 16.294 (60) 7.24 39.08 7.06 5.05 33.18 15.635 (80) 6.47 39.97 7.40 5.29 31.82 15.526 (100) 4.47 39.25 7.71 5.08 32.73 15.237 (neg) 7.01 6.98 7.06 5.15 74.89 5.92*Mean values of 2 replicate samplesTable 5Amino acid composition (per 100 g sample) of soybean meal, feed pea meal and experimentaldiets aAmino acidSoybean Feed pea % Replacement in the dietmeal meal 0 40 80 100Aspartic acid 5.32 3.15 2.29 2.28 2.38 2.69Methionine b 0.63 0.19 0.27 0.29 0.31 0.33Threonine b 1.72 1.06 0.84 0.86 0.83 0.86Serine 2.46 1.33 0.91 0.94 0.95 0.97Glutamic acid 8.64 4.67 4.06 3.97 3.77 3.71Proline 2.11 1.1 1.16 1.21 1.26 1.32Glycine 2.36 1.17 1.29 1.32 1.34 1.32Alanine 2.39 1.15 1.24 1.26 1.28 1.36Valine b 1.41 1.26 1.02 1.04 1.05 1.08Isoleucine b 1.42 1.22 1.22 1.23 1.23 1.71Leucine b 4.07 2.2 1.93 1.95 1.96 1.97Tyrosine b 1.84 0.9 0.87 0.88 0.89 0.89Phenylalanine b 1.93 1.32 0.96 0.98 1.09 1.05Lysine b 2.73 2 1.84 1.86 1.84 1.89Histidine b 1.05 0.57 0.54 0.54 0.53 0.55Arginine b 2.56 1.9 1.45 1.57 1.69 1.68a Values are mean of two replicate samplesb Essential amino acids for shrimp, Penaeus monodon (Kanazawa andTeshima, 1981; NRC, 1993; FDS Manual 1994; Millamena et al., 1996-1999)7

Table 6Response of juvenile P. monodon over a 90-d feeding trial to experimental diets containingreplacement levels of soybean meal protein with feed pea meal protein*Diet No.Initial mean Final mean Weightwt (g) wt (g) gain a SGR b FCR c PER d Survivale(%)(%)1 (0) 0.02±0.01 1.30±0.08 5598±309 a 4.49±0.06 a 1.23±0.14 a 1.23±0.11 a 832 (20) 0.02±0.01 1.13±0.06 5162±92 a 4.40±0.01 a 1.77±0.04 a 1.44±0.03 a 1003 (40) 0.02±0.01 1.45±0.04 5980±165 a 4.61±0.01 a 1.36±0.12 a 1.43±0.01 a 754 (60) 0.02±0.01 1.37±0.01 5839±65 a 4.54±0.12 a 1.89±0.03 a 1.35±0.02 a 925 (80) 0.02±0.01 1.21±0.18 5187±395 a 4.38±0.15 a 1.39±0.32 a 1.34±0.08 a 1006 (100) 0.02±0.01 1.19±0.07 5223±205 a 4.41±0.04 a 1.39±0.33 a 1.66±0.08 a 1007 (neg) 0.02±0.01 0.10±0.02 364±82 b 1.67±0.20 b 7.02±1.86 b 0.11±0.01 b 75* Means of 5 replicate samples. Values in the same row with different superscripts are not significantlydifferent (P

Table 10Percent recovery of experimental diets in seawater*4 8No. of hours1 2 16 24Diet No.Diet 1 (0) 89 87% Recovery85 83 80Diet 2 (20) 91 85 83 82 80Diet 3 (40) 92 91 90 86 85Diet 4 (60) 94 93 93 92 91Diet 5 (80) 92 91 91 90 90Diet 6 (100) 91 89 87 88 86Diet 7 (neg.) 88 83 80 77 76*Values are means of triplicate samplesAcknowledgementThe authors acknowledge the United States Department of Agriculture and the USA Dry Pea and LentilCouncil for the research and travel funding support; M. Mallare and J. Vera Cruz for the technicalassistance; F. Jarder for the proximate analysis; J. Bangcaya and M. Arnaiz for the amino acid analysis,and M.J. Bernas for the Cr 2 O 3 analysis.References:Akiyama, D.M. 1991. Soybean meal utilization by marine shrimp. In: D.M. Akiyama and R.K.H. Tan (eds). Proceedings of theAquaculture Feed Processing and Nutrition Workshop. Thailand and Indonesia. September 19-25, 1991. American SoybeanAssociation. Singapore. pp 207-225.Alava, V. R. and Lim, C. 1983. The quantitative dietary protein requirement of Penaeus monodon juveniles in a controlledenvironment. Aquaculture. 53:229-242Allan, G., Stone, D.A.J., Booth, M.A., 1999. Alternative protein sources: plant proteins. Book of abstracts of the World AquacultureSociety Annual Meeting ’99. April 26-May 2, 1999. Sydney, Australia. WAS, Baton Rouge. I.A. USA, 18.Allan, G. 1998. Protein sources.potential for pulses. .International Aquafeed 2:.17-20Allan, G.L. ,Smith, D.M. 1998. Recent Nutrition Research with Australian Penaeid. Reviews in Fisheries Science. pp. 113-127.Allan, G., 1997. Potential for pulses in aquaculture systems. Proceedings of International Food Legume Research Conference III,Sept. 22-26, 1997, Adelaide, Australia. 13 pp.AOAC (Association of Official Analytical Chemists), 1990. Official methods of analysis, 15 th edition. Association of Official AnalyticalChemists. Arlington, Virginia, USA.Bautista, M.N. 1986. The response of Penaeus monodon juverniles to varying protein/energy ratio in test diets..Aquaculture.,53:229-242Booth, M.A., Allan, G.L., Stone, D.A.J., 1999. Utilization of four agricultural aingredients by silver perch. Book of abstracts of the WorldAquacultue Society Annual Meeting ’99, April 26-May 2, 1999. Sydney, Australia. WAS, Baton Rouge. I.A. USA, 20.Burel, C., Boujard, T., Boeuf. G., Evrard, J., Peyronnet, C., Kaushik, S.J., 1996. Utilisation de proteins d’origine vegetale (pois, lupin,colza) dans l’alimentation de la truite arc-en-ciel: valeur nutritionelle et effets sur l’axe thyreotrope. In: CRITT Valicentre (Eds),Proceedings of Colloque Annuel Valicentre, Ardon, France, 28 Nov. 1996, pp 47-58.Carter, J.F., Bolin, D.W., Erikson, P., 1960. The evaluation of forages by the Agronomic “difference” method and chromogen chromicoxide “indicator” technique. N.D. Agric.Castell, A.G., Guenter, W., Igbasan, F.A. 1996. Nutritive values of peas for non-ruminant diets Animal Feed ScienceTechnology.60:209-227Cho, C.Y., Slinger, S.J., Bayly, M.S., 1982. Bioenrgetics of salmonid fishes; enrgy intake, expenditure and productivity. Comp.Biochem. Physiol. 73B, 25-41.Cruz-Ricque, L.E. Guilllaume, J. Cuzon, G. AQUACOP, Taravao ( French Polynesia) 1987. Squid protein prfotien effect on growthof four penaeid shrimp. 16 ref. Journal of the world Aquaculture Society. Baton Rouge L.A. Vol 18,No.4. pp209-217Cruz-Suarez, L.E., Ricque-Marie, D., Tapia-Salazar, M., McCallum, I.M., Hickling, D., 2001. assessment of differently processed feedpea (Pisum sativum) meals and canola meal (Brassica sp.) in diets for blue shrimp ( Litopenaues stylirostris). Aquaculture 196,87-101.Duncan, D.B., 1955. Multiple-range and multiple F-tests. Biometrics, 11, 1-42.Eusebio, P.S., 1991. Effect of dehulling on the nutritive value of some leguminous seeds as protein for tiger prawn, Penaeusmonodon juveniles. Aquaculture 99, 297-308.Feed Development Section, 1994. Feeds and feeding of Milkfish, Nile Tilapia, Asian sea bass, and Tiger Shrimp. SEAFDECAquaculture Extension Manual No. 21.SEAFDEC Aquaculture Department, Tigbauan, Iloilo, Philippines. 97 pp.Gomes, L.O.A.and Honculada Primavera, J. 1993. Reproductive quality of male Penaeus monodon . Aquaculture. 112:157-164Gomes, E.F., Rema, P., Kaushik, S.J., 1993. Effects of dietary incorporation of co-extruded plant protein (rapeseed and peas) ongrowth, nutrient utilization and muscle fatty acid composition of rainbow trout (Oncorhynchis mykiss). Aquaculture 113, 339-353.Gomes, E.F. Rema, P. Kaushik, S., 1995. Replacement of fish meal by plant protein in the diet of rainbow trout (Oncorhynchismykiss). Digestibility and growth performance. Aquaculture 130, 177-186.Gouveia, A., Davies, S.J., 1998. Preliminary evaluation of pea seed meal in diets for juvenile European sea bass (Dicentrarchuslabrax). Aquaculture 166, 311-320.Gouveia, A., Davies, S.J., 2000. Inclusion of an extruded dehulled pea seed meal in diets for juvenile European sea bass(Dicentrarchus labrax). Aquaculture 182, 183-193.Kanazawa, A., Teshima, S., 1981. Essential amino acids of the prawn. Bulletin of Japanese Society of Scientific Fisheries 17, 1375-1379.9

Kaushik, S.J., Vachot, C., Aguirre, P., 1993. Potential utilization of extruded peas. 6 thInternational Symposium on Fish Nutrition,October 4, 1993. Hobart, Australia.Millamena, O.M., Teruel, M.B., Kanazawa, A., Teshima, S., 1999. Quantitative dietary requirements of postlarval tiger shrimp,Penaues monodon, for histidine, isoleucine, leucine, phenylalanine and tryptophen. Aquaculture 179, 169-179.Millamena, O.M., Bautista-Teruel, M.N., Reyes, O.S., Kanazawa, A., 1998. Requirements of juvenile marine shrimp, Penauesmonodon (Fabricius) for lysine and arginine. Aquaculture 164, 95-104.Millamena, O.M., Bautista, M.N., Reyes, O.S., Kanazawa, A., 1997. Threonine requirement of juvenile marine shrimp Penaeusmonodon. Aquaculture 151, 9-14.Novoa, N.M.A., Castillo, L.O., 1998. Potentiacialidad del uso de las leguminosas como fuent proteica en alimentos para peces. IVSimposium Internacional de Nutricion Acuicola. La Paz, B.C.S. Mexico, Noviembre 15-18, 1998.NRC (National Research Council), 1983. Nutreint requirements of warmwater fishes and shellfishes. National Academic Press.Washington D.C.Olivera-Novoa, M.A. Olivera-Castillo,L. (Potential use of legumes as protein source in foodstuff of fish)Santos, J.M., Gomes, E., 1997. Carbohydrates in sea bass (Dicentrarchus labrax) diets: effect of the replacement of fish meal bydifferent sources of carbohydrates on growth, body composition and digestibility. Proc. 3 rdInt. Symp. On Research forAquaculture: Fundamental and Applied Aspects, 24-27 August 1997, Barcelona, Spain.186.Smith, D.M., Tabrett, S.J., Sarac, H.Z. 1999. Fish meal replacement in the diet of prawn, Penaeus monodon. Book of Abstracts of theWorld Aquaculture Society Annual Meeting ’99. April 26-May 2, 1999. Sydney, Australia. WAS, Baton Rouge, LA, USA, 707.Spyridakis, P., Metailler, R., Gabaudan, J., Riaza, A., 1989. Studies on nutrient digestibility in European sea bass (Dicentrarchuslabrax). I. Methodological aspects concerning feces collection. Aquaculture 77, 61-70.UNIP-ITCF, 1995. In: Carrouee, B., Gatel, F. (eds.) Peas utilization in animal feeding. Union Nationale Interprofessionelle desPlantes Riches en Proteines, Paris, France, 99 pp.10

Feed Pea (Pisum Sativum) As An Alternative Protein SourceIn The Diets Of Nile Tilapia, Oreochromis Niloticus (L.)Corazon B. Santiago, Perla S. Eusebio, and Timothy P. WelshAbstractA study was conducted to test feed pea (Pisum sativum) as a protein source in Nile tilapia diets and todetermine its digestibility. In experiment 1, fishmeal-based diets were used to allow the maximum level offeed pea as fishmeal protein substitute. Prepared as dry pellets, the test diets were isonitrogenous (30%crude protein) and approximately isocaloric (14 kJ g -1 ). Fish meal supplied about 28% protein in the diets.Feed peas (12.7-63.3% of the diet) substituted up to 50% of fishmeal protein. The control diet did notcontain peas. Manually sexed male Nile tilapia, weighing 32-39 g per fish at stocking, were used. The fishwere reared in polyethylene tanks (58x37x27 cm) with aeration. Water was static but was partially changeddaily. Water temperature for the whole duration of the experiment ranged from 22-27°C. Fish were allowedto feed to satiation twice daily. The various inclusion levels of feed peas did not affect the body weight,feeding activity and feed efficiency of the tilapia. Weight gain after 9 weeks (range: 21.7+5.6-34.0+9.3 g) didnot differ significantly among treatments (P>0.05). Survival rates were highly variable (40-75%) andmortality was not related to treatment. For experiment 2, plant-based tilapia diets were also designed to beisonitrogenous and isocaloric. Feed peas in the diets ranged from 5.9-41.0%. Feed peas substituted up to35% of the plant protein (or up to 29% of total dietary protein). Separate feeding trials were conducted ontwo strains of all-male tilapia (CLSU and BFAR strains) using the same test diets and larger tanks(90x78x43 cm). Water temperature during the two trials ranged from 23-27°C. After 12 weeks of feeding,weight gain did not differ significantly among treatments in each tilapia strain (45.5+7.8-57.4+12.4 g for theCLSU strain; 52.5+4.4-74.1+9.2 g for the BFAR strain). FCR and PER were not significantly differentamong treatments. Survival was 100% in all tanks in both trials. Determined with the use of chromic oxideas a marker, the apparent protein digestibility of feed pea as ingredient was 87.2+2.0%. Diets inexperiments 1 and 2 that contained peas had more than 90% protein digestibility. Overall, feed pea is analternative protein source that can be used routinely in the diets of Nile tilapia.Key words: Tilapia nutrition; Peas; Alternative protein source; Fishmeal substitute; DigestibilityIntroductionFish meal is a major source of highly digestible nutrients and is a palatable ingredient in fish diets. It can bethe cheapest protein source, at times, based on the price per kilogram of protein (Hardy, 2000). However,the demand for fish meal by various food production sectors (e.g., poultry, livestock and aquaculture) isincreasing while the supply is stagnating or even decreasing (Starkey, 1994). This causes the rising cost offish meal. In view of the need to minimize dependence of aquaculture feeds on fish meal, the search foralternative protein sources has been an international concern.The most commonly used plant protein sources that could partially substitute fish meal in fish diets are oilseed meals, some leaf meals, and cereals and their by-products (Gerpacio and Castillo, 1979; Zamora andBaguio, 1984; Tacon, 1987; NRC, 1993; Hertrampf and Piedad-Pascual, 2000). Various feedstuffs,including grain legumes, have been tested as fishmeal substitutes in farmed tilapia (El-Sayed, 1999). Thegrain legumes are also called pulses, which refer to edible seeds of plants with pods belonging to the familyLeguminocae (Hertrampf and Piedad-Pascual, 2000). Lupins, beans, black and green gram, (feed or field)pea and several other peas (cow pea, chick pea) belong to this category. Although they are used in poultry,swine, and cattle feeds, peas are not among the ingredients ordinarily added in fish diets. However, thedigestibility of several species of legume seeds has been studied in young tilapia (Oreochromis niloticus)(De Silva et al. 1988), juvenile rainbow trout (Oncorhynchus mykiss) (Gomes et al., 1995; Pfeffer et al.,1995; Burel et al., 2000), turbot (Psetta maxima) (Burel et al., 2000), and Australian silver perch (Bidyanusbidyanus) (Allan et al., 2000). Feed pea (Pisum sativum), in particular, has been tested to replace fish mealin the diets of rainbow trout (Gouveia et al., 1993), European sea bass (Dicentrarchus labrax) (Gouveia andDavies, 1998), and Atlantic salmon (Salmo salar) (Carter and Hauler, 2000). Because of its high potentialas a fish feed ingredient, feed pea has to be tested in other important food fish, including tilapia. Thus, thepresent study was conducted to determine the effect of levels of dietary feed peas on the growth andsurvival of juvenile Nile tilapia and on feed efficiency. The digestibility of feed peas as an ingredient and ofdiets containing feed peas was also determined.Materials and Methods1. Experimental dietsTwo sets of diets in dry pellet form were tested in two separate experiments. For experiment 1, six fishmealbaseddiets (Table 1) were designed to contain 30% crude protein and digestible energy of about 14 kJ g -1 .The computed amino acids in the diets, based on the amino acid contents of the ingredients, met or11

exceeded amino acid requirements of young Nile tilapia (Santiago and Lovell, 1988). A fishmeal-based dietallowed the incorporation of high levels of feed peas in the test diets. The feed peas (12.7-63.3% of thediet) substituted 10-50% of fishmeal protein, or about 9-48% of total dietary protein. The diet without feedpeas served as a control. Because of the high nitrogen-free extract (NFE) in feed peas, the diets had aboutthe same energy content, but the supplemental starch and oil decreased as the feed pea in the dietincreased. Based on the actual analysis of protein, lipid, and NFE, however, the digestible energy of thediet with the highest feed pea (63.3%) was slightly lower (Table 1).In experiment 2, eight plant-based diets containing only 8% fish meal were used (Table 2). The diets werealso designed to be isonitrogenous and isocaloric as in experiment 1. Dietary feed peas (5.9-41.0%)substituted 5-35% of plant protein or about 4-30% of total dietary protein. Substitution of protein fromsoybean meal, copra (coconut) meal and rice bran by the feed peas was maintained at a ratio of 2:1:1.The fish meal used in experiments 1 and 2 was obtained in two batches. The commercial fish meal,soybean meal and copra meal were ground and sieved using a No. 45 standard testing sieve before use.The dry feed peas were ground whole, including hulls, into powder form. Analyses showed that the peascontained 22.32% crude protein, 1.17% crude fat, 3.36% ash, 3.25% crude fiber, and 58.32% nitrogen-freeextract. These values are close to those reported for feed peas (Muehlbauer and Tullu, 1997; PulseCanada, 1999a; Racz, 1999).2. Experimental fish and tanksThree strains of tilapia juveniles were obtained for the study. The young fish were reared in the laboratoryfor 6-8 weeks before use. During this time, the fish were fed dry pellets without feed peas. Each batch offish was sorted three times during acclimatization to ensure uniformity in size at stocking.The fish in experiment 1 were manually sexed male Nile tilapia juveniles (BFS strain) produced in theStation. Each fish weighed 32-39 g at stocking. Twenty-four polyethylene tanks measuring 58x37x27 cmwere filled with 40 l of water and randomly stocked with five fish each.In experiment 2, two all-male tilapia strains were used in separate feeding trials. The CLSU strainindividually weighed 24-31 g at stocking; the BFAR strain, 25-37 g each. Five tilapia juveniles (CLSU strain)were stocked in each of 24 polyethylene tanks (90x78x43 cm) filled with 180 l of water. In the other feedingtrial, 16 tanks were randomly stocked with six juveniles (BFAR strain) per tank.3. Feeding treatments, sampling and water managementEach of the feeding experiments was conducted in a completely randomized design. In experiment 1, therewere six dietary treatments with four replicates each. The treatments represented levels of fishmeal proteinsubstitution by the feed peas. In each of the two feeding trials in experiment 2, there were eight treatmentsor levels of plant protein substitution by feed peas. Each of the dietary treatments had three replicates forthe CLSU strain or two replicates for the BFAR strain.Feeding was done twice daily at 0900 and 1400h for 9 weeks in experiment 1 and for 12 weeks inexperiment 2. The amount of feed given to the fish at each feeding exceeded satiation level to ensure thatthe feed was not limiting. Excess feed was collected 1-1.5 hours after each feeding, dried in an oven,weighed and used in the calculation of feed consumption by difference. The amount of feed consumed wasback calculated to be equivalent to about 5% of fish biomass.In all feeding trials, body weight of fish was measured weekly for the first 4 weeks and biweekly in thesucceeding weeks except in experiment 1 when the final measurements were done after 9 weeks. Totallength of fish was measured at stocking and during harvest. Feed conversion ratio (FCR, the amount offeed consumed divided by weight gain of fish), protein efficiency ratio (PER, weight gain divided by weightof protein consumed), and survival rate were determined for each tank.Deep well water stored in a reservoir was used for the growth trials. Water in the rearing tanks was staticbut was partially changed (1/4-1/3 of the volume) daily. All rearing tanks were provided with aeration.Temperature was determined daily in the morning and afternoon. Dissolved oxygen was monitored everymorning before water was partially changed with the use of YSI DO meter (Model 55/25 FT). The pH wasmeasured with a pH meter (Beckman, Model PHI 50). Total ammonia nitrogen (NH 3 -N) was determinedweekly before changing of water in the morning by the Nessler method with the use of a Hach kit(DREL/2010).12

Water temperature for the whole duration of experiment 1 ranged from 22-27°C, being at the lower rangefor most of the days. Dissolved oxygen in the morning before partial water change ranged from 3.5-6.2 mg l -1 . The total NH 3 -N ranged from 0.63-2.39 mg l -1 and pH, 7.7-7.9. In experiment 2, water temperature for thefeeding trial with the CLSU strain of tilapia was 23-27°C; pH, 7.4-8.1; total NH 3 -N, 0.21-2.42 mg l -1 . For thefeeding trial using the BFAR strain, water temperature also ranged from 23-27°C; pH, 7.6-8.7; and totalNH 3 -N, 0.28-1.74 mg l -1 . For each sampling time, water quality was similar among treatments.4. Biochemical analysesSamples of feed ingredients, experimental diets, and some fish at stocking and during harvest wereobtained for proximate analysis by standard methods (AOAC, 1990). At the end of the each feeding trialand after a 24-hour fasting, fish samples were obtained and dissected. The appearance of the fish and theinternal organs were examined visually for any abnormality. Other samples of fish that received the dietswhose digestibility was determined were subjected to cold shock, weighed and kept frozen until they wereprocessed further. The fish samples were then autoclaved, homogenized, dried in a vacuum oven at 60°C,and ground for analysis.5. Digestibility measurements5.1. Digestibility of feed peas as ingredientThe apparent dry matter digestibility (ADMD) and crude protein digestibility (APD) of feed peas asingredient were measured indirectly using chromic oxide (Cr 2 O 3 ) as external indicator (Cho et al., 1982;Spyridakis et al., 1989). The reference diet for the measurement of digestibility of feed peas was the sameas the control diet used in experiment 1 (Table 1), except that 1% Cr 2 O 3 and 1% carboxymethylcellulose(CMC, binder) replaced some amounts of the filler. The test diet contained 70% reference diet and 30%feed peas, following the method of Cho et al. (1982). It also contained 1% Cr 2 O 3 and CMC.Each of the diets was fed to tilapia in three replicate tanks stocked with 10 juvenile tilapia (37.96 ± 0.42 gfish -1 ). The tilapia came from the same source as those used in experiment 1. The fish were fed at 5-8% offish biomass daily with two feedings a day (0830 and 1430h). Water temperature ranged from 24-26°C.A modified Guelph system (Cho et al., 1982; Eusebio and Coloso, 2000) was used in the digestibility trials.Each conical fiberglass tank (250 L capacity) was provided with filtered, flow-through water. Flow rate of thewater was 780-850 ml min -1 . Water passed through a fecal decantation column into an attached clearplastic bottle where the fecal materials settled until collected. The tanks and the fecal collection apparatuswere cleaned twice daily before feeding in the morning and 2 hours after feeding in the afternoon.Collection of feces released from 1730-0730 h started after a 5-day initial feeding and lasted for 25consecutive days. Fecal material were carefully rinsed with distilled water and stored in a freezer to retardbacterial decomposition. Samples were pooled and freeze-dried. Dry fecal samples and test diets wereanalyzed for Cr 2 O 3 (Carter et al., 1960) and crude protein (AOAC, 1990).5.2. Digestibility of diets containing feed peasThe digestibility of diets with 0, 10, 30 and 50% replacement of fishmeal protein (Table 1), as well as dietswith 0, 15, 25 and 35% replacement of plant protein by feed peas (Table 2), was determined in tilapia inseparate trials. Celufil (filler), starch, and rice bran were adjusted to accommodate 1% Cr 2 O 3 and the binderin the diets. Each feeding period, including the 5-day adjustment with the marked diets, lasted for 30 days.Fish used in determining the digestibility of the two sets of diets containing feed peas were bigger tilapiajuveniles (CLSU strain, 82.7+9.3 g) that came from the same batch used in the growth trial (experiment 2).The digestibility tank system, and the procedure for collection of feces and processing of samples were thesame as in the previous digestibility trial for peas as an ingredient. The apparent digestibility for dry matterand crude protein in the diets was likewise computed using the formula of Spyridakis et al. (1989).6. Statistical analysisData on body weight (weekly and biweekly), weight gain, total length, survival, FCR, PER, carcass proteinand ash, and digestibility coefficients were analyzed by ANOVA using the General Linear ModelsProcedure by SAS for PC (SAS Institute Inc., 1991). When significant difference among treatments wasdetected at P

However, weight gain was highest at the 10% fishmeal protein substitution, or 12.7% feed pea in the diet.The FCR values for the duration of the experiment, as well as the PER, did not differ significantly (Table 3).Survival rates of tilapia in replicate tanks within treatment were highly variable; mean survival rates did notdiffer significantly among treatments (P>0.05). Mortality was caused by the aggressive behavior of the maletilapia in the small rearing tanks and was not treatment-related.In experiment 2, body weight of the CLSU strain of tilapia at weekly or biweekly intervals did not differsignificantly among treatments (P>0.05). The final weight gain and length increment, as well as the FCRand PER, did not differ significantly (Table 4). Survival rate was 100% in all tanks. Likewise, the BFARstrain of tilapia in experiment 2 did not show significant differences in body weight during each sampling, orweight gain and total length increment over a 12-week period (Table 5). Moreover, the FCR and PER didnot differ significantly among treatments. Survival rate in all tanks was also 100%.Carcass protein of fish from four dietary treatments in experiment 1 showed no significant differences(P>0.05) (Table 6). However, body ash increased significantly in fish fed diets with 37.5 and 63.3% feedpeas (or 30 and 50% fishmeal protein substitution). The fish fed with 10% fishmeal protein substitution(12% feed peas) had the highest carcass lipid. Fish in all other treatments had similar carcass lipid. Inexperiment 2, carcass protein as well as body ash differed significantly among treatments (Table 6), but notrend could be established in relation to fish growth and digestibility of the diets. However, as the dietaryfeed peas in the plant-based diets increased and the dietary lipid decreased (Table 2), carcass lipiddecreased (Table 6).There was no visible adverse effect of dietary peas on feeding behavior and the appearance of the fish andthe internal organs. The three tilapia strains used in the feeding trials readily accepted the diets containingfeed peas and consumed almost the same amount of feed in relation to their body weight (about 5% of fishbiomass). Visual examination at the end of each trial revealed that the tilapia were generally lean. Fish liverdid not appear fatty and only few adipose tissues were visible in the body cavity.The apparent dry matter digestibility (ADMD) of feed peas as ingredient was 69.3+1.8% and proteindigestibility (APD) was 87.2+2.0%. The ADMD coefficients for some fishmeal-based diets used inexperiment 1 significantly increased with increasing level of feed peas (Table 7). The APD for the dietstested were all high, exceeding 90%. Although the absolute differences were small for practical purposes,the APD of the control diet (without pea) was significantly higher than that of diet with 12.7% feed pea, butdid not differ significantly from that of diets with 37.5% or 63.3% feed pea (Table 7). The plant-based dietsin which digestibility coefficients were determined also showed a significant increase in ADMD when feedpea in the diet was 29.3% or more (Table 7). The APD coefficients of the plant-based diets were high(>90%), as in the fishmeal-based diets in experiment 1, and did not differ significantly among treatments.DiscussionThe three feeding trials consistently demonstrated that feed peas could be incorporated in tilapia diets at awide range of levels to replace fishmeal protein or other plant proteins without affecting fish growth, feedefficiency, and survival. The FCR values obtained for all diets in the feeding trials were higher than theexpected values (about 2) and, consequently, the PER values were fairly low. These could be attributed tothe water temperature during the experiments, being lower than the optimum (28-31°C) for the tilapia(Luquet, 1991), and to some losses in the collection of the uneaten feed. Because the indoor culture systemhad static water, the dissolved oxygen before partial water change in the morning sometimes dipped closeto the minimum level (3 mg l -1 ) for tilapia (Luquet, 1991). Nevertheless, the tilapia in two of three feedingtrials had 100% survival. The growth response of tilapia to diets with peas in the different feeding trials wasnot affected by the dietary levels of feed pea.In general, carcass protein, lipid and ash did not indicate any adverse effect of increasing dietary feed pea.Although growth did not differ significantly among treatments, significant differences in protein wasobserved in fish fed plant-based diets, but not in fish fed the fishmeal-based diets. Moreover, carcass lipidof the tilapia (CLSU strain) fed plant-based diets decreased with the increase of feed peas in the diet. In acompanion study, feeding of milkfish (Chanos chanos) with varying dietary feed peas caused significantdifferences in growth but it did not influence carcass composition (I. Borlongan, personal communication2002). Similarly, two levels of peas in the diets (20 and 40%) did not affect carcass protein and ash inEuropean sea bass but lipid was significantly lower in fish given diet with 40% pea (Gouveia and Davies,1988).Although high amounts of feed peas could be used in tilapia diets, the actual amount that would beincorporated in practical applications will be influenced largely by the cost of the peas in relation to the cost14

of competing protein sources. The digestibility of the peas is also considered in evaluating its potential as aprotein source. In the present study, the digestibility coefficients for feed peas in Nile tilapia are comparableto those reported for other freshwater fishes (Table 8). The highest digestibility coefficients for P. sativumwere obtained in turbot, a marine species. In a companion study, the crude protein digestibility of feed peasin milkfish was also higher (92%) than in tilapia; however, dry matter digestibility was lower (I. Borlongan,personal communication 2002). Allan et al. (2000) determined the digestibility of peas and other ingredientsin silver perch as well as that of amino acids in the ingredients. The protein digestibility of the ingredientswas a reflection of the availability of amino acids in silver perch. A few exceptions were the animal proteinsources and some oilseed meals whose proteins may have been damaged during processing (Allan et al.,2000). Moreover, the apparent energy digestibility of peas as ingredient in some fish species (Table 8)could also reflect carbohydrate digestibility in the fish. This is because peas contain much higher amountsof carbohydrate than lipid (Muehlbauer and Tullu, 1997; Pulse Canada, 1999a; Racz, 1999) and the proteindigestibility coefficients for peas in different fish species (Table 8) are close. Moreover, the apparent energydigestibility coefficients reported for peas as ingredient are near the values for the carbohydrate digestibilityof soybean meal (54%), wheat grain (61%), and uncooked corn starch (55-61%) in blue tilapia (0. aureus)(NRC, 1993).Information on the use of P. sativum in fish diets is limited. Gomes et al. (1995) used feed pea at very a lowlevel (4% of the diet) together with other plant ingredients (faba bean meal, maize gluten, full-fat soybean,and a co-extruded mixture of peas and rapeseed) to partially substitute fish meal in rainbow trout diet. In astudy by Gouveia et al. (1993), pea seed meal was used at 38.2% of the diet (20% of the dietary protein) ofrainbow trout with or without cooking/expansion (145 º C, 25 kg per cm 2 ). The thermal treatment slightlyimproved the nutritional value of the diets containing the peas. In another study, there was a significantdecrease in the protein digestibility of the diet when the pea (P. sativum) in the diet of rainbow troutincreased from 25–50% (Pfeffer et al., 1995). Furthermore, the digestibility of the diets containing either 25or 50% peas increased significantly when the peas were autoclaved. APD coefficients of rainbow trout dietscontaining 25% feed peas increased from 86-91% when the peas had pre-treatment. For diets containing50% peas, APD increased from 83-86% when the peas were autoclaved (Pfeffer et al., 1995). In asubsequent study by Gouveia et al. (1998) on European sea bass (D. labrax), fishmeal-based dietscontaining 20 and 40% pea had APD coefficients of 88-89%. In the present study, the APD coefficients ofthe tilapia diets containing feed pea were high (90.5-92%) and were practically not affected by the dietarylevel of peas. This suggests that feed pea and the ingredients it replaced had similar protein digestibility.The whole peas with hulls were simply dried and ground. The digestibility of dehulled peas would mostlikely be higher in tilapia since dehulling removes most of the fiber of ingredients (Eusebio, 1991). Whencompared to other feedstuffs tested in tilapia, feed pea has APD coefficient that is comparable to that of fishmeal (Hanley, 1987; NRC, 1993; Jauncey, 1998). However, it is slightly lower than the protein digestibilityvalues reported for soybean meal (91-94%) in tilapia species (Hanley, 1987; Lim, 1987; Luquet 1991; NRC,1993).The protein content of feed peas is close to that of copra (coconut) meal, some cereal grain by-products,brewers' grains, and leaf meals (Gerpacio and Castillo, 1979; Hanley, 1987; Tacon, 1987; NRC, 1993;Hertrampf and Piedad-Pascual, 2000). The relatively low protein content of feed peas compared to fishmeal would limit the amount that could be incorporated in the diets of fish that require high protein levels.However, feed peas could be used as one of the ingredients that would collectively replace fish meal in apractical diet. Feed peas could also replace other plant protein sources, as shown in the present study,especially when its cost is competitive. Feed peas contain high levels of lysine (Pulse Canada, 1999a;Racz, 1999) which could complement the amino acids of other fishmeal substitutes that are deficient inlysine (e.g. canola meal and gluten meals). However, as in soybean meal and other legume seeds, thesulfur amino acids (methionine and cystine) are low and could be the first limiting amino acids when highamounts of plant protein sources are used. For fish requiring high dietary protein, pea protein concentrate(49% crude protein) has been explored as a possible partial replacement of fish meal (Carter and Hauler,2000). Up to 27% pea protein concentrate in the extruded feed did not affect growth of salmon parr. Cowpea (Vigna unguiculata) protein concentrate was also tested in Nile tilapia fry and up to 40% inclusion levelin the diet gave acceptable results (Olvera-Novoa et al., 1997).One important concern in the use of pulses as ingredients in aqua feeds is the presence of anti-nutritionalfactors (Tacon, 1987; Hertrampf and Piedad-Pascual, 2000). However, through genetic selection, presentpea varieties contain no tannins and only low levels of trypsin inhibitors and lectins (Bond and Duc, 1993)that can be deactivated by heat treatment (Melcion and van del Poel, 1993). Pulses are produced mainly fortheir mature seeds and immature pods for human consumption (Muehlbauer and Tullu, 1997), unlikesoybean and other oilseeds that are grown mainly for processing into edible oils and protein concentrates.With the expansion of production of feed peas as human food and animal feed (Pulse Canada, 1999b),more peas would be available in the market.15

The consistent response of the tilapia juveniles to diets containing feed peas in three feeding trials clearlyshowed that feed pea as an alternative ingredient can be routinely included in tilapia diets. Further studieson feed peas as component of tilapia diets (e.g., those related to processing and energy utilization) have tobe conducted.Table 1Composition of fishmeal-based diets for male Nile tilapia in experiment 1 (g per 100g diet)% replacement of fishmeal protein (or total dietary protein)Ingredient0 a 10 (9.3) a 20 (18.6) 30 (28.0) 40 (37.3) 50 (47.6)aFish meal 49.0 44.1 39.2 34.3 29.4 24.5Peas - 12.7 25.3 37.5 50.7 63.3Rice bran 15.0 15.0 15.0 15.0 15.0 10.2Starch 15.0 12.0 9.0 6.0 - -Soybean oil 5.0 5.0 5.0 5.0 2.9 -Vit-min mix 2.0 2.0 2.0 2.0 2.0 2.0Celufil (filler) 14.0 9.2 4.5 0.2 - -Analyzed (as fed)Crude protein (%) 31.9 31.6 31.0 29.9 29.2 28.9Crude fat (%) 11.2 11.0 10.4 10.4 7.7 2.5Ash (%) 9.7 9.0 8.9 8.1 7.7 6.7Crude fiber (%) 6.7 6.5 6.2 6.8 7.0 6.4NFE (%) 33.9 35.7 37.2 37.8 41.3 48.6Digestible energy (kJ g -1 ) 14.5 14.7 14.6 14.4 13.8 12.9a Diet whose digestibility in tilapia was also determined in a separate feeding trial.Table 2 Composition of plant-based diets for male tilapia (CLSU and BFAR strains) in experiment 2(g per 100g diet)% replacement of plant proteins (or total dietary protein)Ingredient 0 a 5 (4.2) 10 (8.4) 15 (12.7) a 20 (16.9) 25 (21.1) a 30 (25.3) 35 (29.6) aFish meal 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00Soybean meal 40.00 38.63 37.25 35.88 34.50 33.13 31.75 30.38Copra meal 20.00 18.37 16.73 15.10 13.47 11.83 10.20 8.57Rice bran 24.00 21.49 18.98 16.46 13.95 11.44 8.93 6.41Peas - 5.86 11.72 17.57 23.43 29.29 35.15 41.00Soybean oil 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00Vitamin mix 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40Mineral mix 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70Starch 2.90 2.56 2.23 1.89 1.55 1.21 0.88 0.54Analyzed (as fed)Crude protein (%) 29.7 32.4 32.4 31 32 29.8 31.8 29.9Crude fat (%) 10.1 9.5 8.7 7.1 8.2 7.5 7.1 6.9Ash (%) 7.7 6.3 7 5.6 7.2 7 6.2 6.6Crude fiber (%) 7.9 7.4 7.7 6.5 7.2 6.6 6.6 6.3NFE (%) 37.4 37.2 37.4 42.9 38.3 41.8 41.2 43Digestible energy (kJ g -1 ) 14.2 14.4 14.2 14.1 14.1 13.9 14 13.9a Diet whose digestibility in tilapia was also determined in a separate feeding trial.Table 3Mean initial weight and total length, gain in weight and length, survival, FCR and PER of male Niletilapia fed diets containing feed peas at varying fishmeal protein substitution for 9 weeks(experiment 1) aFishmeal protein Dietary feed Body weight (g) Total length (cm) Survivalsubstitution (%) peas (%) Initial Gain Initial Gain (%)FCR PER- - 35.5+0.9 24.0+7.7 12.6+0.7 3.0+0.5 40+23 4.1+0.7 0.8+0.110 12.7 35.3+0.6 34.0+9.3 12.5+0.6 3.4+0.4 70+26 3.2+0.5 1.0+0.220 25.3 35.8+0.3 25.3+7.3 12.6+0.5 2.8+0.4 70+26 4.2+1.6 0.8+0.330 37.5 34.9+1.1 21.7+5.6 12.5+0.6 2.4+0.1 55+30 4.2+0.6 0.8+0.140 50.7 35.5+0.6 25.1+4.6 12.7+0.6 3.0+0.6 45+19 3.4+0.7 1.0+0.250 63.3 35.5+0.6 25.2+9.6 12.4+0.7 2.8+0.7 65+25 4.0+1.4 1.0+0.4a Column means are not significantly different from each other (P>0.05). Mean+SEM16

Table 4Mean initial weight and total length, gain in weight and length, FCR and PER of tilapia (CLSUstrain) fed diets containing feed peas at varying plant protein substitution for 12 weeks(experiment 2) aPlant protein Dietary feed Body weight (g) Total length (cm)substitution (%) peas (%) Initial Gain Initial GainFCR PER0 0 28.0+3.0 53.6+12.8 11.6+0.1 4.7+1.4 3.4+0.6 1.0+0.25 5.9 28.2+2.8 57.4+12.4 11.6+0.3 5.0+0.2 3.2+0.4 1.0+0.110 11.7 28.5+3.2 49.5+ 7.2 11.5+0.2 5.1+0.6 3.6+0.4 0.9+0.115 17.6 28.3+2.9 57.1+ 8.5 11.7+0.2 5.2+0.9 3.3+0.3 1.0+0.120 23.4 28.5+2.6 56.7+ 2.1 11.7+0.2 5.1+0.3 3.3+0.3 1.0+0.125 29.3 28.4+3.2 45.5+ 7.8 11.7+0.1 4.6+0.4 4.0+0.4 0.9+0.130 35.2 28.4+3.0 54.0+ 0.7 11.6+0.2 5.1+0.2 3.5+0.2 0.9+0.035 41 28.1+2.4 56.7+13.5 11.5+0.1 5.4+1.0 3.4+0.6 1.0+0.2a Column means are not significantly different from each other (P>0.05). Mean+SEMTable 5Mean initial weight and total length, gain in weight and length, FCR and PER of male tilapia (BFARstrain) fed diets containing feed peas at varying plant protein substitution for 12 weeks(experiment 2) aPlant protein Dietary feed Body weight (g) Total length (cm)substitution 0.80 peas (%) Initial Gain Initial GainFCR PER0 0 31.1+4.3 52.5+4.4 12.1+0.6 5.0+0.6 4.3+0.4 0.8+0.15 5.9 31.0+3.6 71.6+22 12.1+0.5 5.8+0.8 3.3+0.6 0.9+0.210 11.7 30.9+4.3 60.2+0.7 12.1+0.6 5.0+0.4 3.6+0.2 0.8+0.015 17.6 30.9+4.3 69.0+11.5 12.1+0.5 4.8+1.5 3.7+0.6 0.9+0.220 23.4 30.8+3.9 62.4+10 12.2+0.6 5.1+0.3 3.6+0.5 0.9+0.125 29.3 30.9+3.6 57.0+5.9 12.1+0.5 5.2+0.8 3.6+0.1 0.9+0.030 35.2 31.0+3.8 63.9+1.3 12.1+0.4 5.1+0.3 3.8+0.1 0.8+0.035 41 31.1+4.5 74.1+9.2 12.1+0.6 5.7+0.7 3.2+0.1 1.0+0.0a Column means are not significantly different from each other (P>0.05). Mean+SEMTable 6 Carcass protein and ash of Nile tilapia after 9 weeks of feeding using some diets in experiment 1and after 12 weeks of feeding in experiment 2 aDietDiets in Expt 1Dietary feedpeas (%)% fishmeal protein substitutionCrudeproteinAshLipid0 - 56.5± 0.2 a 18.9± 0.1 b 18.8±0.3 b10 12.7 56.4± 0.6 a 19.5± 0.3 b 19.4±0.0 a30 37.5 57.2+0.4 a 20.8+0.4 a 18.7±0.0 b50 63.3 57.4+0.2 a 20.8+0.1 a 18.9±0.1 bDiets in Expt 2 b% plant protein substitutionDry matter basis (%)0 - 55.7+0.4 c 16.3+0.6 b 23.2±0.4 a15 17.6 59.1+0.4 a 14.7+0.2 c 21.2±0.3 b25 29.3 56.5+0.2 c 16.3+0.3 b 20.9±0.3 b35 41.0 58.2+0.2 b 18.3+0.0 a 20.3±0.1 baFor each experiment, column means with a common superscript are notsignificantly different (P>0.05).bAt stocking of the CLSU tilapia strain, body protein was 58.7+0.5%; ash,26.0+0%; lipid, 11.3+0.2%.17

Table 7Apparent dry matter digestibility (ADMD) and protein digestibility (APD) of diets with peas injuvenile male tilapia (CLSU strain)DigestibilitytrialsDietary feedpeas (%)Trial using diets in Expt 1% fishmeal protein substitutionADMD (%) aAPD (%) a0 0 72.1± 0.6 c 92.5± 0.4 a10 12.7 75.6± 0.4 b 90.5± 0.4 b30 37.5 77.8+1.1 ab 91.5+0.7 ab50 63.3 78.8+0.6 a 92.0+0.2 abTrial using diets in Expt 2% plant protein substitution0 0 74.1+0.2 b 90.6+0.3 a15 17.6 73.4+0.59 b 91.5+0.3 a25 29.3 76.2+0.67 a 91.2+0.3 a35 41 76.9+0.42 a 91.6+0.3 aa For each digestibility trial, column means with a commonsuperscript are not significantly different (P>0.05).Table 8Digestibility coefficients (%) for P. sativum in different fish speciesFish Dry matter Protein Energy ReferenceRainbow trout, 52 g 66.1 80.4 59.2 Gomes et al. (1995)Rainbow trout, 100g 66.3 87.9 68.9 Burel et al. (2000)Turbot, 110 g 71.5 92.9 77.7 Burel et al. (2000)Silver perch 51.0 81.0 51.0 Allan et al. (2000)Nile tilapia, 38 g 69.3 87.2 no data present studyAcknowledgementsThis study was funded by the US Department of Agriculture and sponsored by the USA Dry Pea and LentilCouncil. Feeds and fish samples were analyzed by the staff of the Central Analytical Laboratory ofSEAFDEC AQD, Tigbauan, Iloilo or the Animal Nutrition Analytical Service Laboratory, University of thePhilippines Los Baños, Laguna.ReferencesAllan, G.L., Parkinson, S., Booth, M.A., Stone, D.A.J, Rowland S.J., Frances, J., Warner-Smith, R., 2000. Replacement of fish meal indiets for Australian silver perch, Bidyanus bidyanus: 1. Digestibility of alternative ingredients. Aquaculture 186, 293-310.Association of Official Analytical Chemists (AOAC), 1990. Official Methods of Analysis, 15th edn. AOAC, Arlington, VA, 1228 pp.Bond, D.A., Duc, G., 1993. Plant breeding as a means of reducing antinutritional factors in grain legumes. In: van der Poel, A.F.B.,Huisman, J., Saini, H.S. (Eds.), Second International Workshop on Antinutritional Factors (ANFs) in Legume Seeds, RecentAdvances of Research in Antinutritional Factors in Legume Seeds, 1-2 December 1993, EAAP Publication, The Netherlands, pp.379-396.Burel C., Boujard, T., Tulli, F., Kaushik, S.J., 2000. Digestibility of extruded peas, extruded lupin, and rapeseed meal in rainbow trout(Oncorhynchus mykiss) and turbot (Psetta maxima). Aquaculture 188, 285-298.Carter, J.F., Bolin, D.W., Erikson, P., 1960. The evaluation of forages by the Agronomic “difference” method and chromogen chromicoxide “indicator” technique. N.D. Agric. Exp. Stn., Techn. Bull. 426, 55 pp.Carter, C.G., Hauler, R.C., 2000. Fish meal replacement by plant meals in extruded feeds for Atlantic salmon, Salmo salar L.Aquaculture 185, 299-311.Cho, C.Y., Slinger, S.J., Bayley, M.S., 1982. Bioenergetics of salmonid fishes: energy intake, expenditure and productivity. Comp.Biochem. Physiol. 73B, 25-41.De Silva, S.S., Keembiyahetty, C.N., Gunasekera, R.M., 1988. Plant ingredient substitutes in Oreochromis niloticus (L.) diets:Ingredient digestibility and effect of dietary protein content on digestibility. J. Aqua. Trop. 3, 127-138.El-Sayed, A.-F. M., 1999. Alternative dietary protein sources for farmed tilapia, Oreochromis spp. Aquaculture 179, 149-168.Eusebio, P., 1991. Effect of dehulling on the nutritive value of some leguminous seeds as protein sources for tiger prawn (Penaeusmonodon) juveniles. Aquaculture 99, 297-308.Eusebio, P.S., Coloso, R.M., 2000. Nutritional evaluation of various plant protein sources in diets for Asian sea bass Lates calcarifer.J. Appl. Ichthyol. 16, 56-60.Gerpacio, A.L., Castillo, L.S., 1979. Nutrient Composition of Some Philippine Feedstuffs. Technical Bulletin 21, Extension Division,Department of Animal Science, University of the Philippines at Los Baños, Philippines, 117 pp.Gomes, E.F., Rema, P., Kaushik, S.J., 1995. Replacement of fish meal by plant proteins in the diet of rainbow trout (Oncorhynchusmykiss): digestibility and growth performance. Aquaculture 130, 177-186.Gouveia, A., Oliva Teles, A., Gones, E., Rema, P., 1993. Effect of cooking/expansion of three legume seeds on growth and foodutilization by rainbow trout. In: Fish Nutrition in Practice, Biarritz (France), June 24-27, 1991. Ed. INRA, Paris (Les Colloques,no61), pp. 933-938.Gouveia A., Davies, S.J., 1998. Preliminary nutritional evaluation of pea seed meal (Pisum sativum) for juvenile European sea bass(Dicentrarchus labrax). Aquaculture 166, 311-320.18

Hanley, F., 1987. The digestibility of feedstuffs and the effects of feeding selectivity on digestibility determinations in tilapia,Oreochromis niloticus. Aquaculture 66, 163-179.Hardy, R.W., 2000. Fish feeds and nutrition- Fish feeds & nutrition in the new millenium. Aquaculture Magazine, Jan/Feb 2000, pp.85-89.Hertrampf, J.W., Piedad-Pacual, F., 2000. Handbook on Ingredients for Aquaculture Feeds. Kluwer Academic Publishers, TheNetherlands, 573 pp.Jauncey, K., 1998. Tilapia Feeds and Feeding. Pisces Press Ltd., Stirling, Scotland, 241 pp.Lim, C., 1987. Practical feeding – tilapias. In: Lovell, T., Nutrition and Feeding of Fish. Von Nostrand Reinhold, NY, pp 163-183.Luquet, P., 1991. Tilapia, Oreochromis spp. In: Wilson, R.P. (Ed.), Handbook of Nutrient Requirements of Finfish. CRC Press, Inc.Florida, USA, pp. 169-179.Muehlbauer, F.J. and Tullu, A., 1997. Pisum sativum L. http://www.hort.purdue.edu/cropfactsheets/pea.htm (2 July 2002).Melcion, J.-P., van der poel, A.F.B., 1993. Process technology and antinutritional factors: principles, adequacy and processoptimization. In: van der Poel, A.F.B., Huisman, J., Saini, H.S. (Eds.), Second International Workshop on Antinutritional Factors(ANFs) in Legume Seeds, Recent Advances of Research in Antinutritional Factors in Legume Seeds, 1-2 December 1993, EAAPPublication, Wageningen, The Netherlands, pp. 419-434.National Research Council (NRC), 1993. Nutrient Requirements of Fish. National Academy Press, Washington, DC., 114 pp.Olvera-Novoa, M.A., Pereira-Pacheco, F., Olivera-Castillo, L., Pérez-Flores, V., Navarro, L., Sámano, J.C., 1997. Cowpea (Vignaunguiculata) protein concentrate as replacement of fish meal in diets for tilapia (Oreochromis niloticus) fry. Aquaculture 158, 107-116.Pfeffer, E., Kinzinger, S., Rodehutscord, M., 1995. Influence of the proportion of poultry by-products and of untreated orhydrothermically treated legume seeds in diets for rainbow trout, Oncorhynchus mykiss (Walbaum), on apparent digestibilities oftheir energy and organic compounds. Aquaculture Nutr. 1, 111-117.Pulse Canada, 1999a. Nutrient Composition. http://www.pulsecanada.com/FeedPeas/NutrientComposition.htm (29 June 2002).Pulse Canada, 1999b. Production Info. http://www.pulsecanada.com/FeedPeas/ProductionInfo.htm (29 June 2002)Racz, V.J., 1999. Feed pea nutrient composition. In: Feed Peas, Selected Publications and Articles for Animal Nutritionists andLivestock Producers. USA Dry Pea and Lentil Council, Bangkok, Thailand, pp. 5-8.Santiago, C.B., Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. J. Nutr. 118, 1540-1546.SAS Institute Inc., 1991. SAS System for Linear Models. Third Edition. SAS Institute Inc., Cary, North Carolina, 329 pp.Spyridakis, P., Metailler, R., Gabaudan, J., Riaza, A., 1989. Studies on nutrient digestibility in European sea bass (Dicentrarchuslabrax). I. Methodological aspects concerning feces collection. Aquaculture 77, 61-70.Starkey, T. J., 1994. Status of fish meal supplies and market demand. Miscellaneous report. H.J. Baker and Bro. Inc., Stamford, CT.,USA, 28 pp.Tacon, A.G.J., 1987. The Nutrition and Feeding of Farmed fish and Shrimp - A Training Manual 2. Nutrient Sources and Composition.FAO, Brazil, 129 pp.Zamora, R.G., Baguio, S.S., 1984. Feed Composition Tables for the Philippines. PCARRD Book Series No. 1/1984, Philippine Councilfor Agriculture and Resources Research and Development, Los Baños, Laguna, Philippines, 264 pp.19

Potential Of Feed Pea (Pisum Sativum) As An Alternative Protein SourceIn Practical Diets For Milkfish (Chanos Chanos Forsskal)Ilda G. Borlongan, Perla S. Eusebio, and Timothy P. WelshAbstractA 12-week feeding trial was conducted to evaluate the use of feed pea as a potential dietary protein sourcefor juvenile milkfish, Chanos chanos Forsskal. Six isonitrogenous (30% crude protein) and isocaloric (3940kcal/kg) practical diets were formulated. The control diet contained fish meal, soybean meal, meat andbone meal, and copra meal as principal protein sources. Feed pea was progressively substituted at 0, 5,10, 15, 20, 25 and 30% of total protein. A leading commercial milkfish feed was also tested as an additionalcontrol. The experimental diets were fed to triplicate groups of milkfish fingerlings (mean initial weight of0.42 ± 0.01g) at 10% body weight/day. Growth performance (expressed as % weight gain and SGR),survival, feed conversion ratio (FCR) and protein efficiency ratio (PER) of milkfish fed diets with up to 10%substitution of the dietary protein with feed pea were not significantly different (P >0.05) compared to fishfed the control diet. Replacement with feed pea at 15% and higher levels led to milkfish fed these dietsshowing a significantly lower growth response compared to fish fed the control without any feed pea.Nevertheless, it was observed that milkfish fed diets with up to 20% of total dietary protein substitution withfeed pea showed better growth rates and feed conversion ratios than the commercial feed control. Wholebody composition (crude protein, crude fat, crude fiber, nitrogen free extracts, and ash content) of milkfishfed the various test diets was not significantly different. Apparent digestibility coefficients of feed pea andexperimental diets in milkfish were also determined. Results indicate that feed pea is an acceptable proteinsource and can substantially replace up to 20% of the total dietary protein in milkfish diets.Keywords:Aquaculture feeds; alternative protein source; feed pea (Pisum sativum);milkfish (Chanos chanos Forsskal)IntroductionThe current trend in milkfish culture is toward increased intensification of culture systems whereby provisionof feeds becomes necessary and success therefore depends significantly on the availability of wellbalanced,nutritionally complete and cost-effective feeds. For many years, SEAFDEC AQD has doneconsiderable research on the nutrient requirement of milkfish, assessment of nutritive value of availableingredients, and development of simple and appropriate feeding technology. These are all importantfactors toward the development of cost-effective feeds and feeding strategy. SEAFDEC AQD has alsoendeavored to develop cost-effective feeds for milkfish culture. There is a need, however, for these feedsto be continuously refined, improved, and tested for technical and economic feasibility.In recent years, the rising cost, uncertain availability, and fluctuating quality of fish meal has led to thesearch for alternative protein sources for fish feed to sustain fish production. The use of plant proteinsources to completely or partially replace fish meal in fish diets has been studied for many years by severalworkers for different fish species (De la Higuera et al., 1988; Moyano et al., 1992; Borlongan and Coloso,1994; Sanz et al., 1994; Gomes et al., 1995; Kaushik et al., 1995; Hardy, 1996; Robaina et al., 1997;Watanabe et al., 1997; Carter and Hauler, 2000; Kissil et al., 2000 and Farhangi and Carter, 2001).Feed pea (Pisum sativum), an abundant agricultural product, is a potential feed ingredient. It is a highenergy, medium protein ingredient with average protein content (%CP) of 22 to 24% and digestible energy(DE) of 3420 kcal/kg. Lysine is particularly high at 1.6%. The efficiency of feed pea as a feed ingredienthas been evaluated for cattle, swine, and poultry. However, there is a paucity of information on the use ofthis ingredient as dietary protein source for aquaculture species.The aim of the current study was to assess the potential of feed pea (Pisum sativum) as an ingredient inpractical feeds for milkfish (Chanos chanos Forsskal). This was compared with solvent-extracted soybeanmeal because soybean meal is the most often used plant alternative to fish meal in milkfish feeds. The firstphase of the experiment was conducted to establish the nutritive value of feed pea through measurement offish growth, feed utilization and carcass composition. The second phase assessed the apparentdigestibility coefficients for dry matter and dietary protein.Materials and MethodsExperiment 1: Growth performance and feed utilizationExperimental DietsThe test diets were formulated to be isonitrogenous and isocaloric. The control diets were the existingSEAFDEC milkfish formulation that had been previously tested in intensive ponds as having an FCR of 1.5(D-1), and a leading commercial milkfish feed (D-8). The composition and proximate analyses of the20

experimental diets are presented in Table 1. All diets were formulated to contain 30% protein, 10% lipidand energy of about 394kcal/100g diet. Analyses however, showed that experimental diets contained 33%protein and 10% lipid, while the commercial milkfish feed control contained 37% protein and 9% lipid. Thebasal formulation contained fish meal, defatted soybean meal, meat and bone meal, and copra meal asdietary protein sources. The levels of feed pea incorporation tested were 0, 5, 10, 15, 20, 25, and 30% ofthe total crude protein. Dietary essential amino acid profiles (Table 2) were calculated based on analysesof ingredients and published data (NRC, 1977) and compared to that of requirements for milkfish juvenile(Borlongan and Coloso, 1993) which was used as the reference. Analyzed values of the essential aminoacids are likewise presented. No supplemental amino acids were added to the diets. Cod liver andsoybean oils served as lipid sources. Vitamin and mineral premixes were kept constant in all diets.Commercial feed peas of US origin were sourced from a local agricultural distributor. The whole dry peawas oven-dried at 60 0 C for 4 hours and finely ground to homogenized flour. No specialized ingredientprocessing method was used. Diets were prepared by first mixing all dry ingredients in the Hobart mixer.Oils were then blended with the dry ingredient mixture. An equal portion of bread flour was gelatinized bycooking in 600 ml water and added to the mixture. The semi-moist mixture was then passed through theHobart food grinder to form 2 mm diameter pellets. The pellets were dried in an air convection oven at40 0 C. The dry pellets were then ground, sieved to uniform sizes, and stored at 4 0 C until used for feeding.Experimental fish and feeding managementThe experiment was conducted at the Feed Development Section of the Aquaculture Department of theSoutheast Asian Fisheries Development Center (SEAFDEC/AQD), Tigbauan, Iloilo, Philippines. Hatcherybredmilkfish juveniles were acclimated for two weeks under laboratory conditions and to a dry diet (controldiet) prior to the experiment. The feeding trials were conducted in an indoor flow-through system. Sixty-litercapacity, oval fiberglass tanks containing 50L seawater with aeration were used. Milkfish juveniles (meanwt. = 0.42 g) were randomly distributed at a stocking rate of 10 fish per tank and in four replicate tanks pertreatment. Fish in each treatment were then fed three times daily at 0830, 1130, and 1400H at a feedingrate of 10% body weight per day for 12 weeks. Feeding ration was adjusted at every 3 weeks samplinginterval. Water quality parameters (temperature, salinity, DO, pH, ammonia, nitrite) were monitoredfollowing the methods of Strickland and Parsons (1972) to ensure water quality remained well within limitsrecommended for milkfish culture (Bagarinao, 1991).Statistical AnalysesSurvival, % weight gain, specific growth rate (SGR), food conversion ratio (FCR), and protein efficiencyratio (PER) were calculated and subjected to statistical analyses. Data were subjected to analysis ofvariance (ANOVA) and Duncan’s Multiple Range test (DMRT). Differences between means wereconsidered significant at P

1% chromic oxide to replace an equivalent amount from the filler (Celufil). The fish were acclimated withthe control diet/reference diet (without Cr 2 O 3 ) for 1 week prior to feeding them test diets containing 1%Cr 2 O 3 . Diets were fed to satiation twice daily (0900 and 1400 h). Fecal collection was started at day 5 afterthe fish were acclimated to the green diets, or when the fecal matter became greenish. The tanks and fecalcollection apparatus were cleaned twice daily, 2 hours after feeding in the morning and afternoon. Fecalcollection bottles were then attached, and feces collected. Feces were collected from the plastic bottles,rinsed 3 times with distilled water; freeze-dried and prepared (Eusebio, 1991) for dry matter and crudeprotein (AOAC, 1990) and Cr 2 O 3 (Carter et al., 1960) analyses. The test diets were likewise analyzed fordry matter, crude protein and Cr 2 O 3 .In vivo apparent protein digestibility (APD) and apparent dry matter digestibility (ADMD) of feed pea werecomputed using the formula of Spyridakis et al. (1989) and Cho et al. (1982). ADMD and APD offormulated diets were computed using the formula of Spyridakis et al. (1989). The data were analyzedusing ANOVA for completely randomized design (CRD). Treatment means were compared by Duncan'sMultiple Range Test (DMRT) using SAS computer software. Differences were considered significant atP

The results of the digestibility trial did not support the assumption of reduced protein availability from feedpea. In the present study, dietary crude protein digestibility values of the experimental diets were above80%. A comparison with the control diet supports the observation that pea protein is well digested bymilkfish. In fact, apparent protein digestibility value was superior with the partial inclusion (10% of dietaryprotein) of this plant material compared to the control without feed pea. Feed pea at this level may onlyshow marginal effects of anti-nutritional factors and limiting amino acids due to its relatively low content ofsuch compounds and its fairly good protein biological value for fish nutrition. Another consequence of usingfeed pea in place of soybean meal in the diet formulation for milkfish is the reduction of bread flour in dietscontaining high levels of feed pea. This is because of the higher carbohydrate content of feed peacompared to soybean meal. At this inclusion level, in practical conditions, feeding costs can be dramaticallydecreased.In general, feed pea meal proved to be an acceptable ingredient for consideration in practical diets forgrower stage production of milkfish. Future work showed be directed towards evaluating extruded peaproducts where pre-gelatinization of the starch would be possible, or testing various pea proteinconcentrates which have been processed to remove unwanted components such as fiber, tannin, etc.These products contain much higher protein levels that could be used to substitute increasing amounts offish meal in fish feed formulations. Further studies are needed to find out the maximum inclusion level offeed pea using other ingredient processing methods (e. g. dehulling, autoclaving, pressure cooking, proteinconcentrating, etc) and different types of pellet making (e. g. extrusion); in consideration of the effect ofthese processing methods on efficient utilization of essential nutrients by the fish.Previous experiments using cold-pressed pellets for Atlantic salmon suggested that 40% fish meal proteinreplacement with pea or lupin protein concentrate was feasible (Carter, 1998). Carter and Hauler (2000)confirmed that at least 33% fish meal protein replacement with pea protein concentrate was possible. Amajor problem in the use of plant meals is their relatively low protein content. In the study of Carter andHauler (2000), the protein concentrates used were produced by air separation and resulted in lupin and peaconcentrates with 46% and 49% crude protein, respectively. This represented an increase of between 44and 133% over the protein in the raw lupin and peas, respectively. Protein at this level is comparable tosoybean meal, but still far lower than that in fish meal. Gouveia et al., (1993) compared the efficacy of threelegume seed meals, e. g., lupin seed meal (Lupinus albus), faba beans (V. faba) and pea seed meal (P.sativum) in a 3-month feeding trial with juvenile rainbow trout. They reported that trout performed well ondiets that had up to 20% of the protein content being supplied by each of these ingredients. In fact, growthand feed utilization was superior with the partial inclusion of these plant materials. The inclusion of a coextrudedplant protein made from rapeseed and field pea had no effect at up to 15% replacement of theprotein. However, at 45% inclusion, growth performance of rainbow trout was significantly lower than thecontrol diet (Gomes et al., 1993). The low digestible energy from both raw and autoclaved field peas waspredicted to limit their use in rainbow trout feeds (Pfeffer et al., 1995; Gouveia and Davies, 1998). Energydigestibility of legumes is considerably lower than for protein due to their high carbohydrate content. Theuse of extrusion is important in increasing the nutrient availability of plant meals, especially in relation toincreasing the amount of digestible energy available through greater gelatinization of starch (Pongmaneeratand Watanabe, 1993).Since the objective of this study was to evaluate the direct nutritive value of feed peas, the experiment wascarried out in an in-door clear water flow-through system. However, experience shows that milkfish inponds grow better than milkfish given the same feed under controlled laboratory conditions (Sumagaysay etal., 1991; Sumagaysay and Borlongan, 1995). It is expected that higher growth rates and better FCR andPER may result if the milkfish are reared in ponds. It is recommended that feeding experiments using thebest diets be done under natural pond conditions and an economic assessment of the feed pea-basedfeeds be conducted.23

Table 1Composition (g/100g diet) and proximate analyses of the experimental dietsD-1 D-2 D-3 D-4 D-5 D-6 D-7 D-8Ingredients0% 5% 10% 15% 20% 25% 30% *CMFWhite fish meal 10.0 10.0 10.0 10.0 10.0 10.0 10.0Soybean meal 35.0 31.5 28.0 24.5 21.0 17.5 14.0Copra meal 13.0 13.0 13.0 13.0 13.0 13.0 13.0Meat and bone meal 11.0 11.0 11.0 11.0 11.0 11.0 11.0Vitamin mix 3.0 3.0 3.0 3.0 3.0 3.0 3.0Mineral mix 2.0 2.0 2.0 2.0 2.0 2.0 2.0Cod liver oil 2.0 2.0 2.0 2.0 2.0 2.0 2.0Soybean oil 2.0 2.0 2.0 2.0 2.0 2.0 2.0Bread flour 21.2 17.3 13.9 10.6 7.2 3.8 0.4Filler (Celufil) 0.8 1.7 2.0 2.3 2.6 3.0 3.3Green peas - 6.5 13.1 19.6 26.2 32.7 39.3Total 100 100 100 100 100 100 100Proximate compostion (%dry basis) of the test dietsCrude protein 33.52 33.44 33.20 33.42 33.29 33.22 33.35 37.38Crude fat 10.24 10.22 10.10 10.08 10.02 10.08 10.07 9.14Crude fiber 4.66 4.96 4.42 4.39 4.94 4.51 4.63 5.20Nitrogen-free extracts 43.54 43.34 43.96 43.37 43.33 43.79 43.70 39.18Ash 8.04 8.04 8.32 8.74 8.42 8.40 8.25 9.10*CMF = Commercial Milkfish FeedTable 2Amino acid composition of various test diets and a comparison of the amino acid requirements ofmilkfishEssential amino acid Diet 1 Diet 3 Diet 5 Diet 7 Milkfish Req’t*EAA content: Calculated (g/100g diet)Arginine 1.57 1.43 1.29 1.43 1.58Histidine 0.68 0.60 0.54 0.46 0.60Isoleucine 1.05 0.95 0.85 0.75 1.20Leucine 1.79 1.64 1.48 0.77 1.53Lysine 1.56 1.45 1.32 1.21 1.20Methionine + Cystine 0.70 0.64 0.57 0.51 0.98Phenylalanine + Tyrosine 1.61 1.46 1.27 1.08 1.57Threonine 0.96 0.88 0.79 0.71 1.35Tryptophan 0.15 0.15 0.15 0.13 0.18Valine 1.17 1.08 1.00 0.90 1.06EAA content: Analyzed Value (g/100g diet)Arginine 1.21 1.61 0.94 1.30Histidine 0.63 0.54 0.33 0.32Isoleucine 0.96 0.98 0.64 0.74Leucine 1.43 1.70 1.26 1.27Lysine 1.31 1.37 1.23 0.96Methionine** 0.27 0.31 0.2 0.22Phenylalanine + Tyrosine 1.58 1.88 1.31 1.36Threonine 0.92 0.82 0.72 0.64Tryptophan nd nd nd ndValine 1.04 1.09 0.91 0.85* Based on Borlongan and Coloso, 1995** Cystine not determined24

Table 3Composition (g/100g diet) of the reference and test diets for the digestibility determinationIngredientsReferenceDietTest Diet70:30:00White fish meal 10.0 7.0Soybean meal 35.0 24.5Copra meal 13.0 9.1Meat and bone meal 11.0 7.7Vitamin mix 3.0 2.1Mineral mix 2.0 1.4Cod liver oil 2.0 1.4Soybean oil 2.0 1.4Bread flour 20.2 13.6Filler (Celufil) 0.8 0.8Cr 2O 3 1.0 1.0Green peas - 30.0Total 100 100Table 4Response of milkfish juveniles to the various test diets after 12 weeks of feedingDiet No.% % Weight SGRSurvival Gain (%/day)FCR PER1 (0%) 90 c 834.8 e 1.58 e 2.15 e 1.44 d2 (5%) 90 c 833.1 e 1.51 e 2.10 e 1.46 d3 (10%) 90 c 835.3 e 1.50 e 2.10 e 1.44 d4 (15%) 80 b 691.0 d 1.32 d 2.25 d 1.37 c5 (20%) 80 b 680.0 d 1.29 d 2.39 c 1.35 c6 (25%) 70 a 512.6 b 0.89 b 2.50 b 1.21 b7 (30%) 70 a 464.0 a 0.80 a 2.60 a 1.15 a8(CMF) 80 b 581.4 c 1.00 c 2.46 b 1.23 bSGR (specific growth rate) = ln (final wt.- initial wt)/84 days x 100FCR (feed conversion ratio) = total dry feed given (g) / total wet wt. gain (g)PER (protein efficiency ratio) = wet wt.gain (g) / amount of protein fed (g)CMF = Commercial Milkfish FeedTable 5Carcass proximate composition (%) of milkfish juveniles fed the various test dietsDiet No.MoistureCrudeProteinCrudeFatCrudeFiberNFE*Ash1 (0%) 76.96 59.06 23.92 0.86 5.92 10.242 (5%) 75.02 60.02 23.69 0.44 4.41 10.443 (10%) 75.16 60.64 23.18 0.79 4.78 10.614 (15%) 76.24 60.46 23.16 0.58 5.56 10.245 (20%) 77.31 60.08 23.87 0.48 4.21 11.366 (25%) 76.92 59.66 22.12 0.62 6.39 11.217 (30%) 76.63 59.96 23.74 0.74 4.05 11.518(CMF) 75.00 60.56 23.71 0.44 4.25 11.04Table 6Apparent digestibility coefficients (%) for dry matter (ADMD) and crude protein (APD) ofexperimental diets in milkfish (Chanos chanos Forsskal)Diet No. ADMD APD1 (0%) 61.1 a 86.4 b3 (10%) 68.1 a 89.2 a5 (20%) 65.4 a 86.3 b7 (30%) 60.1 a 84.8 b25

AcknowledgementThe authors gratefully acknowledge the United States Department of Agriculture (USDA) through the USADry Peas and Lentil Council (USADPLC), Moscow, Idaho, USA for funding support. The excellent technicalassistance of Lucia Jimenez, Marthena Joyce Bernas and Niel Tibubos is also appreciated.ReferencesAssociation of Official Analytical Chemists (AOAC), 1990. 15 th ed. K. Helrich (ed) AOAC, Arlington, VA, USA.Bagarinao, T. U. 1991. Biology of milkfish, Chanos chanos Forsskal. Aquaculture Department, Southeast Asian FisheriesDevelopment Center, Tigbauan, Iloilo, Philippines, p.94.Borlongan, I. G., Coloso, R. M., 1993. Requirement of milkfish (Chanos chanos Forsskal) juveniles for essential amino acids. J. Nutr.123, 125-132.Borlongan, I. G., Coloso, R. M. 1994. Leafmeals as protein sources in the diets for milkfish (Chanos chanos Forsskal). In: de Silva, S.S. (ed). Fish Nutrition Research in Asia, Proceedings of the 5 th Asian Fish Nutrition Network Workshop, Asian Fish. 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Nutritional evaluation of various plant protein sources in diets for Asian sea bass Lates calcarifer.J. Appl. Ichthyol. 16, 546-560.Eusebio, P.S., 1991. Effect of dehulling on the nutritive value of some leguminous seeds as protein for tiger prawn, Penaeus monodonjuveniles. Aquaculture 99, 297-308.Farhangi, M., Carter, G., 2001. Growth, physiological and immunological responses of rainbow trout (Oncorhynchus mykiss) todifferent dietary inclusion levels of dehulled lupin (Lupinus angustifolius). Aquacult. Res. 32(Suppl.1), 329-340.Gomes, E. F., Corraze, G., Kaushik, S., 1993. Effects of dietary incorporation of a co-extruded plant protein (rapeseed and peas) ongrowth, nutrient utilization and muscle fatty acid composition of rainbow trout (Oncorhynchus mykiss). Aquaculture 113, 339-353.Gomes, E. F., Rema, P., Kaushik, S. J., 1995. Replacement of fish meal by plant proteins in the diet of rainbow trout (Oncorhynchusmykiss): digestibility and growth performance. Aquaculture 130, 177-186.Gouveia, A., Davies, S. J., 1998. Preliminary nutritional evaluation of pea seed meal (Pisum sativum) for juvenile European sea bass(Dicentrarchus labrax). Aquaculture 166, 311-320.Gouveia, A., Oliva Teles, A., Gomes, E., Rema, P., 1993. Effect of cooking/expansion of three legume seeds on growth and foodutilization by rainbow trout. In: Kaushik, S. J., Luquet, P. (Eds.), Fish Nutrition in Practice, INRA, Paris, pp.933-938.Hardy, R. W., 1996. Alternative protein sources for salmon and trout diets. Anim. Feed Sci. Technol. 59, 71-80.Kaushik, S. J., Cravedi, J. P., Lalles, J. P., Sumpter, J., Fauconneau, B., Laroche, M., 1995. Partial or total replacement of fish mealby soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh qualitry inrainbow trout. Oncorhynchus mykiss. Aquaculture 133, 257-274.Kissil, G. Wm., Lupatsch, I., Higgs, D. A. and Hardy, R. W. 2000. 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Fishmeal replacers: Review of antinutrients within oilseeds and pulses- a limiting factor for the aquafeed greenrevolution? In: Tacon, A., Basurca, B. (Eds.), Feeding Tomorrow’s Fish. Cashiers Options Méditerranéenes, Vol. 22, pp.154-182.Watanabe, T., Verakunpiriya, V., Watanabe, K., Kiron, V., Satoh, S. 1997. Feeding of rainbow trout with non-fish meal diets.Fisheries Science 63(2), 258-266.26

USA Dry Pea& Lentil CouncilHead Office2780 W. Pullman Rd.Moscow, Idaho, 83843-8024 USATel: 208-882-3023 Fax: (208) 882-6406;E-mail: pulse@pea-lentil.comWeb: www.pulse.comSoutheast Asia RepresentativeAttention: Mr. Tim WelshAgriSource Co. Ltd.76/1 Soi Lang Suan, Ploenchit Rd.Bangkok 10330 ThailandTel: 66-2-251-8655 to 6 Fax: 66-2-251-0390Email: agsource@loxinfo.co.thSoutheast Asian Fisheries Development CenterAquaculture DepartmentTigbauan, Iloilo 5021 PhilippinesTel: (6333) 362965 Fax: (6333) 3351008E-mail: clmarte@aqd.seafdec.org.phWeb: www.seafdec.org.ph