Meeting the Challenge of no Fishmeal in Practical Diets for Litopenaeus vannamei: Case Studies from LABOMAR, Brazil

Alberto J.P. Nunes Associate Professor

X Simposio Internacional de Nutrición Acuícola Monterrey, Nuevo Leon, Mexico November 9, 2010 – Session 3 Aquaculture: largest consumer of fishmeal

ƒ In 2006, aquafeeds used 3.7 million MT of fishmeal, 68.2% of the estimated global production¶ MT x 1,000 60,014 Production of finfish and crustaceans* 45,557 Total fed production

23,851 (76%) 15,072

(63%)

2006 2020E ¶ Source: Tacon and Metian, 2008 In 10 years, fed-raised finfish and crustaceans will account for ¾ of world production

*MT x 1,000. Excludes filter-feeding fish Fishmeal use is reducing in shrimp feeds ƒ Shrimp are the largest consumer of fishmeal within the aquaculture industry, ahead of marine fish and salmon

10,000 MT x 1,000 FIFO 2.5 9,000 Farm-raised marine shrimp production 8,000 1.9 2.0

7,000

6,000 1.5 Fish IN : Fish OUT Ratio 5,000

4,000 1.0

3,000

Source: Tacon Source: and Metian, 2008 Pelagic forage fish equivalent ¶ 2,000 0.5 0.3 1,000 Projections 0 0.0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2010 2015 2020

Over the past 15 years, fishmeal inclusion in shrimp feeds reduced from 28% (1995) to 12% (2010). FIFO more efficient than salmon, trout, eel and marine fish¶ . Drivers for fishmeal reduction

2,000 (1) PRODUCTION

1,800 capture fisheries production remains stagnant compared to 1,600 an 8.8% annual growth rate in 1,400 Fishmeal aquaculture output 1,200 (2) PRICES CIF Price(USD/MT) 1,000 fishmeal prices have risen 800 significantly compared to other Soybean meal 600 agricultural commodity protein

400 ingredients

200 (3) SUSTAINABILITY as shrimp farming moves into 0 Jan-2005 Jan-2006 Jan-2007 Jan-2008 Jan-2009 Jan-2010 more intensive systems and Year production rises, there is a Five-year market price (2005-2010) for fishmeal and soybean meal. growing demand for formulated Source: Oil World. diets dependent on static supplies of fish meal Fishmeal (64/65% CP, CIF Hamburg). Soybean meal (pellets 44/45% CP Argentina, CIF Rotterdam). Farmers are raising a less nutrient- dependent shrimp species

3,399 MT 3,500 Grand Total Harvest (MT x 1,000) 3,000

2,500 2,259 MT 1,135 MT 66% 2,000 631 MT Litopenaeus vannamei 56% 1,500 145 MT 13% Penaeus monodon 1,000 722 MT Source: FAO (2010) 21% 500 Other species 0 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08

Production of L. vannamei increased 16x in 8 years (2000 vs. 2008) compared to 14% for the tiger shrimp About aquaculture at LABOMAR, Brazil

ƒ 50-year old marine Lane snapper, Lutjanus synagris sciences institution located in NE Brazil ƒ Part of the Federal University of the

State of Ceará Mutton snapper, Lutjanus analis ƒ Owns 5-ha facility where applied OUTDOOR SYSTEM research on (Marine Finfish) reproduction, nutrition, disease and genetics of marine fish and crustaceans

is carried out Fat and common snook, Centropomus Cobia, Rachycentron canadum parallelus and C. undecimalis Rearing system: shrimp

GREEN WATER

CLEAR WATER

Clear water Green water Round tanks of 500-L volume Round tanks of 1.000-L volume 0.57 m2 bottom area 1.02 m2 bottom area 12-h sand filtering 25% weekly water exchange Shrimp rearing: standard protocol

1 2 3

4 5 6 1. PL10 rearing: 2 PLs/L – 30 -40 days 4. Fed twice a day on a consumption basis 2. Juvenile stocking (2-4 g shrimp) 5. Meals calculated individually ƒ Green water: 40 – 70 shrimp/m2 6. Shrimp samples every 3.5 weeks ƒ Clear water: 70 - 100 shrimp/m2 7. Harvest after 10 weeks (10 – 20 g shrimp) Sources of Rendered Animal Protein Have Low Stimulatory Power for L. vannamei Source: Source: Nunes al et Attractant* +choices (%)* % rejection CON 2.9g 100.0 MBM 54.5af 8.3 SM 59.1ad 0.0 2006 . Aquaculture, Aquaculture, . FMPO 75.6a 0.0 FMBO 65.9ac 0.0 BM 45.7abcd 25.0 260 : 244 b -

FO 25.7 44.4 254 FS 58.5ae 8.3 . Chi-square P <0.001 ---

*Values in the column which do not share a same superscript are statistically different between them by the z-test (P<0.05); Y-maze system to evaluate feeding effectors in shrimp *control (CON) without SEM; meat and bone meal (MBM); squid meal (SM); fishmeal–Peruvian origin (FMPO); fishmeal–Brazilian origin (FMBO); blood meal (BM); fish oil (FO); fish solubles (FS) Replacing Fishmeal by Ingredients with Low Feeding Stimulation

Fish meal Soybean meal Meat meal

~ +

+

Natural and synthetic feeding effectors Feeding Effectors not a Feed Perfume Evidence that AA pools are better attractants % +choices than isolated ones % rejection 80,0 High content of water-soluble AA. Rich in water soluble 70,0 substances with boosted feeding stimuli

60,0 Whole squid is an effective attractant. 50,0 Protein hydrolysis process can promote 40,0 even better responses

30,0

20,0

10,0

0,0 CON VDB80 VDB68 CAA CFSP SLM Bet DFSLH DFSHH WSPH

Supplementation of vegetable sources with certain amino acids (glutamate and betaine) may prove useless to stimulate feeding responses

(1) 80%-crude protein (CP) vegetable dried biomass (VDB80); (2) 68%-CP vegetable dried biomass + glutamate + betaine (VDB68); (3) complex of amino acids (alanine, valine, glycine, proline, serine, histidine, glutamic acid, tyrosine and betaine) with enzymatically digested bivalve mollusk (CAA); (4) condensed fish soluble protein (CFSP); (5) squid liver meal (SLM); (6) betaine (Bet); (7) dried fish solubles - low biogenic amines (DFSLB); (8) dried fish solubles - high biogenic amines (DFSHB); (9) whole squid proteinPhoto hydrolysate credit: (WSPHAlberto); Nunes**Soybean meal = experimental control Aminoacid Profile of Commercial Feeds

Mean How important +18% +37% 0% Minimum is MET to shrimp 9.00 Maximum 8.00 Required* biological

7.00 +9% performance? +16% 6.00 +12% +6% 5.00 +11% -26%

4.00 +1% -33% 3.00 +17% 2.00

1.00

0.00 ARG HIS ISO LEU LYS MET CYS M+C PHE TYR P+T THR TRY VAL

g of EAA/100 g of crude protein*

Analyzed feeds met marine shrimp EAA requirements, but METHIONINE was the most limiting EAA in all diets

*Source: Lemos and Nunes (2008). Aquaculture Nutrition 2008 14; 181–191 Methionine Crucial to Growth Performance Performance of L. vannamei in clear water after 56 days of rearing fed commercial diets. Temp. 29.5 qC; sal. 33.4 ‰; stocking density. 114 ind./m2; initial weight 3.28 (± 0.31). Source: Lemos and Nunes (2008). Aquaculture Nutrition 2008 14; 181–191.

Parameters T3 T4 T5 T6 T7 T8 Survival (%) 92.7a (1.94) 91.5a (5.10) 81.9b (9.26) 93.8a (2.18) 91.2a (2.31) 90.8a (3.32) Yield (Kg/m2) 0.50a (0.12) 0.44a (0.09) 0.61ab (0.10) 0.60ab (0.13) 0.77b (0.11) 0.78b (0.14) Growth (g/week) 0.63a (0.13) 0.56a (0.10) 0.91b (0.04) 0.73a (0.14) 0.97b (0.13) 0.98b (0.14) Feed cons. (g) 755.9a (23.6) 691.9b (55.9) 879.7c (62.0) 915.4c (32.7) 887.9c (23.7) 977.9d (31.6) Biomass gain (g) 286.2a (68.0) 252.2a (50.0) 349.1ab (58.7) 342.9ab (71.5) 439.2b (64.8) 444.1b (81.3) FCR 2.75 (0.63) 2.80 (0.41) 2.56 (0.37) 2.75 (0.49) 2.05 (0.27) 2.26 (0.44) Crude Protein 371 (1.2) 348 (0.9) 361 (0.4) 350 (1.2) 356 (0.1) 359 (1.3) Met. (g/100 CP) 1.38 1.47 1.91 1.46 1.75 1.73 Met (%, dw) 0.51% 0.51% 0.69% 0.51% 0.62% 062% ͻ High correlation between shrimp growth rate and methionine levels (R2 = 0.73) ͻ Higher growth achieved when feed showed: 1. Lower number of EAA below recommended levels 2. Methionine: 1.70 -1.75 g/100 g of crude protein 3. Lysine: > 6.0 g/100 g of crude protein 4. Methionine+cystine: > 2.68 g/100 g of crude protein Intact methionine affects performance

Ingredient (%) 80 A 70 A 60 A Soybean meal, 46% 32.0 33.3 30.3 Wheat flour 25.0 25.0 25.0 Fishmeal, Anchovy 13.0 7.3 0.0 Fishmeal, by-catch 10.0 10.0 5.1 Corn gluten meal 5.0 5.0 10.4 Lower aminoacid levels Rice, Broken 3.7 1.8 1.8 Dicalcium Phosphate 3.6 3.2 2.3 Ingredient (%) 80 A 70 A 60 A Fish oil 2.8 2.3 0.4 Crude Protein 35.50 35.50 35.50 Crude Fat 8.00 8.50 8.50 Lecithin, Fluid 1.7 1.9 2.2 Crude Fiber 1.86 1.96 1.84 Salt 1.0 1.0 1.0 Ash 11.94 12.57 13.27 Vitamin-Mineral Pmx 1.0 1.0 1.0 Pegabind (Pellet Binder) 0.5 0.5 0.5 Lysine 1.85 1.72 1.41 Magnesium Sulfate 0.16 0.00 0.00 Met+Cys 1.09 1.01 0.93 Potassium Chloride 0.14 0.00 0.00 Methionine 0.67 0.59 0.50 Cholesterol 0.12 0.11 0.11 Stay C 0.03 0.03 0.03 Commercial attractant 0.2 0.3 0.4 Meat and bone meal 0.0 7.2 19.6 Formula cost (US$/MT) 658 593 505

Cost savings in formulation -11.0% -33.3% AA Profile Significantly Impacts Growth and FCR

72-day rearing trial with L. vannamei in indoor tanks (clear water) at LABOMAR, Brazil.

Feeds Survival % Yield (g/m2) Growth (g/wk) 60A 91.2 ± 4.8 884 ± 74.9 0.98 ± 0.06 a Initial Stocking Density: 70A 93.0 ± 3.8 1,094 ± 192.0 1.17 ± 0.13 a 57 shrimp/tank or 2 80A 91.6 ± 1.5 1,085 ± 78.0 1.19 ± 0.10 b 100 shrimp/m ANOVA P NS NS < 0.05

Feeds Weight In. (g) Weight Fn. (g) FCR 60A 4.14 ± 0.31 14.3 ± 0.64 a 2.75 ± 0.17 b 70A 3.93 ± 0.16 16.0 ± 1.39 ab 2.30 ± 0.24 a 80A 4.09 ± 0.46 16.3 ± 1.12 b 2.47 ± 0.07 a ANOVA P NS < 0.05 < 0.05 Experimental Design ƒ Fifty 500 L clear water tanks: 10 replicate tanks per diet NV100_C+ ƒ 40 shrimp/tank: 70 NV50_C- animals/m2 NV50_C+ ƒ Started with juveniles of 2.22 ± 0.19 g (n = 50) in NV100_C- wet body weight NV_B ƒ Two harvests: 72 days and 96 days (stress MERA™ Met Ca* event) NV_B: basal diet with 150 g/kg of Anchovy fishmeal (FML) ƒ Five diets prepared with NV50_C+: positive control diet with 50 g/kg of FML + 1g/kg MERA™ Met Ca NV50_C-: negative control diet with 50 g/kg of FML and no MERA™ Met Ca laboratory equipment NV100_C+: positive control diet without FML + 2 g/kg MERA™ Met Ca NV100_C-: negative control diet without FML and no MERA™ Met Ca

*84% 2-hydroxy-4-(methylthio)butanoic acid (HMTBa) Ingredient (g/kg, as is) NV_B NV50_C+ NV50_C- NV100_C+ NV100_C- Soybean meal 350.0 457.6 450.0 487.0 485.2 2 1 % reduction in formula cost % to formula cost in reduction comparison the in control diet 84

Wheat flour 235.6 217.0 221.7 210.0 210.0 % 2

Fish meal, Anchovy 150.0 75.0 75.0 0.0 0.0 - hydroxy Poultry by-product meal 60.0 60.0 65.7 60.0 60.0 Rice, broken 50.0 21.9 21.8 0.0 0.0 - 4 -

Soy protein concentrate 43.1 30.0 30.0 93.3 96.4 ( methylthio Squid meal, whole 0.0 20.0 20.0 20.0 20.0 Fish oil 15.0 30.0 30.0 44.0 44.0 ) Soybean oil 19.4 8.5 7.9 0.0 0.0 butanoic MERA™ Met Ca1 0.0 1.0 0.0 2.0 0.0 L-lysine 0.0 0.0 0.0 0.4 0.3 acid Other micro ingredients 76.8 79.8 77.8 83.3 84.2 ( Proximate Composition (g/kg, dry matter basis) HMTBa Moisture 92.6 95.7 91.0 88.1 92.9 Crude protein 392.2 383.5 391.8 393.2 406.6 ); Lipids 70.5 75.9 70.7 78.0 60.7 Total fiber 22.7 26.6 28.3 27.1 31.7 NV_B Ash 98.7 97.6 97.9 95.5 88.4 Amino Acids (g/kg, dry matter basis) HMTBa 0.0 0.65 0.0 1.14 0.0 Methionine 6.0 5.4 5.2 4.5 4.8 Cystine 5.4 5.3 5.4 5.6 5.7 Methionine + cystine 11.4 10.7 10.6 10.1 10.5 Lysine 19.7 20.4 18.8 19.4 22.4 Formulation Cost2 -- 11.7% 12.5% 22.2% 23.2% Overall Performance

Diet % Survival Yield (g/m2) FCR NV_B 93.8 ± 6.7 849 ± 72 2.13 ± 0.14 NV50_C+ 91.5 ± 5.3 841 ± 119 2.27 ± 0.23 NV50_C- 92.3 ± 4.9 768 ± 81 2.13 ± 0.20 days NV100_C+ 91.5 ± 3.6 776 ± 65 2.21 ± 0.17 NV100_C- 92.5 ± 5.3 806 ± 64 2.11 ± 0.17 72 Mean 92.3 ± 5.1 808 ± 86 2.17 ± 0.19 NV_B 78.3 ± 12.1 526 ± 72 3.26 ± 0.52 NV50_C+ 82.5 ± 7.1 563 ± 88 3.23 ± 0.33 NV50_C- 82.0 ± 9.6 519 ± 61 3.11 ± 0.45 NV100_C+ 82.3 ± 5.3 534 ± 31 3.09 ± 0.22 NV100_C- 82.0 ± 4.0 552 ± 51 2.94 ± 0.20 96 days Mean 81.4 ± 8.0 539 ± 70 3.12 ± 0.37

No statistical differences in shrimp final survival, yield and FCR among harvested shrimp fed the different experimental diets Final shrimp body weight

A higher body weight was observed when shrimp were fed the basal diet with 150 g/kg of fish meal (NV_B) or when diets were supplemented with HMTBa Weekly shrimp growth

On day 72, a higher weekly growth rate was found for shrimp fed either the basal diet, NV_B or diets NV50_C+ and NOV100_C+. Feed intake

The reduction of fishmeal in experimental diets had no detrimental effect over shrimp feed intake. Indication of a higher feed intake in diets with

MERA™ MetCa as seen for NV50_C+. Feed attractability

The addition of poultry by-product meal, squid meal and HMTBa may have helped prevent reductions in palatability typically associated with reductions in fish meal content. Fishmeal forecast study

ƒ Fifty 500 L clear water tanks: 6-7 replicate tanks per diet Soy protein Anchovy fishmeal ƒ 40 shrimp/tank: 70 concentrate animals/m2 ƒ Started with juveniles of 2.02 ± 0.51 g (n = 500) in wet body weight ƒ Shrimp reared for 72 days ƒ Two sets of diets: (1) 2% fish oil (2) 1% FO ƒ Each set varied Anchovy fishmeal, SPC and ƒ Eight diets prepared with SBO inclusion laboratory equipment Year 2010: 12% fishmeal (0% replacement) Year 2015: 8.5% fishmeal (30% replacement) Year 2020: 5.0% fishmeal (60% replacement) Year 2025: NO fishmeal (100% replacement) Experimental Diets

Experimental Diets/Composition (g/kg, as is) Ingredient 2010 2015 2020 2025 2010 2015 2020 2025 2.0% Fish OIL 1.0% Fish OIL Fish meal, Anchovy 120.0 85.0 50.0 0.0 120.0 85.0 50.0 0.0 Soy protein concentrate 0.0 38.5 77.5 133.4 0.0 38.4 77.5 133.2 Variable Broken rice 41.5 35.1 25.8 11.9 41.5 35.4 25.9 12.7 Soybean oil 10.5 13.3 18.0 25.1 20.4 23.0 27.9 34.5 Fish oil 20.0 20.0 20.0 20.0 10.0 10.0 10.0 10.0 L-lysine 1.2 1.3 1.5 1.7 1.2 1.3 1.5 1.7 DL-methionine 0.0 0.4 0.8 1.4 0.0 0.4 0.8 1.4 Soybean meal 330.0 330.0 330.0 330.0 330.0 330.0 330.0 330.0 Wheat flour 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 Poultry by-product meal 150.0 150.0 150.0 150.0 150.0 150.0 150.0 150.0 Vitamin-mineral premix 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 Soybean lecithin 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 Fixed Bicalcium phosphate 13.0 13.0 13.0 13.0 13.0 13.0 13.0 13.0 Common salt 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Potassium chloride 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Synthetic binder 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 Magnesium sulfate 1.1 0.7 0.7 0.8 1.2 0.7 0.7 0.8 Vitamin C 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 2010 2015 2020 2025 2010 2015 2020 2025 Nutritional Composition 2.0% Fish OIL 1.0% Fish OIL Proximate Compostion (g/kg, dry matter basis) Crude protein 388.1 384.1 393.9 390.8 393.5 384.9 385.9 388.4 Crude fat 99.8 89.5 94.8 97.0 93.0 89.3 93.7 97.8 Crude fiber 14.7 17.3 17.0 19.2 17.9 15.5 13.4 17.4 Ash 104.7 97.6 96.1 88.9 105.6 97.1 94.6 91.3 Calcium 15.9 15.1 14.2 13.0 15.9 15.1 14.2 13.0 Gross energy (MJ/kg) 19.7 19.6 19.9 20.1 19.6 19.8 19.9 20.1 Essential amino acids (g/kg, dry matter basis) Lysine 23.6 24.3 24.3 25.0 23.6 24.1 24.9 25.2 Methionine 7.6 7.5 7.7 7.7 7.2 7.4 7.8 7.9 Cystine 4.1 4.9 4.3 5.1 5.0 5.2 4.3 4.3 Methionine + cystine 11.7 12.0 12.0 12.8 12.2 12.6 101.5 105.7 Essential Fatty Acid (g/kg, dried matter basis) Linoleic (18:2 n-6) 31.9 30.0 33.1 36.7 36.1 35.3 38.2 42.4 Arachidonic (20:4 n-6) 0.5 0.4 0.3 0.3 0.1 0.1 0.2 0.1 Total n-6 PUFA 32.4 30.4 33.4 37.0 36.2 35.4 38.4 42.5 Linolenic (18:3 n-3) 5.2 4.4 4.9 5.2 5.1 4.9 4.7 5.0 Eicosatrienoic (20:3 n-3) 0.5 0.3 0.3 0.3 0.1 0.2 0.2 0.2 Eicosapentaenoic (20:5 n-3) 7.7 5.3 4.2 3.1 1.9 1.8 2.6 1.6 Docosahexaenoic (22:6 n-3) 5.8 4.3 3.5 2.9 1.7 1.6 2.2 2.1 Total n-3 PUFA 19.2 14.3 12.9 11.5 8.8 8.5 9.7 8.9 Total PUFA 2 51.6 44.7 46.3 48.5 45.0 43.9 48.1 51.4 n-3/n-6 0.59 0.47 0.39 0.31 0.24 0.24 0.25 0.21 Overall performance

Fish Oil Level Variable FM (%) Year 2.0% 1.0% 12.0 2010 91.3 ± 2.2 94.0 ± 2.2 8.5 2015 90.0 ± 5.0 94.2 ± 3.0 Survival (%) 5.0 2020 93.3 ± 4.1 89.6 ± 5.1 0.0 2025 94.6 ± 4.3 91.3 ± 5.4

12.0 2010 0.69 ± 0.06 A 0.74 ± 0.07 A Growth Rate 8.5 2015 0.65 ± 0.04 AB 0.70 ± 0.09 A (g/week) 5.0 2020 0.62 ± 0.09 AB 0.60 ± 0.05 B 0.0 2025 0.58 ± 0.07 B 0.53 ± 0.06 B

12.0 2010 547.5 ± 29.2 Aa 592.7 ± 47.1 Aa 8.5 2015 540.0 ± 13.0 Aa 597.4 ± 46.2 Ab Yield (g/m²) 5.0 2020 538.5 ± 47.5 Aa 505.5 ± 52.5 Ba 0.0 2025 524.5 ± 63.0 Aa 477.7 ± 59.1 Ba

At 2% fish oil, shrimp growth rates were reduced only when diets had not fishmeal. At 1% fish oil, growth rates dropped at 5% fishmeal inclusion. Final shrimp body weight

The lowest possible combinations of dietary inclusion levels of fish meal (FM) and fish oil (FO) level were 5%FM-2%FO (diet 2020) and 8.5%FM-1% FO (diet 2015). Feed intake

Feed intake was consistent among experimental diets, except when fishmeal was removed in the 1% fish oil diet FCR

At 1% fish oil, FCR significantly increased, starting at 5% fishmeal and below Fishmeal, Anchovy

EAA (as is) ARG 4.07% HIS 1.57% 24.74% ISO 3.04% Other versus 67.65% LEU 5.07% 7.61% Protein LYS 5.01% Lipid MET 1.92% PHE 2.76% Krill meal THR 2.55% EAA (as is) VAL 3.51% 14.90% ARG 6.11% HIS 2.61% EFA (% of total lipid) Other ISO 3.85% HUFA n-3 4.32% TL Ɨ LEU 6.61% ƗHUFA n-6 1.80% TL 60.10% 25.00% Protein LYS 7.22% Lipid MET 2.66% EFA (% of total lipid) PHE 3.81% 22.11% TL ƗHUFA n-3 THR 3.19% 4.99% TL ƗHUFA n-6 VAL 3.99% > 40% phospholipid Objectives

1. To evaluate the growth performance of juveniles of L. vannamei when fed diets containing Krill meal and Krill oil under partial or full replacement of fishmeal, fish oil, soy lecithin and cholesterol

2. To determine optimum Major protein and lipid ingredients used in inclusion levels of Krill meal experimental diets for growth trials with L. in diets for the white vannamei shrimp in regards its growth and economical performance Diet composition (g/kg as is) Ingredients CLT KM10 KM50 KM110 Fish meal, Anchovy 150.0 100.0 50.0 0.0 Fish meal, by-catch 37.5 25.0 12.5 0.0 Broken rice 130.4 115.6 108.0 113.5 Soybean meal 300.0 340.2 338.6 349.4 Meat and bone meal 23.1 55.0 100.0 99.5 Wheat flour 250.0 250.0 250.0 250.0 Fish oil 20.0 15.2 17.7 0.1 Soybean lecithin 15.0 15.0 0.0 0.0 Krill meal 0.0 10.0 50.0 115.0 Cholesterol 1.5 1.5 0.8 0.0 Others* 322.5 322.5 322.5 322.5 USD/MT** / % Savings 662.8 8.0% 12.8% 8.7% Proximate composition (g/kg dried matter basis) Crude protein 325.5 336.0 327.3 338.6 Total lipids 103.8 103.5 107.5 107.5 Ash 99.5 98.3 99.5 96.7 Crude fiber 1.65 1.49 1.64 2.22 Gross energy (kJ/g) 17.5 17.5 16.9 16.8 Moisture (% as fed) 11.1 10.7 10.2 8.8 *Others included: 1.5 g/kg magnesium sulfate, 4.0 g/kg potassium chloride, 5.0 g/kg synthetic binder, 10.0 g/kg common salt, 10.0 g/kg vitamin-mineral premix, 12.0 g/kg bicalcium phosphate, 30.0 g/kg of corn gluten meal and 250.0 g/kg wheat flour. **FOB prices (Fortaleza, Brazil, Dec. 2007). Formula savings (USD/MT) compared to the CLT (basal) diet. Growth response (mean ± standard deviation) of L. vannamei juveniles fed diets containing Krill meal for 72 days in 500-L indoor (clear water) and 1,000-L outdoor (green water) tanks. Source: Nunes et al. (2010). Aquaculture Nutrition. Experimental Diets Parameter Water CLT KM10 KM50 KM110 Mean ± SD Initial Clear 2.8 ± 0.7 2.9 ± 0.7 2.8 ± 0.6 3.0 ± 0.8 2.9 ± 0.7 Weight (g) Green 3.5 ± 0.8 3.5 ± 0.7 3.5 ± 0.7 3.4 ± 0.8 3.5 ± 0.8 Final Weight Clear 13.1 ± 2.2 13.3 ± 1.9 12.9 ± 1.8 13.3 ± 2.1 13.2 ± 2.0 (g) Green 14.7 ± 2.1 13.9 ± 2.1 14.0 ± 2.5 14.2 ± 2.4 14.2 ± 2.3 Growth Clear 0.98 ± 0.02 1.01 ± 0.07 0.98 ± 0.07 1.01 ± 0.08 1.0 ± 0.05 (g/week) Green 1.06 ± 0.08 1.01 ± 0.08 1.02 ± 0.14 1.05 ± 0.07 1.04 ± 0.09 Survival (%) Clear 83.9 ± 10.1 80.0 ± 6.2 81.4 ± 6.9 80.7 ± 6.0 81.4 ± 7.1 Green 91.8 ± 4.6 94.4 ± 1.9 91.5 ± 3.6 85.9 ± 8.7 91.3 ± 5.7 Yield Clear 0.80 ± 0.13 0.77 ± 0.14 0.77 ± 0.13 0.78 ± 0.04 0.78 ± 0.12 (kg/m2) Green 0.58 ± 0.05 0.58 ± 0.04 0.56 ± 0.09 0.53 ± 0.09 0.57 ± 0.07 AFI* (kg/m2) Clear 1.58 ± 0.18 1.65 ± 0.21 1.66 ± 0.22 1.76 ± 0.14 0.95 ± 0.10 Green 1.28 ± 0.23 1.12 ± 0.24 1.17 ± 0.12 1.25 ± 0.14 1.23 ± 0.15 FCR* Clear 1.99 ± 0.25 2.18 ± 0.37 2.18 ± 0.33 2.27 ± 0.18 2.16 ± 0.28 Green 2.47 ± 0.26 1.96 ± 0.10 2.13 ± 0.18 2.44 ± 0.54 2.15 ± 0.34 *AFI, apparent feed intake per area of culture (kg/m2); FCR, food conversion ratio. BFT creates new perspectives in the way shrimp feeds are formulated

Biofloc system in a commercial shrimp farm in NE Brazil

Bioflocs can spare protein and the dependence on fishmeal in shrimp diets

Going heterotrophic under lab conditions

Bioflocs formation

C:N ratio of 20:1

Application in water Poultry feed

Bacterial flocs

Dried mollasses Phytoplankton bloom Innoculation Experimental diets Low protein diet

INGREDIENTS INCLUSION Poultry feed, 15.7% CP 54.03% Shrimp feed, 35% CP 40.73% Molasses, dried 5.00% Synthetic binder 0.24% 23.5% CP 36.9% CP

NUTRIENT LEVELS Weighing feed and molasses C:N Ratio 12.30 Fat 4.25% Fiber 4.38% Protein 23.52% Moisture 7.99% Under floc conditions, shrimp can grow well with low protein diets

Shrimp/m2 In. WGT (g) Fn. WGT (g) Grams/wk Survival (%) Yield (g/m2) 50 – INT 3.99 ± 0.35ac 21.22 ± 1.10a 1.68 ± 0.12a 92.8 ± 7.6 771 ± 116 75 – INT 3.28 ± 0.22bc 18.57 ± 1.26c 1.49 ± 0.11ab 72.7 ± 10.7 751 ± 158 100 – INT 3.58 ± 0.14abc 17.27 ± 1.29c 1.33 ± 0.12b 67.2 ± 21.7 766 ± 308 50 – BFT 3.70 ± 0.36ab 20.22 ± 0.43b 1.61 ± 0.04ac 81.6 ± 15.6 629 ± 167 75 – BFT 3.26 ± 0.75c 17.99 ± 1.67c 1.33 ± 0.05bc 85.1 ± 10.4 883 ± 152 100 – BFT 3.31 ± 0.25bc 16.95 ± 0.35c 1.48 ± 0.16b 80.0 ± 15.7 1,002 ± 225 ANOVA < 0.05 < 0.05 < 0.05 ns ns

Growth of P. vannamei under an autotrophic versus heterotrophic system over 72 days of culture Data: Fonseca (unpublished)

Biofloc system allowed reducing feed protein content without any detriment to shrimp growth 180 100-BFT 160 Volume (mL/L)

140 75-BFT

120

100 80 50-BFT 60 100-INT

40 50-INT 75-INT 20

0 -2 3 6 9 10 12 16 19 24 27 30 34 38 46 48 52 55 59 62 67 70 Days of Rearing

Biofloc production higher at BFT than conventional intensive system, but reduced at low stocking density Why shrimp feeds still rely on fishmeal? (1) ECONOMICS: use remains economically competitive at strategic inclusion levels, for specialty diets (starters, anti- stress/transition, premium) and certain markets (2) CONVENIENCE: few ingredients available capable of replacing the single value of fishmeal. It contains a highly attractive package from the nutrition standpoint ƒ Source of multiple essential nutrients (protein, AA, fatty acids, cholesterol, phospholipids) ƒ Highly digestible, few anti-nutritional factors, feeding effectors, unidentified growth factors (3) MARKET PERCEPTION: feeds with high levels of fishmeal are still perceived as high performers CONCLUSIONS 1. On methionine supplementation: Supplementation of crystalline amino acids provides a viable cost effective alternative for innovative nutritional strategies focusing on maintaining ideal protein ratios in the diet while providing increased flexibility in ingredient selection.

2. On fishmeal reduction: Effective fishmeal reduction in shrimp diets is dependent on methionine supplementation and an adequate supply of fish oil or another source of n-3 HUFA. Reduction beyond 5% fishmeal inclusion with 1% fish oil caused detriment to shrimp performance. Acknowledgements

Financial support Novus International Inc. (USA) EMBRAPA – Empresa de Pesquisa Agropecuária (Brazil)

Co-workers Dr. Marcelo Sá, Hassan Sabry-Neto, students and staff at LABOMAR