Balancing Nutrient Levels Through the Application of Functional Additives: Survey of Nutrient Levels in Commercial Shrimp Feeds in India

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1 Balancing Nutrient Levels Through the Application of Functional Additives: Survey of Nutrient Levels in Commercial Shrimp Feeds in India Alexander van Halteren, Business Development Manager Aquaculture Nutrition Peter Coutteau, PhD, Business Unit Director Aquaculture

2 Understanding the local conditions : Survey of nutrient levels in commercial shrimp feeds in India The numer of shrimp feed producers has increased significant in recent years, from 5 main producers the market has grown to more then 25 feed-mills producing shrimp feed Increased numer of feed rands 8 rands have een selected and the feeds have een collected from the market during the second quarter of 2016 from farmers and feed distriutors. All feed samples were produced during Q2 of 2016 and stored under typical la conditions, efore sending for analysis. Three different samples of each type of feed was pooled into one representative sample.

3 Understanding the local conditions : Survey of nutrient levels in commercial shrimp feeds Crude protein was analysed following the Kjeldahl method (Commission Directive 93/28/EEC.OJ No L ). Crude fat has een determined with acid hydrolysis following the Soxhlet method (AOAC ). Cholesterol was determined y direct saponification using the gas chromatographic method (AOAC ; expressed as g/kg) Phospholipids were analysed with 31 P-NMR spectroscopy using the internal standard method (SAA-MET002-03, expressed as % as is) The analyses of amino acids and nitrogen were performed y Evonik Degussa GmH (official European method of amino acid analysis in feed, COMMISSION DIRECTIVE 98/64/EC of 3 Septemer 1998; official method code of the AOAC International 2000, and expressed % as is)

4 CRUDE PROTEIN (%) Proximate composition : Crude protein versus Crude lipid 43 CRUDE PROTEIN VERSUS CRUDE FAT y = x R² = CRUDE FAT % Crude protein levels etween 33.9 % and 40.7 %, specification 35 % - 36% 5/8 samples higher protein level compared to the specifications on the lael Crude fat levels > 5 %; on average 6.18 % ± 0.54 %, fat specification 4.5 %-6 % No clear relation etween crude fat and protein

5 Crude Protein : Amino acid profile Amino acid profile : Indicator for protein quality Indicator for nutrient density Protein digestiility? Lysine 2.11% ± 0.17% Methionine 0.63% ± 0.09%

6 Lipids, essential nutrients for shrimp Lipids are essential components for shrimp growth : Cholesterol Phospholipids n-3 HUFA Source of energy Mainly sourced from marine raw materials Shrimp are not efficient in lipid digestion

7 Lipid composition : Crude fat / n-3 HUFA Five out of the 8 feeds had n-3 HUFA values around 5 g/kg One feed > 7g/kg n-3 HUFA and two feeds <3 g/kg n-3 HUFA

8 Lipid composition : n-3 HUFA Requirements on n-3 HUFA Marine shrimp do not have the aility to iosynthesize n-3 HUFA Indicator for the quantity of marine fats/meals used in the feed The dietary requirements of Litopenaeus vannamei were found to e at least 10 g/kg (Kanazawa et al., 1979; Kontara et al., 1995; Shiau, 1998)

9 Lipid composition : Crude fat / Phospholipids Phospholipid level : 1.13 % % (avg ± sd; 1.74 % ± 0.43)

10 Requirements : Phospholipids Dietary phospholipid is required for optimal growth of penaeid shrimp including L. vannamei (Glencross et al. 1998; Paiulkichakul et al. 1998; Thongrod and Boonyaratpalin 1998). Recommended levels of dietary phospholipids from soyean sources range from 1.25% to 6.5%, depending on shrimp species, developmental stage, as well as purity of the lecithin (Coutteau et al., 1997). The average levels of phospholipids : 1.74 % ± 0.43 The lowest level eing 1.13% and the highest 2.5%

11 Lipid composition : Crude fat / Cholesterol The cholesterol levels found in the analysed feeds were all well elow 1 g/kg The average cholesterol level was 0.64 g/kg ± 0.16, with half of the feeds around g/kg and one feed sample as low as 0.34 g/kg.

12 Requirements : Cholesterol Cholesterol requirement : 0.5 to 5 g/kg for L. vannamei (Chen, 1993, Duerr and Walsh, 1996; Gong et al., 2000) Dietary cholesterol levels elow 1 g/kg limit growth in L. vannamei.duerr and Walsh (1996) Morris et al. (2011) reported the cholesterol requirement for L. vannamei grow-out to e somewhere etween 0.76 and 1.1 g/kg, however, a regression analysis predicted the cholesterol requirement for maximum growth to e 1.5 g/kg At 1.5% and 3% phospholipids, dietary cholesterol requirements reduced to 1.4 and 1.3 g/kg, respectively The level of the essential fat nutrients, cholesterol, n-3 HUFA and phospholipids, were not correlated at all with the total level of dietary fat

13 Trends revealed from comparing surveys 2014 and Average ± s.d. (n=4) Average ± s.d. (n=8) Crude fat (%) 6.28 ± ± 0.54 Crude protein (%) ± ± 2.25 n-3 HUFA (mg/g) 5.23 ± ± 1.74 Cholesterol (mg/g) 0.84 ± ± 0.16 Phospholipids (%) 1.26 ± ± 0.43 Average crude protein increased from 35.8% to 36.4% in 2016 Average crude fat levels are similar in oth surveys, around 6.2%. n-3 HUFA and cholesterol levels have dropped with 16% and 24%, respectively, The average inclusion of phospholipids increased with 38 % etween 2014 and 2016.

14 Discussion and possile solutions The changes in the lipid profiles reflects the possily replacement of fish meal and fish oil in 2016 the domestic supply of these raw materials was insufficient Average levels indicate that overall reduced levels of cholesterol and n-3 HUFA compensated with increased levels of phospholipids and crude protein Reduced levels of essential lipids (cholesterol, phospholipids and n 3 HUFA) significantly affected growth, FCR and PER in white shrimp (van Halteren et al., 2016) PROMOTE THE APPLICATION OF DIGESTIBILITY ENHANCING ADDITIVES

15 Digestiility enhancing additives : Improve the asorption efficiency of the increasingly limited levels of cholesterol and n-3 HUFA Natural product ased on purified ile salts 1. FUNCTIONAL digestive enhancer Enhance hepatopancreas function Increase digestive capacity for essential lipids Compatile with the shrimp s digestive system 2. NUTRITIONAL supplement Alternative source of cholesterol 15

16 Bile acids : a natural emulsifier capale of enhancing the digestive capacity for lipids in the digestive system of shrimp natural emulsifier Optimal emulsifier properties (HLB 25) Essential for lipid digestion/asorption (micelle formation) co-factor lipase Co-factor for the action of BS dependent lipase (BSDL, triacyl glycerol lipase) Bridge function to contact enzyme and sustrate (ile acid hydrophilic group comines with lipase, hydrophoic group comine with lipid) Transport and asorption of the fat-solule vitamins & carotenoid pigments

17 Bile acids : Alternative source for the steroid ring which shrimp cannot synthesize, as precursor for molting hormones Common steroid ring structure with cholesterol : Essential for shrimp ecdysteroids (molting hormones) NRC, 2011 Essential for shrimp ecdysteroids (molting hormones) Lin et al. (2017)

18 Location of ecdysteroid signaling response genes (Yu-Hung Lin, 2017) Black Box reactions Chol. 1 5β-diketo? 2 ecdysone

19 Ecdysteroid signaling response genes Gene Function Location of gene Ecdysteroid receptor Retinoic acid X receptor Hemocyanin Chitin synthase Chitinase isoenzyme Myosin heavy chain Actin Receptor heterodimer EcR RXR directly activates a small set of early response genes Increased oxygen requirements of the molting process Involved in synthesis of chitin to uild the exoskeleton and peritrophic memrane. Involved in the hydrolysis of chitin from the exoskeleton and peritrophic memrane. Elevated muscle protein degradation or muscle atrophy Conserved proteins that participate in muscle contraction, cell motility, cell division, and cytoskeletal structure ecdysteroid ecdysteroid hepatopancreas ecdysteroid ecdysteroid muscle muscle Qian et al., 2014

20 Effects of dietary cholesterol replaced y ile salt on performance and molting gene expression in white shrimp Ingredient FM SBM SMB+BS 0,1 % SMB+BS 0,2 % Fishmeal 50,28 % 15,94 % 15,94 % 15,94 % SBM 0 % 48,51 % 48,51 % 48,51 % FO 1,40 % 3,45 % 3,45 % 3,45 % Cholesterol 0,41 % 0,19 % 0,19 % 0,19 % Bovine BS 0,00 % 0,00 % 0,10 % 0,20 % Yu-Hung Lin, 2017

21 Effects of dietary cholesterol replaced y ile salt on performance 250 Weight gain (%) a a 0 FM SBM SBM+BS0.1 SBM+BS0.2 Diet Growth performance can e improved y adding ile salts in SBM-ased diet for white shrimp Yu-Hung Lin, 2017

22 Gene expression Gene expression Gene expression Molting signal gene expression improved y adding ile salt in SBM-ased diet for white shrimp Ecdysteroid receptor Retinoic acid X receptor c a c a 0 FM FM SBM SBM+CA0.1 SBM+BS0.1 SBM+CA0.2 SBM+BS0.2 Diets code FM SBM SBM SBM+BS0.1 SBM+CA0.1 SBM+BS0.2 SBM+CA Hemocyanin Diets code a a a FM FM SBM SBM SBM+CA0.1 SBM+BS0.1 SBM+CA0.2 SBM+BS0.2 Diets code Yu-Hung Lin,

23 Gene expression Gene expression Chitin metaolism gene expression improved y adding ile salt in SBM-ased diet for white shrimp Chitinase isoenzyme a FM FM SBM SBM+BS0.1 SBM+CA0.1 SBM+BS0.2 SBM+CA0.2 Diets code Chitin synthase a FM FM SBM SBM SBM+CA0.1 SBM+BS0.1 SBM+CA0.2 SBM+BS0.2 Diets code Yu-Hung Lin, 2017

24 Gene expression Gene expression Gene expression Muscle growth gene expression improved y adding ile salt in SBM-ased diet for white shrimp Alpha-actin Myosin heavy chain 1.2 c a FM SBM SBM SBM+BS0.1 SBM+CA0.1 SBM+BS0.2 SBM+CA0.2 FM SBM SBM SBM+BS0.1 SBM+CA0.1SBM+BS0.2 SBM+CA0.2 Diets code Diets code Beta-actin a a 1 a FM SBM SBM SBM+BS0.1 SBM+CA0.1 SBM+BS0.2 SBM+CA0.2 Yu-Hung Lin, 2017 Diets code

25 Source and composition of Bile salts can effect the performance of shrimp EXPERIMENTAL DESIGN: 5 treatments 1. CON HI (positive control): 0.10% cholesterol (marine ingredients FM/FO/SQM + purified 0.25 kg/mt of feed) 0.9% n-3 HUFA provided y fish meal and fish oil 1.1% phospholipids (marine ingredients + 1.5% lecithin) 2. CON LO (negative control): reduced level of fish oil (-0.5%) and lecithin (-0.5%) compared to CON HI No cholesterol supplementation Experimental treatments: CON LO supplemented with different types of ile acids at 250 mg ile salts/kg diet : 3. LIPOGEST: CON LO + Commercial product, purified from ovine origin 4. PURE CHOLIC ACID: CON LO + Pharmaceutical product, purified cholic acid from ovine origin 5. BILE SALTS PIG: CON LO + Commercial product, purirfied from pig origin

26 Source and composition of Bile salts : Effects on performance

27 Understanding the local conditions : Conclusions The survey of shrimp feeds in India during 2016, average (± s.d. for eight samples) Crude protein 36.4 ± 2.3% Lysine 2.11 ± 0.17% Methionine 0.63 ± 0.09% n-3 HUFA 4.42 g/kg ± 1.74 g/kg Cholesterol 0.64 ± 0.16 g/kg Phospholipids 1.74 ± 0.43% Industry trend etween 2014 and 2016 : lower levels of cholesterol and n-3 HUFA, Crude protein and phospholipid levels were increased over the same period Application of digestiility enhancers can support the variation in nutrient density : increased AWB increased gene expression in the metaolic pathways involved in growth BS source and composition affects performance

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