larvi 2013 Optimum phospholipids and antioxidant levels in microdiets for gilthead seabream larvae Reda Saleh & Marisol Izquierdo

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1 lrvi 213 th fish & shellfish lrviulture symposium Optimum phospholipids nd ntioxidnt levels in mirodiets for gilthed serem lrve Red Sleh & Mrisol Izquierdo ghent university, elgium, 2- septemer 213

2 Optimum phospholipids nd ntioxidnt levels in mirodiets for gilthed serem lrve Red Sleh1,2 & Mrisol Izquierdo1 1Grupo de Investigión en Auiultur (GIA), Grn Cnri, Spin. 2Oenogrphy Dpt., Fulty of Siene, Alexndri University, Egypt.

3 Contents Introdution Mteril nd methods Effet of level nd type of PL Comined effet of PL nd vit E Comined effet of PL nd Se Conlusions

4 Introdution Growth nd survivl (Knzw et l. 13, Coutteu et l. 1, Fontgne et l. 2, Wold et l. 2) Lipoproteins synthesis (Liu et l., 22, Hds et l., 23) Redue mlformtions (Knzw et l., 11, Guerden et l., 1; Chu et l., 23) Soure of ftty ids (Izquierdo & Koven 2) nd energy (Rinuzzo et l., 12) Feeding tivity (Koven et l., 1) Emusifiers (Olsen & Ringø, 1) Digestion nd trnsport of lipids (Izquierdo et l., 21; Moris et l., 2), gut mturtion (MQueen Leifson et l., 23; Wold et l., 2; Sleh et l., 212)

5 Introdution Speies Phospholipid Optiml supplemented requirement Common rp Hen egg leithin 2% Red serem soyen leithin (SBL) % Knife jw SBL.4% SBL % SBL 12% SBL 1% Jpnese flounder Europen se ss Serem Referene Geurden et l. (1) Knzw et l. (13) Knzw et l. (13) Knzw (13) Chu et l. (23) Seiliez et l. (2)

6 Introdution O2 Se AOE ROS Cell Interior SOD O2- O2- As As-H CAT H2O2 H2O 2GSH Fe+ 3 Fenton GR GSSG Fe+ 2 GPX OH ++OH- H2O αto- αtoh αtoh Cell Exterior Betnoret l., Lrvi 213

7 Introdution Mrine fish lrve re under high oxidtion risk High metoli rte & oxygen requirements High wter ontent nd wter resorption t metmorphosis High PUFA requirements Lipid ontent nd lipid moiliztion from yolk s Feed with high surfe/volume Long wter exposure of feed

8 Introdution Ojetives 1. How muh? Whih type? How will it ffet digestive enzymes? How will it ffet skeleton development? How will it ffet oxidtive sttus? 2. Will PL requirements e ffeted y the ntioxidnt vit E? 3. Se levels improve the performne of lrve fed optimum PL levels?

9 Contents Introdution Mterils nd methods Effet of level nd type of PL Comined effet of PL nd vit E Comined effet of PL nd Se Conlusions

10 Mterils & Methods studies pulished in Aquulture Nutr. & Br. J. Nutr. Tnk volume 2 l Wter renewl 2%h Aertion 12 ml/min 2 Serem lrve 14 dph (T.L. mm & µg DBW) Photoperiod 12L:12D -2 lux Feeding 2.- g/dily

11 Mterils & Methods Zootehnil Prmeters Totl length, Dry ody weight, Survivl, Hndling stress Test, Feed eptne Exp 1. Optimum krill phospholipids Exp 2. Optimum soy phospholipids Exp 3. Comprison etween KPL or SBL Exp 4. Comined PL/vit E levels Exp. Rnged Se levels dph 3 1 Sprus urt 22 31

12 Mterils & Methods Ingredients, feeds nd lrve Protein (AOAC, 1) Totl Lipids (Folh et l., 1) Moisture & Ash (AOAC, 1) TBARS (Burk et l., 1) Neutrl & polr lipids (Olsen & Henderson, 1 ) Ftty Aids (Christie, 12, Izquierdo et l.,1) Selenium Collision/retion ell ICP-MS

13 Mterils & Methods Digestive Enzymes Alkline Phosphtse (Gee et. l.,1) Trypsin (Rotllnt et l., 2) Spetrofluorometer Lipse (Rotllnt et l., 2) Phospholipse A2 (Hung et. l., 2)

14 Mterils & Methods Whole mount stining Deformities studied ording to Boglione et l. (21) Alizrin red (Izquierdo et l., 212, modified from Vndewlle et l., 1)

15 Mterils & Methods Gene expression (RT-PCR) AOE mrkers Osteo mrkers CAT BMP4 SOD RUNX2 GPX Alkline phosphtse Osteolin Osteopontin Osteonetin Mtrix Gl Mens ompred y Dunn s test (P <.) using SPSS softwre (SPSS for Windows 11.; SPSS In., Chigo, IL, USA).

16 Contents Introdution Mterils nd methods Effet of level nd type of PL Comined effet of PL nd vit E Comined effet of PL nd Se Conlusions

17 Exp. I Krill phospholipids Finl Survivl Survivl % 4 4 High survivl & stress resistne with -% KPL Polr Lipids (% of dry diet) 11 Survivl fter hndling stress Survivl % 12 Polr Lipids (% of dry diet) Sleh et l., 212. Aquulture Nutr. 11 Lower requirements thn previous uthors for Europen sess using 12 SBL (Chu et l., 23)

18 Exp 2. Soyen leithin levels Survivl % Diet (% Polr Lipids) Poor effet of SBL on survivl nd 11 stress resistne Survivl % Finl Survivl Survivl fter hndling stress Diet (% Polr Lipids) Sleh et l, 213. Aquulture Nutr. 11 Up to 12% SBL did not ffet pikeperh lrvl survivl (Hmz et l., 2)

19 Exp 3. Krill phospholipids vs soyen leithin 4 d 4 Survivl % 3 2 d Polr Lipids (% of dry diet) Survivl fter hndling stress Survivl % Krill phospholipid s good PL soure (Betnor et l. 212) SBL Better survivl nd stress resistne y KPL thn SBL even t lower dietry levels 2 MPL 3 1 Finl Survivl MPL SBL d Polr Lipids (% of dry diet) Sleh et l, in press. Aquulture Nutr.

20 Expphospholipids 3. Krill phospholipids vs soyen leithin Exp. I Krill MPL y = 2,1443ln(x) SBL + 3,4 Log. (MPL) R² =, Log. (SBL) 13 Totl Length (mm) Totl Length (mm) Totl Length (mm) Polr Lipids (% of dry diet) d Exp 2. Soyen leithin levels d High growth (TL& BW) Better totl with -% length nd KPL ody weight y KPL thn SBL even t lower dietry levels Inresed growth (TL & Higher effetiveness of BW) with out mrine PL (Slhi et y = 2,3ln(x) + 4,4 R² =,44 % SBL l. 1; Izquierdo et l. 21; Wold et l. 2) Sleh et l, PolrLipids (% dry diet) 11 Diet (% Polr Lipids) in press. Aquulture Nutr.

21 Exp. I Krill phospholipids Alkline Phosphtse (RFU/Lrve) y = 131,4ln(x) -, R² =,23 11 Polr Lipids (% of dry diet) Alkline Phosphtse (RFU/Lrve) 3 Exp 2. Soyen phospholipids y = -4.43x2 +.34x R2 =. 1 1 Diet (% Polr Lipids) Sleh et l., 212 & 213. Aquulture Nutr Stronger positive effet of KPL on Alkline phosphtse nd thus on mturtion of digestive trt ould ontriute to the etter growth

22 Trypsin (RFU/Lrve) 3 Exp. I Krill phospholipids Trypsin (RFU/Lrve) Polr Lipids (% of dry diet) Exp 2. Soyen phospholipids Diet (% Polr Lipids) Sleh et l., 212 & 213. Aquulture Nutr. Very similr effet of oth PL on Trypsin tivity, so not relted with the HUFA ontent of 11 the PL soure

23 Exp. I Krill phospholipids 34 y = 1,1ln(x) - 1, Lipse (RFU/Lrve) R² =, Stronger effet of KPL on Neutrl lipse tivity Polr Lipids (% of dry diet) Lipse (RFU/Lrve) Exp 2. Soyen leithin levels y =,1ln(x) + 4,2 R² =, Neutrl lipse hs high ffinity for n-3 HUFA esterified lipids (Izquierdo et l., 22) Diet (% Polr Lipids) Sleh et l., 212 & 213. Aquulture Nutr. 11

24 Exp. I Krill phospholipids Phospholipse A2 (U*/ Lrve) y = 4,2ln(x) - 3,2 R² =,21 Polr Lipids (% of dry diet) Exp 2. Soyen leithin levels 14 Phospholipse A2 (U*/Lrve) y = 13,ln(x) - 113, R² =, Diet (% Polr Lipids) Sleh et l, 213. Aquulture Nutr. Very similr effet of oth PL soures on PLA2 only depending on the level of PL 12 Trnsriptionl regultion of PLA2 y dietry PL (ZmoninoInfnte nd Chu, 1). SBL enhne gut nd liver lipid trnsport tivity (Liu et l., 22) 11

25 Exp 3. Krill phospholipids vs soyen leithin Numer of minerlized vertere mm Control MPL MPL SBL SBL Size lss (mm) Control Lordosis 1 Kyphosis MPL Anomlies % 12 d 4 2 SBL Control MPL MPL Mndiul/Mxill SBL Sleh et l, in press. Aquulture Nutr. SBL 14 Both PL enhned one minerliztion, ut KPL ws more effetive nd t % one MPL redued nomlies Improved minerliztion nd redued deformities y n-3 HUFA ut very high levels inresed rnel deformities SBL(Izquierdo et l., 212 Br.J.Nutr.)

26 Endohondrl one rtilge Sox Intrmemrnous one IGF osteolsts Mesenhyml stem ell Runx2 BMP-2 PPARϒ Collgen II Collgen I ALP Osteopontin Osteolin hondrolst osteolsts Mtrix GLA hondroyte dipoyte osteoyte

27 Exp 3. Krill phospholipids vs soyen leithin OP d 1 ON At 3 dph OP (SPP1) is more sensitive to DHA other levels thn in one moleulr mrker studied (Izquierdo unpulished dt) Control MPL MPL SBL SBL Control MPL MPL SBL SBL Diet OC d 1 Sleh et l, in press. Aquulture Nutr. ALP The three minerliztion protein genes were up1 ykpl regulted nd to 13 lower extent y SBL, 11 ut Osteopontine expression ws etter orrelted with the minerliztion oserved 1 11

28 Exp 3. Krill phospholipids vs soyen leithin 4 2 RUNX2 BMP N-3HUFA nd DHA up regultes BMP4 nd OC in serem lrve (Izquierdo et l., 212) Control MPL MPL SBL Control SBL Control MPL MPL SBL Diet Sleh et l, in press. Aquulture Nutr. MPL MPL SBL SBL Diet Diet Mtrix GLA 3 SBL BMP ws up-regulted y KPL nd it ws negtively relted to lordosis inidene. The other two mrkers of erly nd lte one differentition were lso ffeted y KPL

29 Exp 3. Krill phospholipids vs soyen leithin TBARs MPL SBL Log. (MPL) Log. (SBL) nm MDA/g Lrve nd % SBL nd % KPL inresed ROS prodution in the lrve Diet (PL %) Sleh et l, in press. Aquulture Nutr.

30 Exp 3. Krill phospholipids vs soyen leithin 4 3 SOD CAT GPx Control MPL MPL SBL SBL Control MPL MPL SBL Diet Sleh et l, in press. Aquulture Nutr. d 4 SBL AOE, prtiulrly CAT, gene expression ws up-regulted y SBL nd % KPL

31 Contents Introdution Mterils nd methods Effet of level nd type of PL Comined effet of PL nd vit E Comined effet of PL nd Se Conlusions

32 Exp 4. Comined Vit E nd PL levels Finl Survivl Survivl % KPL/1 SBL/3 PL/ KPL/3 PL/ 4KPL/1 KPL/4KPL/3 KPL/KPL/1 11KPL/KPL/3 11KPL/ 11SBL/ Dietry vit E improved growth in KPL diets 3 Dry Weight (ug)/lrve Vit E ddition ws not le to improve survivl or growth in SBL diets Dry Body Weight d d PL/ PL/ KPL/ KPL/ 11KPL/ 11KPL/ 11SBL/ KPL/1 KPL/3 4KPL/1 4KPL/3 KPL/1 KPL/3 SBL/3 Sleh et l, sumitted. Aquulture Nutr.

33 Exp 4. Comined Vit E nd PL levels Inresed dietry Vit E rised the perentge of minerlized one in given size lss 2 Bone minerliztion % <..-1 >1 2 1 KPL/1 KPL/3 PL/ PL/ 11SBL/ 1 3 4KPL/1 KPL/ 4KPL/3 KPL/ Diet 1 Sleh et l, sumitted. Aquulture Nutr. 3 KPL/1 11KPL/ KPL/3 11KPL/ 1 3

34 KPL/1 KPL/1 KPL/3 PL KPL/3 PL 4KPL/1 KPL 4KPL/3 KPL 11KPL 11KPL/SBL/3 11SBL/ Diet OP Vit E ddition did not ffet ny of the one iomrkers studied, lthough KPL nd, thus n-3 HUFA, upregulted them, ut not through ROS!! BMP4 Exp 4. Comined Vit E nd PL levels KPL/1 KPL/3 4KPL/1 4KPL/3 KPL/1 KPL/3 SBL/3 PL PL KPL KPL 11KPL 11KPL/ 11SBL/ Diet 1 3 Sleh et l, sumitted. Aquulture Nutr

35 nm MAD/g Lrve Exp 4. Comined Vit E nd PL levels PLKPL/3 PL4KPL/1 KPL KPLKPL/1 11KPLKPL/3 11KPL/SBL/3 11SBL/ KPL/1 4KPL/ Diet Sleh et l, sumitted. Aquulture Nutr.

36 Exp 4. Comined Vit E nd PL levels 4 CAT KPL/1 KPL/3 4KPL/1 4KPL/3 KPL/1 KPL/3 SBL/3 Diet SOD KPL/1 KPL/3 4KPL/1 4KPL/3 KPL/1 KPL/3 SBL/3 Diet GPX d d d KPL/1 KPL/3 4KPL/1 4KPL/3 KPL/1 KPL/3 SBL/3 Diet Sleh et l, sumitted. Aquulture Nutr. CAT gene expression is etter orrelted thn GPX to oxidtive risk y DHA (Izquierdo et l., 212).

37 Contents Introdution Mterils nd methods Effet of level nd type of PL Comined effet of PL nd vit E Comined effet of PL nd Se Conlusions

38 Survivl % Survivl % Exp. Effet of Se levels Finl Survivl 12 Diet (mg Se/ Kg Diet) Addition of Se up 1 to -12 mg/kg in diets ontining % KPL inresed lrvl survivl nd stress resistne Survivl fter hndling stress 4 Sleh et l, sumitted. Br. J. Nutr

39 Exp. Effet of Se levels Lrvl Se nd n-3 HUFA were orrelted Lrvl ftty ids % * R² = N-3HUFA DHA 3 * 3 2 R² = (μg Se/g lrve)* Sleh et l, sumitted. Br. J. Nutr.

40 Exp. Effet of Se levels nm MDA/g lrve R² =.4 1 Diet (mg Se/Kg Diet) Gene Expression 12 CAT 4 Dietry nd lrvl Se ws negtively orrelted with TBARs nd AOE gene expresion GPX (ug Se/g Lrve)* Sleh et l, sumitted. Br. J. Nutr.

41 ALP Exp. Effet of Se levels SE 4SE SE SE 12SE Diet Dietry nd lrvl Se, or redution in ROS ws etter orrelted with ALP gene expresion thn the other one moleulr mrkers In greement, there ws n inresed minerliztion nd redued soliosis inidene (Poster session, Bénitez et l.) Antioxidnts my intert with ellulr reeptors nd trnsriptionl ftors thtetmy further led hnges in mrna levels (Olsvik et l. 211) Sleh l, sumitted. Br. J.to Nutr.

42 Oxidtive lne in mrine lrve ROS ROS Vit C PUFA Pro-oxidnts Fe Stress Light Photoperiod Cnilism Anti-oxidnts Se rotenoids

43 Contents Introdution Mterils nd methods Effet of level nd type of PL Comined effet of PL nd vit E Comined effet of PL nd Se Conlusions

44 1. Serem from 1-4 dph require out -% HUFA rih PL to ompletely sustitute live preys. KPL improves digestion, trnsport nd deposition of dietry lipids nd ontriute to redue skeleton nomlies y up-regulting one moleulr mrkers, prtiulrly OP nd BMP, induing erly minerliztion nd resistne of verterl odies to redue nomlies suh s lordosis nd kyphosis. 2. SBL is not le to promote survivl nd growth s effetively s KPL, inreses oxidtive risk in the lrve nd up-regultes AOE genes. 3. Dietry α-toopherol promoted the enefiil effets of dietry PL, promoting growth, denoting its protetive role ginst oxidtion nd reduing lrvl TBARs nd gene expression of SOD nd CAT. In reltion to oxidtive risk y HUFA, CAT gene expression is etter moleulr mrker. 4. Inrese Se up to -12 mg/kg improved lrvl survivl nd stress resistne, proteting the lrvl tissues from oxidtive risk

45 Aknowledgments