THE EFFECT OF GRADED DIETARY LEVELS OF VITAMIN A, GIVEN TO EARLY SEA BREAM (Sparus aurata) LARVAE ON SKELETAL DEFORMITIES AND GENOMIC EXPRESSION

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1 THE EFFECT OF GRADED DIETARY LEVELS OF VITAMIN A, GIVEN TO EARLY SEA BREAM (Sprus urt) LARVAE ON SKELETAL DEFORMITIES AND GENOMIC EXPRESSION B. Ginzourg, W.M. Koven, S. Fontgne, A. Sgi, D. M. Power nd A. Tndler

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3 Aims of the study: To develop vitmin A enrichment technology sed on the use of liposomes. To evlute the effect of different vitmin A dietry levels on fish development, in terms of optiml growth, nd incidence of skeletl mlformtions. To provide n insight into dietry lrvl requirements. To study the reltive mrna gene expression undnce of RBP RALDH2, LRAT, STRA6 during Se rem lrvl morphogenesis,

4 Metolism Mri Moren 2004

5 Diets: Mterils nd methods Artemi nd rotifers enriched with incresing vitmin A levels with liposomes. Lrvl performnce: Anlysis: Mesurement of optiml growth nd survivl rte. HPLC - vitmin A nlysis. Gs Chromtogrphy ftty cids nlysis. X RAY nlysis for one deformtion. Potthoff stin for crtilge nd one formtion. Gene expression: Semi-quntittive RT-PCR for RALDH2, LRAT, STRA6 nd RBP expression.

6 Rotifers enrichment ) /gr (ww μg T0 D AlgMc vit' A isomers D D Lipo 0 Lipo10 C *** Tretment B Lipo 50 ** A Lipo1000 * Retinol Retinyl cette Retinyl plmitte μg/ /gr (ww) Totl vit' A T0 c d d d AlgMc lipo 0 lipo 10 Tretment lipo 50 lipo 100 The pttern of use nd ccumultion of vitmin A isoforms in rotifers (B. rotundiformis), s function of its concentrtion in the culturing medium, fter 6 hours of enrichment. () Vitmin A ccumultion in the rotifers. () Vitmin A isoforms distriution in the rotifers. (Sttisticl nlysis: Retinol- -, Retinyl cette- A-B, Retyinl plmitte- * - ***).

7 50µm 25µm B A 10µm 25µm D C

8 Artemi nuplii enrichment μg/g gr (ww) Vit' A isomers 20 A B 10 0 TO D AlgMc Lipo 0 C Lipo10 Lipo 50 Lipo100 Retinol Retinyl cette μg/gr r (ww) T0 e AlgMc d Totl vit' A Lipo 0 c e Lipo 10 Lipo 50 Lipo 100 Tretment Tretment The pttern of ccumultion of vitmin A isoforms in rtemi nuplii s function of its concentrtion in the culturing medium, fter 8 hours of enrichment. () Vitmin A ccumultion in rtemi nuplii. () Vitmin VitminAiofom isoforms distriution dititioninin rtemi nplii fter the ove conditions. (Sttistics: Retinol --,Retinyl cette - A-D).

9 Experimentl Yolk sc Rotifers Artemi 4 19 DPH DPH Erly Rotifers enriched with incresing vitmin A levels Artemi NO vitmin A enrichment Lte Rotifers NO vitmin A enrichment Artemi enriched with incresing vitmin A levels Entire Rotifers enriched with incresing vitmin A levels Artemi enriched with incresing vitmin A levels

10 Mterils nd methods Experimentl systems

11 Erly lrvl odily ccumultion of vit A nd it s sisoforms μg/gr (ww) AlgMc Retinol Retinyl cette c Lipo 0 Lipo 10 B Lipo 50 Tretment A Lipo 100 lrve - vitmin A co ontents μg/gr (ww w) Rotifer- vitmin A contents μg/gr (ww) The reltionship etween rotifers vitmin A content on lrvl ody ccumultion of Vit A nd its isoforms in 19 dph se rem lrve, (Sprus urt) fter 15 dys of feeding (4-19 DPH). () Vitmin A ccumultion in 19 dph lrve. () Vitmin A isoforms distriution in the lrve. (Sttistics: A-B Retinyl ccette, -c Retinol).

12 Entire lrvl odily ccumultion of vit A nd it s isoforms μg g/gr (ww w) 5 Retinol 4 Retinyl cette * Retinyl plmitte A AlgMc Lipo 0 Lipo 10 ** B c c c Lipo 50 Tretment Lipo 1000 n A (ww) e - vitmin nts μg/gr ( lrve conten Rotifer stge Artemi stge Rotifer & Artemi - vitmin A contents μg/gr (ww) The reltionship etween vitmin A content in rotiers nd rtemi to the ccumultion nd isoforms distriution of this vitmin in 34 dph se rem (Sprus urt) ) lrve, fter 30 dys of feeding (4-34 DPH) with rotifers nd rtemi contining incresing vitmin A levels. () Vitmin A ccumultion in 34 DPH lrve. () Vitmin A isoforms distriution in the 34 DPH lrve.(sttistics: -c - Retinol, A-B - Retinyl cette, *-** -Reyintl plmitte).

13 The effect of dietry Vit. A supplementtion on lte lrvl performnce: Length Length (m mm) α A Dys Dys 4-19 Dys A A A 34 DPH 70 DPH AB AB α α α C BC α Length (m mm) B Dy 4-19 Dys 4-34 B c 0 0 Control Lipo 0 Lipo 10 Lipo 50 Lipo 100 Control lipo 0 lipo10 lipo 50 lipo 100 Tretment Tretment Length (mm) of 34 DPH (Grph ) nd 70 DPH (Grph ), fter feeding se rem lrve, with incresing vitmin A levels, t 3 different time periods during lrvl metmorphosis. (Sttistics: A-B: 4-19 DPH, α-β: DPH, -: 4-34 DPH)

14 The effect of lrvl dietry vit. A supplementtion on lte skeletl deformities in serem fingerlings

15 Types of skeletl deformities

16 X ry nlysis

17 B A D C F E

18 Skeletl stining

19 Gill deformities nd swimldder infltion % Defo ormtio on no deformity gill deformtion non- inflted swim ldder A α A A A α α c β Control Lipo 0 Lipo 10 Lipo 50 Lipo 100 Tretment t B β Gill deformities distriution nd non inflted swim ldder distriution versus undmged fingerling g t 115 DPH, fter feeding se rem lrve, with rtemi contining incresing vitmin A levels, t DPH, (A-B: Gills deformity, α-β: non inflted swim ldder, -: undmged fingerling)

20 Deforme ed % A 10 0 α control Skeletl deformities AB B C D lipo 0 lipo 10 lipo 50 lipo 100 lipo 0 lipo 10 c lipo 50 α Tretment α β γ lipo 0 lipo 10 lipo 50 lipo 100 control dys 4-34 lipo dy 4-19 lipo dys The reltionship etween vitmin A ody content in serem lrve t 3 different time periods during the lrvl metmorphosis on reltive presence of skeletl deformities in 115 dph fingerlings. (Sttistics: A-D: 4-19 DPH, α-γ: DPH, -c: 4-34 DPH).

21 Crnil deformities % Deformed C α A control AB AB BC lipo 0 lipo 10 lipo 50 lipo100 lipo 0 lipo 10 lipo 50 Tretment α α α α lipo 0 lipo 10 lipo 50 lipo 100 control dys 4-34 lipo dy 4-19 lipo dys The reltionship etween vitmin A ody content in serem lrve t 3 different time periods during the lrvl metmorphosis on reltive presence of crnil deformities in 115 dph fingerlings. (Sttistics: A-D: 4-19 DPH, α-γ: DPH, - c: 4-34 DPH).

22 STSM funded y the fish welfre COST ction 867

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24 0.75 ST TRA6/18S Ari itrry units TZ C BV G K HK D SP L B WM SK 18s STRA6 500 p 211 p STRA6 expression in dult tissues. Reltive undnce of STRA6 determined y semi-quntittive RT-PCR, with 18S to normlize quntity of totl RNA in the different tissues. Tissues: TZ, thyroid zone; C, crtilge; BV, one verterl; G, gond; K, kidney; Hk, hed-kidney; D, duodenum; S, spleen; L, liver; B, rin; WM, white muscle; S, skin

25 3 STRA A6/18S Aritr ry units Dys post-htching Dys 18s p STRA6 211 p STRA6 expression in lrve. Reltive undnce of STRA6, determined y semi-quntittive RT-PCR, with 18S to normlize quntity of totl RNA in different lrve stge: 1, 4, 8, 10, 24, 46 dys post htching.

26 7.5 RAL LDH2/18S Arit trry units Dys post-htching Dys s RALDH p 239 p RALDH2 expression in lrve. Reltive undnce of RALDH2, determined y semi-quntittive RT-PCR, with 18S to normlize quntity of totl RNA in different lrve stge: 1, 4, 8, 10, 24, 46 dys post htching.

27 Conclusions This study developed vitmin A enrichment technology sed on the use of liposomes. No reltionship ws found etween the reltive presence of operculr deformities or swim ldder presence nd dietry vitmin A. This study demonstrted tht during the first stges of the metmorphosis (4-19 DPH) higher h dietry level l of vitmin i A cused high numer of crnil deformtions, while during the lte stges of the metmorphosis (20-34 DPH) extreme level l of vitmin i A cused high h numer of norml vertere. These results indicte tht in Sprus urt lrve the effect of vitmin A is development stge specific.

28 Conclusions The reltive mrna gene expression undnce of RALDH2, LRAT, STRA6 ws found to e higher during the first 3 weeks of Se rem lrvl morphogenesis. STRA6, multi-trnsmemrne trnsmemrne domin protein, is widely expressed in emryonic development nd in dult ovry which emphsizes the importnce of vitmin A in the mternl yolk sc nd during the emryonic development. The level of vitmin A tht is ssocited with the est performnce in terms of growth promotion nd reduced rte of deformities is within the rnge of μg retinoid/gr ww in the live food nd 0.4 μg retinoid/gr ww in the lrve

29 Thnks