A MORE EFFICIENT METHOD OF FATTY ACID ANALYSIS AND THE DETERMINATION OF CAROTENOID CONTENT IN THE STUDY OF BROOD STOCK AND JUVENILE NUTRITION IN MARINE ORNAMENTAL FISHES CoSMoS 2006 Stephen O Shea, Nancy Breen, Brad Bourque and Skip Pomeroy
Clownfish are one of the most common residents in private saltwater aquariums kept by hobbyists around the world, creating a high demand for high quality fish.
Current Status of Clownfish Market Effects on environment dynamite cyanide wild populations Prefer aquacultured fish to wild caught better suited to aquarium environment Free from disease
Production Balancing market demands with cost and time of production Successful breeding pairs 2 year old fish followed by 6 month courtship Fecundity and egg quality High survival rate through to juveniles Size Fast growth rate for quick sale (fish > 1 ) Good proportions Coloration Vibrant deep orange/red Bright white stripes Bold black accents Health Strong immune system Well formed with no abnormal deformities
Broodstock Nutrition Broodstock nutrition plays an important role in egg quality and fecundity Fish have essential fatty acid requirements. PUFAs - polyunsaturated fatty acids HUFAs - highly unsaturated fatty acids This study focuses on HUFAs, the omega-3 and omega-6 fatty acids The importance of essential fatty acids in fish nutrition has been extensively investigated during the past 20 years
Fatty Acids are Essential Gonadal development Egg and sperm quality Sperm motility Egg fertilization Embryo development Fecundity Offspring survival
Essential Fatty Acids Arachidonic Acid (AA) 20:4n-6 Docosahexaenoic Acid (DHA) 22:6n-3 Eicosapentaenoic Acid (EPA) 20:5n-3 Docosapentaenoic Acid (DPA) 22:5n-6
Essential Fatty Acids DHA high concentrations are found the in brain and retinal polar lipids; neural membrane structure and function EPA and AA important structural components, precursors to biologically active compounds that regulate growth DHA and AA - increase larval resistance to stress and pathogen infections The n-3 HUFAs are also regarded as major energy sources during early embryonic development
Current Study Verification of LC/MS ESI Technology in characterization fatty acid content in embryos Determination of the effect of broodstock diet on egg quality: 15 brood pairs (5 replicate pairs per treatment) rotated through 3 treatment diets 4 months on each diet, with each 5 replicate pairs receiving each treatment diet. Determine variation of fatty acid content in single eggs from a clutch
Gas Chromatography Standard Technique requires fatty acid methyl ester formation separation essentially on boiling point with FID or Mass Spec detection
Common Technique 1. C14:0 2. C16:0 3. C18:0 4. C18:1n9 5. C18:2n6 6. C18:4n3 7. C20:0 8. C20:1n9 9. C20:5n3 10. C22:0 11. C21:5n3 12. C23:0 13. C22:5n3 14. C24:0 15. C22:6n3 16. C24:1 Column: 30m Carrier Gas: He, 30cm/sec. Column Temp: 150-205 C Detector: FID, 260 C Retention Time: Note 34 minutes before emergence of C22:6n3 (DHA). Can we process samples quicker?
HPLC and ESI Mass Spectrometry
Waters HPLC and Mass Spectrometer Operated with Mass Lynx Software HPLC: Waters Alliance: XTerra C 18 Reverse Phase column - 50mm length -3.5μm diameter silica bead Isocratic Solvent Flow (90% MeOH, 10% Water) Mass Spec: Waters Micromass ZQ Electrospray Ionization (ESI).
Mixed Standards Docosapentaenoic Acid (DPA) 22:5n-6 Docosahexaenoic Acid (DHA) 22:6n-3 Arachidonic Acid (AA) 20:4n-6 Eicosapentaenoic Acid (EPA) 20:5n-3 Heptadecanoate (internal standard) TIC Note retention times are < 5 minutes, not 34 minutes
100 Arachidonic Acid (AA) 303.3 100 Docosahexaenoic Acid (DHA) 327.3 % Mass spec shows purity of AA at R T of 3.58 min % 304.3 328.4 283.3 108.9 122.9 144.8 162.9 201.0 212.0 259.2 269.3 325.3 357.4 231.2 379.4 433.5 478.4 401.4 421.4 465.4 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 108.9 122.9 144.8 284.3 199.1 349.4 162.9 212.1229.2 255.2 325.3 381.4 459.2 363.4 403.4 425.5 445.5 487.5 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Eicosapentaenoic Acid (EPA) Docosapentaenoic Acid (DPA) 100 301.3 100 329.4 % % 302.3 330.3 257.3 108.8 122.8 144.8 162.9 203.1 212.0 255.3 258.3 283.3 323.3 355.3 377.4 399.4 419.4 433.4 463.5 49 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 108.9 122.8 331.3 144.8 351.4 168.9 269.3 285.3 194.9 212.0 231.2 311.3 383.4 397.5 419.3 459.6 447.3 489 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480
Calibration Curve Data DPA-329 Area (AU) 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 Conc (microg)/ml
Calibration Curve Data EPA-301 10 Peak Area (AU) 8 6 4 2 0 0 2 4 6 8 10 Conc. (microg/ml)
Combined Calibration Curve Data Calabration Curve Data Peak Area 10 9 8 7 6 5 4 3 2 1 0 EPA AA DHA DPA 0 2 4 6 8 10 Conc. (microg/ml) Great linear response From 0-10μg. Our Studies used 0-1μg
Combined Calibration Curve Data Calabration Curve Data 1 Peak Area 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 EPA AA DHA DPA 0 0.2 0.4 0.6 0.8 1 Conc. (microg/ml)
Proof of Method Artemia Investigation Matthew R. Palmtag, Cynthia Faulk and G.J.Holt University of Texas Marine Science Institute
Enriched Artemia Feeding Study Artemia fed live algae Isochrysis galbana Fatty acid composition compared to a previous study using GC/MS Palmtag, Faulk and Holt in press
Tissue Grinding Extraction into chloroform methanol solution (2:1) Saponification Methanolic base hydrolysis 70 o C (15 mins) [not 30 mins classical] Esterfication BF 3 catalyzed esterfication 70 o C (2 mins) [not 15 mins classical] back extraction into hexane HPLC Preparation Holt s Methodology Dried under nitrogen flow and resuspended in LC/MS grade methanol with standard (C 17 heptadecanoate)
Enriched Artemia Feeding Study Artemia fed live algae Isochrysis galbana % Total Fatty Acids GC Analysis EPA/AA LCMS Analysis EPA/AA 3.0 + 0.2 3.09 + 0.2
Clownfish diet study each 5 brood pair group was rotated through all treatments for 4 months on each treatment Five Brood Pairs Five Brood Pairs Five Brood Pairs Number of spawns, hatch rate, survival to 30 days Determination of fatty acid content in eggs
Tetra Marine Flake gradient TM1426 100 % 0 TM1426 100 % 0 TM1426 100 % 0 TM1426 100 % 0 TM1426 100 % 8 0.41 0.66 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 3.36 3.40 3.56 3.54 3.58 3.69 3.78 DPA DHA AA EPA TIC 1: Scan ES- 329 6.00e6 1: Scan ES- 327 2.54e7 1: Scan ES- 303 4.76e6 1: Scan ES- 301 2.17e7 1: Scan ES- TIC 9.92e7 Time
Food Fatty Acid Content Formula 1 EPA 1 AA 0.140 DHA 0.678 DPA 0.119 Gelly Belly 1 0.222 0.494 0.141 Tetra Marine Flake 1 0.243 1.335 0.236
Spawns Picture taken of nest analyzed with NIH Image J to determine counts and size distribution of eggs Sample of 20 eggs collected, weighed, and analyzed for fatty acids Remaining eggs were returned to hatch tank for determination of hatch rate.
Eggs from Formula One Diet Docosapentaenoic Acid (DPA) 22:5n-6 Docosahexaenoic Acid (DHA) 22:6n-3 Arachidonic Acid (AA) 20:4n-6 Eicosapentaenoic Acid (EPA) 20:5n-3 Heptadecanoate (internal standard) TIC
Cis-Trans Isomerization Arachidonic Acid (AA) Trans Cis
Diet Study Results EPA content in eggs was significantly different for brood stock fed on TMF AA, DHA and DPA did not vary significantly with the three diets On average, of the 4 fatty acids studied in eggs DHA (56%) and EPA (28%) TMF gave smallest clutch number but largest size eggs with poor hatch survival quality Breeding pairs fed the same diet did not show distinct differences in accumulation of fatty acids in eggs
Determination of variation of fatty acid content in a clutch Twenty Eggs Two Egg Selection
Fatty Acid Content Variation with Sample Size μg/ml EPA AA DHA DPA AV 20 eggs Std. Dev. AV 2 eggs Std. Dev. 0.14 +.004.004 0.10 +.028 0.05 +.001.001 0.03 +.01 0.32 +.042.042 0.21 +.063 0.06 +.009.009 0.05 +.019.019
Carotenoids Biological Activity
Background Carotenoids are essential nutrients important in maintaining good health in marine fish Diets supplemented with astaxanthin result in higher survival rates in atlantic salmon juveniles and in goldfish Astaxanthin has been shown to be the key determinant of color in clownfish
O OH H O O all trans Astaxanthin β-carotene O H O H O O Canthaxanthin Lutein Zeaxanthin OH OH Important active carotenoids determined to have biological activity H O β-cryptoxanthin OH all trans Retinol
Juvenile Experiment Diet Tetra Marine Flake Wardley Total Color Marine Gourmet Flake Blend Ocean Nutrition TM Formula Cyclop-eeze
Feeding 4 diet treatments 6 replicates per treatment with 30 fish per replicate Tanks stocked with fish at 30 dph All fish fed the same standard larval diet until 30 dph Rotifers Enriched Artemia Golden Pearl Artemia
Digital Pixel Analysis Photo set-up frame Kodak CX 7530 Digital Camera Camera frame Scale Petri dish Ruler
Pixel Analysis Color Photoshop CS Length NIH Image J Software Weight Taken at same time as pictures Flake foods
Photometry Acetone extraction UV/Vis Analysis @450nm Determination of concentration from an average extinction coefficient for carotenoid mixture (130058 M -1 cm -1 ) Flake Foods Poor astaxanthin diet fish Rich astaxanthin diet fish
Waters HPLC and Mass Spectrometer Operated with Mass Lynx Software HPLC: Waters Alliance: XTerra C 18 Reverse Phase column - 50mm length - 3.5μm diameter silica bead Gradient Solvent Flow 25 min. run (Initial MTBE 5% 90%MeOH, 5% Water to MTBE 60% 35%MeOH, 5% Water) Mass Spec: Waters Micromass ZQ Electrospray Ionization (ESI).
Digital Pixel Analysis of Feeds F1 Tetra Marine Wardleys Cyclop-eeze Histogram Significance of red green and blue Percentage of red pigment Area of curve
UV/Visible Spectra Cyclpo-eeze Wardleys Tetra-Marine Formula 1 Relative Concentrations of Caroteniods Determine @450nm Cyclop-eeze Wardleys Tetra-marine Formula 1 0.95mg/g 0.20mg/g 0.14mg/g 0.09mg/g
Pixel Diet Study Tetra Marine Wardley s Formula One Cyclop-eeze Histogram Significance of red green and blue Area of curve Location of colors Percentage of red pigment
Bottom left fish Cyclop-eeze (astaxanthin rich) diet showing intense red coloration. Histogram of bottom left fish showing high amount of red pixels (61.59%). Top right fish (astaxanthin low) diet showing dull coloration Histogram of top right fish showing relatively low amount of red pixels (40.88%).
Photometry Results A.Ocellaris Skin Carotenoid Content Acetone extraction UV/Vis Analysis @450nm Astaxanthin poor feed Carotenoid Content Dull Color 4.0μg/cm 2 Astaxanthin rich feed Carotenoid Content Bright Color 10.4 μg/cm 2
Cyclop-eeze 10microliters 720food1 100 1.92 Astaxanthin 1: Scan ES+ 619 5.27e5 % 0 720food1 100 1.73 1: Scan ES+ 536 1.28e6 β-carotene % 0 720food1 100 1.71 1.88 1: Scan ES+ TIC 9.66e7 % 59 1.49 2.10 2.66 2.96 3.34 3.79 2.372.49 4.24 4.69 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 Time
UV/Vis Mass Spectrum β-carotene 1 0 0 4 5 0. 5 7 % 5 7 4. 5 7 6 1 3. 5 7 6 3 6. 5 7 6 6 2. 5 7 6 6 9. 5 7 6 8 3. 5 7 6 9 5. 5 7 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 5 6 0 5 8 0 6 0 0 6 2 0 6 4 0 6 6 0 6 8 0 7 0 0 n m 100 536.48 % 537.49 567.50 444.41 413.31 445.38 429.22 411.25 415.28 431.25 461.28 475.41 503.12 509.46 523.47 535.47 552.48 551.46 554.50 549.52 568.51 599.49 583.46 615.52 621.49 639.48 0 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640 m/z
Astaxanthine 10microliters 100 474.57 486.57 % 528.57 10microliters 306.57 362.57 0 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 nm 100 302.18 601.53 603.54 % 577.47 387.12 429.12 503.10 304.98 329.98 371.10 388.16 430.12 461.12 551.46 578.57 604.57 418.80 501.32 535.15 0 617.49 629.63 648.79 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 m/z
Cyclop-eeze Cyclop-eeze showed the best results In color, length, and weight No differences in survival In diet switch trials fish became more colorful.
Funding NRAC NRAC Grant Waters Waters Corporation Lead Lead Scientist Michael Balogh
Rogue s Gallery