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 9. C20:5n3
2. C16:0 10. C22:0
3. C18:0 11. C21:5n3
4. C18:1n9 12. C23:0
5. C18:2n6 13. C22:5n3
6. C18:4n3 14. C24:0
7. C20:0 15. C22:6n3
8. C20:1n9 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 C18 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)
Note retention times are
TIC < 5 minutes, not 34 minutes
Arachidonic Acid (AA) 303.3
Docosahexaenoic Acid (DHA) 327.3
100 100
Mass spec shows
purity of AA at
%
RT of 3.58 min
%
304.3 328.4
283.3
108.9 122.9 144.8 325.3 108.9 122.9
162.9 201.0 212.0 259.2 269.3 357.4 144.8 284.3
231.2 379.4 401.4 421.4 433.5 465.4 478.4 162.9 199.1 212.1229.2 255.2 325.3
349.4
459.2
0 363.4 381.4 403.4 425.5 445.5 487.5
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 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)
301.3 329.4
100 100
% %
330.3
302.3
257.3
108.8 122.8 331.3 351.4
144.8 255.3 258.3 108.9 122.8 144.8 168.9
162.9 203.1212.0 283.3 323.3
355.3 377.4 399.4 419.4 433.4 463.5
269.3 285.3
383.4 397.5 419.3 447.3 459.6 489
49 194.9212.0 231.2 311.3
0 0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480
Calibration Curve Data
DPA-329
10
9
8
7
Area (AU)
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
10
9 EPA
8 AA
DHA
7
DPA
Peak Area
6
5
4
3
Great linear response
2 From 0 -10μg. Our
1 Studies used 0-1μg
0
0 2 4 6 8 10
Conc. (microg/mL)
Combined Calibration Curve Data
Calabration Curve Data
1
0.9 EPA
0.8 AA
DHA
0.7
DPA
0.6
Peak Area
0.5
0.4
0.3
0.2
0.1
0
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
Holt’s Methodology
•Tissue Grinding
Extraction into chloroform methanol solution (2:1)
•Saponification
Methanolic base hydrolysis 70oC (15 mins) [not 30 mins classical]
•Esterfication
BF3 catalyzed esterfication 70oC (2 mins) [not 15 mins classical] back
extraction into hexane
•HPLC Preparation
Dried under nitrogen flow and resuspended in LC/MS grade
methanol with standard (C17 heptadecanoate)
Enriched Artemia Feeding Study
Artemia fed live algae Isochrysis galbana
% Total Fatty Acids
GC Analysis LCMS Analysis
EPA/AA 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 1: Scan ES-
3.69 329
100
%
DPA 6.00e6
0
TM1426 1: Scan ES-
3.56 327
100
%
DHA 2.54e7
0
TM1426 1: Scan ES-
3.54 303
100
%
AA 4.76e6
0
TM1426 1: Scan ES-
3.36 301
100
%
EPA 2.17e7
0
TM1426 1: Scan ES-
100
3.40
3.58
3.78 TIC TIC
9.92e7
% 0.41 0.66
8 Time
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Food Fatty Acid Content
EPA AA DHA DPA
Formula 1 1 0.140 0.678 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 0.14 0.05 0.32 0.06
Std. Dev. +.004 +.001 +.042 +.009
AV 2 eggs 0.10 0.03 0.21 0.05
Std. Dev. +.028 +.01 +.063 +.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 all trans Astaxanthin
O O
β-Carotene O
Canthaxanthin
Important
O OH active carotenoids
determined
H Lutein
O OH to have
biological activity
H Zeaxanthin
O
H β-Cryptoxanthin
O
OH
all trans Retinol
Juvenile Experiment Diet
Tetra Marine Flake
Wardley Total Color Marine®
Gourmet Flake Blend
Ocean NutritionTM 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-1cm-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 C18 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 0.95mg/g
Wardleys 0.20mg/g
Tetra-marine 0.14mg/g
Formula 1 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/cm2
Astaxanthin rich feed
Carotenoid Content
Bright Color 10.4 μg/cm2
Cyclop-eeze
10microliters
720food1 1: Scan ES+
100
1.92 Astaxanthin 619
5.27e5
%
0
720food1 1: Scan ES+
100
1.73
β-Carotene 536
1.28e6
%
0
720food1 1: Scan ES+
1.71 TIC
100
1.88 9.66e7
%
1.49
2.10 2.96
2.66 3.34 3.79
2.372.49 4.24 4.69
59 Time
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
UV/Vis Mass Spectrum β-Carotene
4 5 0 .5 7
1 0 0
%
6 6 2 .5 7 6 6 9 .5 7 6 8 3 .5 7 6 9 5 .5 7
5 7 4 .5 7
6 1 3 .5 7 6 3 6 .5 7
0 n m
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
536.48
100
% 537.49 567.50
444.41
535.47
413.31
552.48 568.51
445.38
429.22 551.46
599.49
431.25 475.41 503.12
411.25 415.28 509.46 554.50 583.46 615.52 621.49
461.28 523.47 549.52
639.48
0 m/z
410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640
Astaxanthine
10microliters
474.57 486.57
100
%
528.57
306.57 362.57
0 nm
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
10microliters
601.53
100
302.18
603.54
% 577.47
387.12 429.12
503.10 604.57
304.98 329.98 371.10 388.16 430.12 461.12 551.46 578.57
501.32 535.15
418.80
617.49 629.63 648.79
0 m/z
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
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 Grant
Waters Corporation
Lead Scientist
Michael Balogh
Rogue’s Gallery