the effect of variation in dietary energy and protein levels on the lipid class and fatty acid...
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The effect of variation in dietary energy and protein levels on the
lipid class and fatty acid composition of Atlantic salmon
(Salmo salar) fed at elevated temperature
Matt Miller1&3
Julia Barnes1, Rhys Hauler2 , Chris Carter1 & Peter Nichols3
1School of Aquaculture, TAFI, University of Tasmania,2Skretting, 3CSIRO Marine Research
Why is temperature important?
Salmon Aquaculture under Australian conditions•Australia - higher water temperatures than in the main northern hemisphere
Aquaculture areas
•Salmon threshold of 20oC
The Effect of Temperature on lipids•Little is known for salmon
•Cold water fish need dietary 3
•PUFA have greater flexibility and provide membrane permeability at low
temperature
Why lipids?
Major component of fish diets
Important to fish health?
Storage of energy
Vital structural role of cells
Flesh Quality
Polar Lipids (PL)
Phosphatidy-choline
(PC) 18:0/22:6
Triacylglycerols (TAG)
18:0/22:6/20:5
Sterols Cholesterol
Free Fatty Acids (FFA)
22:6
Major lipid classes in Salmon
Omega 3 (3) polyunsaturated fatty acids (PUFA)
1
2
3
DHA (Docosahexaenoic Acid) 22:63
EPA (Eicosapentaenoic Acid) 20:53
Omega 3 represents first
unsaturation from terminal
methyl carbon
Important in human
physiology and heath (&
farmed fish)
First discovered in 1960s
0
0.3
0.6
0.9
1.2
1.5
1.8
0 5 10 15 20 25
Temperature oC
Max
imu
m S
pec
ific
Gro
wth
Rat
e
(%b
w.d
-1)
The experiment
•Temperature and growth rate•Threshold 20oC
•Julia Barnes Trial
•Energy / Protein ratios
• Atlantic salmon •Average weights 140g to 460g
•15 week (104 days)
•Four feeds
•Elevated temperature (19oC)
Analysis Traditional Methods
• Growth and weight measurements
• FAME by GC and GC-MS
Give information about the length and amount of double
bonds in the lipids
Destructive and non regiospecific
• Lipid Class by TLC-FID
Gives information about the type of lipid
Results•Over 45 FAME identified
•Relationship between Diet and FAME Profile
•Lipid Class 80-95%TAG, 8-14% PL
•No obvious relationship between Lipid class or FAME with diet and growth data
•Decrease in PUFA at higher temperature•FAME
–Shift in the ratio of PUFA/SFA–Reduction in the accumulation or production on PUFA at higher temperatures
0
0.5
1
1.5
2
2.5
10C 11C 12C 19C
Diet
Muscle
PUFA/SFA ratio for muscle & diets of Atlantic Salmon at different temperatures
12oC Results taken from Bransden et al (2003), 11oC from Bell et al (2003), 10oC from Bell et al (2004) and 19oC from Julia’s protein/energy trial
PUFA
/SF
A
Analysis New Methods
Why? –Regiospecific non destructive
LC-MS–Structural (polar) lipid analysis
–Functional
13CNMR –Storage (TAG) Lipid analysis
–Energy
Polar Lipids (PL)Phosphatidy-choline (PC) 18:0/22:6
Triacylglyerols (TAG)18:0/22:6/20:5
Polar lipids
•Phospholipids play a central role in the biochemistry of all
living cells
•Role in biochemistry is only partly understood
•Functional and structural properties
•Evolving area of research
•Respond in a graded manner
Major polar lipids in salmon membranes
PCPI
PS PE
Phospholipids
•Phosphatidylcholine (PC)
•Phosphatidylethanolamine
(PE)
•Phosphatidylinositol (PI)
•Phosphatidylserine (PS)
Sn1
Sn2
Electrospray Ionization Reversed-Phase Liquid Chromatography-Mass Spectrometry (ESI RP-LC-MS)
•Direct, fast, convenient and sensitive technique
•Quantitative and Qualitative
•Regiospecificity is determined
•Very complex (20-50 FA x 2 regiospecific positions x 5 PL head groups)
1
2>5 x possible head groups
LC of a Polar lipid fraction
Major phosphatidylinositol components of red muscle in Atlantic salmon fed a High Protein Diet
Overall Outcomes•First Study•DHA and EPA preferred at the Sn2 position•Generally the larger and/or more unsaturated chain was on the Sn2 position•Major PL for Diet and salmon muscle - PC 16:0/22:6 •Major PL for Gills - PC 16:0/18:1
0%20%
40%
60%
Diet Gill WhiteTissue
Red Tissue
PC (16:0/ 22:6)PE (16:0/ 22:6)
Major phospholipids in salmon Major phospholipids in salmon By ESI-LC-MS of High Protein dietBy ESI-LC-MS of High Protein diet
13C Nuclear Magnetic Resonance (13C NMR)
•Regiospecific (why is it
important?)
•Determination of acyl chain by
chemical shift difference at the C1
position to determine location on
glycerol backbone
•Non destructive
•Very complex (20-50
components)
Triacylglyerol (TAG)18:0/22:6/20:5
C1
TAG Regiospecificity in Salmon% in the Sn2 positions
By 13C NMR spectroscopy
EPA
Gills 48.8
White Tissue 29.9
Red tissue 21.2
Diet 20.6
33% Probability for DHA in each position
DHA
Gills 58.3
White Tissue 58.5
Red tissue 61.8
Diet 66.6
TAG Regiospecificityfor Sn2 position
By 13C NMR spectroscopy
DHA EPA
Salmon White Tissue High
Protein Diet58.5 29.9
Tuna 53.7 44.7
Seal 3.9 7.9
Thraustochytrids 65.4 -
Theoretical proportion 33.3 33.3
Acknowledgementsand the assessment of alternative oils
•Julia Barnes UTAS Aquaculture PhD
•Evan Peacock CSL 13NMR Spectroscopy
•Noel Davies CSL LC-MS
•Skretting
Future PhD work
•Stearidonic acid (18:43) trial
•2005 Thraustochytrid & Temp trial
•2006 possible GM seed oils trial