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