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 Miller 1&3 Julia Barnes 1, Rhys Hauler 2, Chris Carter 1 & Peter Nichols 3 1 School of Aquaculture, TAFI, University of Tasmania, 2 Skretting, 3 CSIRO 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 20 o C 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) DHA (Docosahexaenoic Acid) 22:6  3 EPA (Eicosapentaenoic Acid) 20:5  3  Omega 3 represents first unsaturation from terminal methyl carbon  Important in human physiology and heath (& farmed fish)  First discovered in 1960s

The experiment Temperature and growth rate Threshold 20 o C Julia Barnes Trial Energy / Protein ratios Atlantic salmon Average weights 140g to 460g 15 week (104 days) Four feeds Elevated temperature (19 o C)

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 PUFA/SFA ratio for muscle & diets of Atlantic Salmon at different temperatures 12 o C Results taken from Bransden et al (2003), 11 o C from Bell et al (2003), 10 o C from Bell et al (2004) and 19 o C from Julia’s protein/energy trial PUFA/SFA

Analysis New Methods Why? –Regiospecific non destructive LC-MS –Structural (polar) lipid analysis –Functional 13 CNMR –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 PC PI 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 Major phospholipids in salmon By ESI-LC-MS of High Protein diet

13 C Nuclear Magnetic Resonance ( 13 C 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 13 C NMR spectroscopy EPA Gills48.8 White Tissue29.9 Red tissue21.2 Diet % Probability for DHA in each position DHA Gills 58.3 White Tissue58.5 Red tissue 61.8 Diet 66.6

TAG Regiospecificity for Sn2 position By 13 C NMR spectroscopy DHAEPA Salmon White Tissue High Protein Diet Tuna Seal Thraustochytrids65.4- Theoretical proportion33.3

Acknowledgements and the assessment of alternative oils Julia Barnes UTAS Aquaculture PhD Evan Peacock CSL 13 NMR Spectroscopy Noel Davies CSL LC-MS Skretting Future PhD work Stearidonic acid (18:4  3) trial 2005 Thraustochytrid & Temp trial 2006 possible GM seed oils trial