Fatty acid metabolism in barramundi (Lates calcarifer)
By
Mr Michael J. Salini
BSc (Hons)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
April, 2016
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Acknowledgements I’m going to keep this short because there are another ~200 pages after this one.
However, there are a few people that I need to mention for their support and
encouragement along the way. Firstly, I need to say that the most important person
that has supported and put up with me throughout this process is my wife Gaëlle. Je
ne sais pas comment tu as fait. Merci pour tout ton soutien pendant ces années très
difficiles et parfois compliquées. Je ne l’oublierai jamais Also, many thanks to my
family and Gaelle’s family for supporting both of us during my candidature.
During my PhD experience I enjoyed supervision by arguably two of the world’s
best fish nutritionists. This is something that I am proud of and constantly grateful
for. Their knowledge, expertise and professionalism have helped me to develop the
set of skills I wanted and worked hard for. I don’t need to say anything else about
Brett and Gio as their reputation precedes them everywhere in every aspect of their
lives. Thanks guys. I would like to thank the CSIRO Agriculture for supporting this
PhD both in-kind and through the PhD Top up scholarship award.
I would like to acknowledge all of my friends, family, colleagues and peers at the
CSIRO labs (formally at Cleveland then Ecoscience precinct and hopefully finally at
the Queensland Biosciences Precinct). These include among others: Bruno Araújo
(Brazil), Nick Bourne, Susan Cheers, Mat Cook, Jake Goodall, Nigel Preston and
Nick Wade.
Additionally, I would also like to acknowledge all of my friends, colleagues and
peers at the shared CSIRO and DAF research centre on Bribie Island (BIRC). These
people have helped me along in my aquaculture journey since 2003 and their
contributions are as varied as they are valued. These names are only a short list and
apologies for those who I have missed: Stuart Arnold, David Blyth, Natalie Habilay,
Simon Irvin, Nick Polymeris, Dylan Rylatt, Kinam Salee, Hazra Thaggard, Richard
Thaggard, Trevor Borchert, Leanda Maunder and David Poppi.
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Publications
Salini, M.J., Irvin, S.J., Bourne, N., Blyth, D., Cheers, S., Habilay, N., Glencross, B.D., 2015. Marginal efficiencies of long chain-polyunsaturated fatty acid use by barramundi (Lates calcarifer) when fed diets with varying blends of fish oil and poultry fat. Aquaculture. 449, 48-57.
Salini, M.J., Turchini, G.M., Glencross, B.G., 2015. Effect of dietary saturated and
monounsaturated fatty acids in juvenile barramundi Lates calcarifer. Aquac. Nutr. DOI:10.1111/anu.12389.
Salini, M.J., Wade, N.M., Bourne, N., Turchini, G.M., Glencross, B.D., 2016. The
effect of marine and non-marine phospholipid rich oils when fed to juvenile barramundi (Lates calcarifer). Aquaculture. 455, 125-135.
Salini, M.J., Turchini, G.M., Wade, N., Glencross, B.D., 2015. Rapid effects of
essential fatty acid deficiency on growth and development parameters and transcription of key fatty acid metabolism genes in juvenile barramundi Lates calcarifer. Br. J. Nutr. 114, 1784-1796.
Salini, M.J., Wade, N.M., Araújo, B.C., Turchini, G.M., Glencross, B.D., 2016. Eicosapentaenoic acid, arachidonic acid and eicosanoid metabolism in juvenile barramundi Lates calcarifer. Lipids. 51(8), 973-988
Salini, M.J., Poppi, D.A., Turchini, G.M., Glencross, B.D., 2016. Defining the
allometric relationship between size and nutrient turnover in barramundi Lates calcarifer. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 201, 79-86.
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Abstract The thesis presented herein is an adaptation of a series of published manuscripts. The
manuscripts have been peer reviewed and published in high standing journals
relevant to the field of aquaculture nutrition. The aims of the research were designed
to generate a thorough understanding of lipid metabolism in the barramundi (Lates
calcarifer); extending from a more general applied approach through to a complex
understanding of molecular mechanisms and lipid bioenergetics.
In the first experiment, a series of five diets with blends of fish (anchovy) oil and
poultry fat (F100:P0, F60:P40, F30:P70, F15:P85, F0:P100) were fed to 208 ± 4.1 g
barramundi over a 12-week period. The replacement of fish oil (FO) with poultry fat
had no impact on growth performance (average final weight of 548.3 ± 10.2 g) or
feed conversion (mean = 1.14 ± 0.02). Analysis of the whole body composition
showed that the fatty acid profile reflected that of the fed diet. However, it was also
shown that there was a disproportional retention of some fatty acids relative to others
(notably 18:2n-6 and 18:3n-3). By examining the body mass independent retention of
different fatty acids with differential levels of intake of each, the marginal
efficiencies were able to be determined. The differential retention of fatty acids in the
meat was also examined allowing the determination of oil blending strategies to
optimise meat n-3 long chain – polyunsaturated fatty acid (LC-PUFA) levels.
In the second experiment, the response of juvenile barramundi (47.0 g/fish initial
weight) fed isolipidic and isoenergetic diets with 8.2% added oil was tested. The
experimental test diets were a 2:1 or 1:2 ratio of saturated to monounsaturated fatty
acids (SFA-D and MUFA-D respectively) compared to a control diet (CTRL-D) fed
for eight weeks. The diets containing mostly olive oil (diet MUFA-D) and mostly
refined palm oil (diet SFA-D) did not impact the growth performance or feed
utilisation parameters of the barramundi. The in vivo beta-oxidation activity was
consistent with the diet fatty acid composition with the most dominant fatty acids
being heavily beta-oxidised. Together, the in vivo whole body mass-balance of fatty
acids showed that n-3 long chain polyunsaturated fatty acids (LC-PUFA) were most
efficiently utilised in the SFA-D and MUFA-D fed fish. This study provides
evidence that additional dietary MUFA and SFA are suitable fatty acid classes for
juvenile barramundi and they are both equally efficient at sparing LC-PUFA from an
oxidative fate.
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In the third experiment, the response of juvenile barramundi to four diets containing
either marine- or non-marine derived neutral lipid (NL) or polar lipid (PL) sources
was assessed for eight weeks in a 2x2 factorial design. The four diets contained 8.2%
added lipid composed of a 1% fish oil base with 7.2 % test lipid (n-3 NL: Fish oil, n-
3 PL: Krill oil, n-6 NL: Soybean oil, n-6 PL: Soybean lecithin). The results
demonstrated that the different lipid sources (either n-3 or n-6 omega series from
either NL or PL class) had significant effects on growth performance and feed
utilisation with some interaction terms noted. Growth was negatively affected in the
n-6 NL fish and the feed conversion ratio was highest in the n-6 PL fish. Digestibility
of total lipid and some specific fatty acids (notably 18:2n-6 and 18:3n-3) were also
negatively affected in the n-6 PL fish. Analysis of the whole body NL fatty acid
composition showed that these mirrored those of the diets and significant interaction
terms were noted. However, the whole body PL fatty acids appeared to be more
tightly regulated in comparison. The blood plasma biochemistry and hepatic
transcription of several fatty acid metabolism genes in the n-6 PL fed and to a lesser
extent in the n-6 NL fed fish demonstrated a pattern consistent with modified
metabolic function. These results support that there are potential advantages in using
phospholipid-rich oils, however, there are clear differences in terms of their origin.
In the fourth experiment, the essential fatty acid requirement (EFA) of juvenile
barramundi (47.0 g/fish initial weight) fed isolipidic and isoenergetic diets with 8.2%
added oil was tested. The experimental test diets were either devoid of FO, and thus
with no n-3 LC-PUFA (FO FREE diet) or with a low inclusion of FO (FO LOW
diet). These were compared against a control diet containing only FO (FO CTRL
diet) as the added lipid source, over an eight week period. Interim samples and
measurements were taken fortnightly during the trial in order to define the aetiology
of the onset and progression of EFA deficiency. After two weeks, the fish fed the FO
FREE and FO LOW diets had significantly lower live-weights and after eight weeks
significant differences were detected for all performance parameters. The fish fed the
FO FREE diet also had a significantly higher incidence of external abnormalities.
The transcription of several genes involved in fatty acid metabolism was affected
after two weeks of feeding, showing a rapid nutritional regulation. This experiment
documents the aetiology of the onset and the progression of EFA deficiency in
juvenile barramundi and demonstrates that such deficiencies can be detected within
two weeks.
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In the fifth experiment, barramundi (initial weight = 10.3 ± 0.03 g; mean ± S.D.) fed
one of five diets with graded levels of EPA (1, 5, 10, 15 and 20 g/kg) or one of four
diets with graded levels of ARA (1, 6, 12 and 18 g/kg) were compared against a
control diet containing fish oil. A six-week feeding trial demonstrated that the
addition of EPA or ARA did not impact growth performance or feed utilisation.
Analysis of the whole body fatty acids showed that these reflected those of the diets.
The ARA retention demonstrated an inversely related curvilinear response to either
EPA or ARA. The marginal utilisation efficiencies of EPA and ARA were higher
than expected (62.1 and 91.9% respectively) and a dietary ARA requirement was
defined (0.012 g/kg0.796/d) in juvenile barramundi. The partial cDNA sequences of
genes regulating eicosanoid biosynthesis were identified in barramundi tissues,
namely cyclooxygenase 1 (Lc COX1a, Lc COX1b), cyclooxygenase 2 (Lc COX2) and
lipoxygenase (Lc ALOX-5). Both Lc COX2 and Lc ALOX-5 expression in the liver
tissue were elevated in response to increasing dietary ARA, meanwhile expression
levels of Lc COX2 and the mitochondrial fatty acid oxidation gene Lc CPT1a were
elevated in the kidney. A low level of EPA increased the expression of Lc COX1b in
the liver. Consideration should be given to the EPA to ARA balance for juvenile
barramundi in light of the nutritionally inducible nature of the cyclooxygenase and
lipoxygenase enzymes.
A sixth and final experiment was conducted with barramundi to examine the
allometric scaling effect of individual fatty acids. Six treatment size classes of fish
were deprived of food for 21 days (Treatment A, 10.5 ± 0.13 g; Treatment B, 19.2 ±
0.11 g; Treatment C, 28.3 ± 0.05 g; Treatment D, 122.4 ± 0.10 g; Treatment E, 217.6
± 0.36 g; Treatment F, 443.7 ± 1.48 g; mean ± SD) with each treatment comprising
fifteen fish, in triplicate. The assessment of somatic losses of energy and lipid were
consistent with previous studies, validating the methodology to be extended to
individual fatty acids. Live-weight (LW) exponent values were determined to be
0.817 ± 0.010 for energy and 0.895 ± 0.007 for lipid. There were significant
differences among the fatty acids ranging from 0.687 ± 0.005 for 20:5n-3 and 0.954
± 0.008 for 18:1n-9. The LW exponent values were applied to existing fatty acid
intake and deposition data of barramundi fed with either 100% fish oil or 100%
poultry oil. From this the maintenance requirement for each fatty acid was
determined by extrapolation of the linear regression. The metabolic demands for
maintenance and growth were then iteratively determined for fish over a range of
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size classes. Application of these exponent values to varying levels of fatty acid
intake demonstrated that the biggest driver in the utilisation of fatty acids in this
species is deposition demand and despite their reputed importance, the long-chain
polyunsaturated fatty acids had nominal to no maintenance requirement.
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Contents
Acknowledgements ...................................................................................................... 3
Publications .................................................................................................................. 4
Abstract ........................................................................................................................ 5
List of tables ............................................................................................................... 13
List of figures ............................................................................................................. 17
List of acronyms ......................................................................................................... 20
General Introduction ................................................................................................... 21
1.1 Introduction ....................................................................................................... 21
1.2 The role of lipids in fish nutrition ..................................................................... 25
1.2.1 Lipid classes in fish tissues ........................................................................ 25
1.2.2 Fatty acid biochemical structure ................................................................ 25
1.2.3 Minimum and optimum lipid nutrition ...................................................... 26
1.2.4 Alternative oils ........................................................................................... 29
1.3 Barramundi aquaculture nutrition ..................................................................... 32
1.3.1 Macro nutrient requirements of barramundi .............................................. 32
1.3.2 Alternative oils for barramundi .................................................................. 33
1.3.3 Different fatty acid class use by barramundi ............................................. 34
1.3.4 Different lipid class use by barramundi ..................................................... 36
1.3.5 Essential fatty acids in barramundi ............................................................ 37
1.3.6 EPA, ARA and eicosanoid metabolism in barramundi .............................. 39
1.3.7 Allometric scaling effects of LC-PUFA .................................................... 40
1.4 Thesis structure ................................................................................................. 41
General methodology ................................................................................................. 42
2.1 Diet production ................................................................................................. 42
2.1.1 Ingredient preparation ................................................................................ 42
2.1.2 Diet preparation by screw press technology .............................................. 42
2.1.3 Diet preparation by extrusion technology .................................................. 42
2.1.4 Vacuum infusion of lipid ........................................................................... 43
2.2 Experimental methodology ............................................................................... 43
2.2 Ethical statement ........................................................................................... 43
2.3 Barramundi husbandry .................................................................................. 43
2.4 Sample collection techniques ........................................................................ 44
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2.5 Abnormalities and behaviour assessment ..................................................... 45
2.6.1 Digestibility analysis – settlement ............................................................. 45
2.6.2 Digestibility analysis - abdominal stripping .............................................. 46
2.7 Experimental analyses ...................................................................................... 46
2.7.1 Chemical analysis ...................................................................................... 46
2.8.1 Biochemical analysis – Lipid transesterification ....................................... 46
2.8.2 Biochemical analysis – Plasma chemistry ................................................. 47
2.9.1 Molecular analysis - Cloning of putative prostaglandin G/H synthase (COX) and arachidonate 5-lipoxygenase (LOX) genes ...................................... 48
2.9.2 Molecular analysis - Sequencing analysis ................................................. 48
2.9.3 Molecular analysis - RNA extraction and normalisation ........................... 49
2.9.4 Molecular analysis - Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) ............................................................... 49
2.10 Calculations ................................................................................................. 50
2.11 Statistical interpretation .............................................................................. 51
Experimental Chapters ............................................................................................... 53
3.0 Marginal efficiencies of long chain-polyunsaturated fatty acid use by
barramundi (Lates calcarifer) when fed diets with varying blends of fish oil and
poultry fat ................................................................................................................ 53
3.1 Aims .............................................................................................................. 56
3.2 Materials and Methods .................................................................................. 56
3.3 Results ........................................................................................................... 59
3.3.1. Growth performance and feed utilisation .................................................. 59
3.3.2. Nutrient marginal efficiencies ................................................................... 64
3.3.3. Fillet quality assessment ........................................................................... 64
3.4 Discussion ..................................................................................................... 68
4.0 Effect of dietary saturated and monounsaturated fatty acids in juvenile
barramundi Lates calcarifer ................................................................................... 74
4.1 Aims .............................................................................................................. 77
4.2 Materials and methods .................................................................................. 77
4.3 Results ........................................................................................................... 81
4.3.1 Growth and feed utilisation ........................................................................ 81
4.3.2 Biochemical analysis .................................................................................. 84
4.3.3 Mass-balance computations ....................................................................... 87
4.4 Discussion ..................................................................................................... 90
5.0 The effect of marine and non-marine phospholipid rich oils when fed to
juvenile barramundi (Lates calcarifer) ................................................................... 94
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5.1 Aims .............................................................................................................. 97
5.2 Materials and methods .................................................................................. 97
5.3 Results ......................................................................................................... 103
5.3.1 Growth and feed utilisation ...................................................................... 103
5.3.2 Biochemical analysis ................................................................................ 106
5.3.3 Gene expression ....................................................................................... 110
5.4 Discussion ................................................................................................... 113
6.0 Rapid effects of essential fatty acid deficiency on growth and development
parameters and transcription of key fatty acid metabolism genes in juvenile
barramundi Lates calcarifer ................................................................................. 118
6.1 Aims ............................................................................................................ 121
6.2 Materials and methods ................................................................................ 121
6.3 Results ......................................................................................................... 126
6.3.1 Growth and feed utilisation ...................................................................... 126
6.3.2 Biochemical analysis ................................................................................ 130
6.3.3 Clinical observations ................................................................................ 133
6.3.4 Sub-clinical parameters ............................................................................ 133
6.4 Discussion ................................................................................................... 137
7.0 Eicosapentaenoic acid, arachidonic acid and eicosanoid metabolism in juvenile
barramundi Lates calcarifer ................................................................................. 141
7.1 Aims ............................................................................................................ 144
7.2 Materials and methods ................................................................................ 144
7.3 Results ......................................................................................................... 150
7.3.1 Growth performance and feed utilisation ................................................. 150
7.3.2 Digestibility analysis of the diets ............................................................. 153
7.3.3 Whole-body composition. ........................................................................ 153
7.3.4 Fatty acid retention efficiency .................................................................. 157
7.3.5 Marginal utilisation efficiencies ............................................................... 157
7.3.6 Gene identification and quantitative expression ...................................... 162
7.4 Discussion ................................................................................................... 165
8.0 Defining the allometric relationship between size and nutrient turnover in
barramundi Lates calcarifer ................................................................................. 169
8.1 Aims ............................................................................................................ 172
8.2 Materials and methods ................................................................................ 172
8.3 Results ......................................................................................................... 172
8.3.1 Fish compositional changes ..................................................................... 172
8.3.2 Determination of metabolic live-weight exponents ................................. 179
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8.3.3 Metabolic demands for fatty acids ........................................................... 181
8.4 Discussion ................................................................................................... 186
Conclusions .............................................................................................................. 190
General conclusions ................................................................................................. 193
References ................................................................................................................ 195
Appendix - Supplementary figures ........................................................................... 212
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List of tables Table 1. 1 Current world production (MMT) and value (USD/tonne) data of major
and emerging food grade oils. .................................................................................... 29
Table 3. 1 Chemical composition of key ingredients. All data are g/kg DM unless
otherwise stated. Fatty acid data are expressed as a percentage of total fatty acids
(%). ............................................................................................................................. 57
Table 3. 2 Experimental diet formulation and composition. All data are g/kg DM
unless otherwise stated. Fatty acid data are expressed as a percentage of total fatty
acids (%). .................................................................................................................... 58
Table 3. 3 Growth, feed utilisation and nutrient deposition parameters. ................... 60
Table 3. 4 Whole body chemical composition of the experimental fish on a live-
weight basis. All data are g/kg unless otherwise stated. Fatty acid data are expressed
as a percentage of total fatty acids (%). ...................................................................... 62
Table 3. 5 Summary of marginal efficiencies of specific fatty acids by juvenile
barramundi when fed diets with varying blends of fish oil and poultry fat. .............. 65
Table 3. 6 Fillet (NQC) composition on a live-weight basis. All data are g/kg unless
otherwise stated. Fatty acid profiles of NQC samples from each treatment are
expressed as mg/100g meat. ....................................................................................... 66
Table 4. 1 Chemical composition of key ingredients, all values are presented as g/kg
DM unless otherwise stated. ....................................................................................... 78
Table 4. 2 Formulation and composition of experimental diets. All values are g/kg
DM unless otherwise stated. ....................................................................................... 79
Table 4. 3 Growth performance and feed utilisation of juvenile barramundi fed
experimental diets for eight weeks. All data (n=3 per treatment) are presented as
mean ± SEM. .............................................................................................................. 82
Table 4. 4 Apparent digestibility coefficients of macro nutrients and specific fatty
acids of experimental diets fed to juvenile barramundi. All data (n=3 per treatment)
are reported as apparent digestibility percentage (%) ................................................ 83
Table 4. 5 Whole body composition (n=3 per treatment, g/kg live basis). Whole body
and liver fatty acid data (n=3 per treatment, % total). ................................................ 85
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Table 4. 6 Whole-body fatty acid mass balance computations of β-oxidation,
elongation and desaturation activity of juvenile barramundi fed experimental diets
for eight weeks. All data (n=3 per treatment) are reported on a nmol/g fish/d basis. 88
Table 4. 7 Calculated summary of LC-PUFA flux in juvenile barramundi. All data
are (n=3) reported as a percentage based on the total intake of each fatty acid (nmol/g
fish/d). ......................................................................................................................... 89
Table 5. 1 Chemical composition of ingredients used in experimental diets, all values
are g/kg DM unless otherwise stated .......................................................................... 98
Table 5. 2 Formulation and composition (as analysed) of experimental diets, all
values are g/kg DM unless otherwise stated. ........................................................... 100
Table 5. 3 Neutral and polar lipid composition of experimental diets, all values are
mg/g lipid unless otherwise stated. ........................................................................... 101
Table 5. 4 Real time quantitative PCR primer pairs for fatty acid metabolism and
control genes ............................................................................................................. 102
Table 5. 5 Growth and feed utilisation parameters of juvenile barramundi fed
experimental diets for eight weeks. Data (n=3) are presented as mean ± SEM. ...... 104
Table 5. 6 Apparent digestibility (%) parameters of the diets fed to juvenile
barramundi. Data (n=3) are presented as mean ± SEM. .......................................... 105
Table 5. 7 Neutral and polar lipid composition in the whole body of juvenile
barramundi fed experimental diets. All values are mg/g lipid unless otherwise
stated. Data (n=3) are presented as mean ± SEM ..................................................... 107
Table 5. 8 Whole body fatty acid balance calculations of β-oxidation, elongation and
desaturation of juvenile barramundi fed experimental diets for eight weeks. All
values are presented as nmol/g fish/d. Data (n=3) are presented as mean ± SEM. .. 109
Table 5. 9 Plasma chemistry of barramundi fed experimental diets for eight weeks.
Data (n=3) are presented as mean ± SEM. ............................................................... 111
Table 6. 1 Chemical composition of ingredients used in experimental diets, all values
are g/kg unless otherwise stated. .............................................................................. 122
Table 6. 2 Formulation and composition of experimental diets, all values are g/kg
DM unless stated. ..................................................................................................... 124
Table 6. 3 Real-time qPCR primer pairs for target genes involved in fatty acid
metabolism. .............................................................................................................. 125
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Table 6. 4 Growth performance and feed utilisation of barramundi fed experimental
diets for eight weeks. ................................................................................................ 127
Table 6. 5 Split-plot analysis of variance for repeated measures design of growth
performance and feed utilisation parameters in juvenile barramundi. ..................... 128
Table 6. 6 Apparent digestibility of macro nutrients and fatty acids present in the
experimental diets. .................................................................................................... 129
Table 6. 7 Initial and final fatty acid composition of whole-body and liver tissue from
juvenile barramundi. All fatty acid data are presented as percentage of total fatty
acids (%) unless otherwise stated. ............................................................................ 131
Table 6. 8 Plasma chemistry parameters in juvenile barramundi fed experimental
diets, sampled fortnightly for eight weeks. .............................................................. 134
Table 6. 9 Split-plot analysis of variance for repeated measures design of plasma
chemistry parameters in juvenile barramundi. ......................................................... 135
Table 7. 1 Composition of key ingredients used in diet formulations (g/kg DM). Fatty
acids are presented as mole percentage (mole%). .................................................... 145
Table 7. 2 Formulation and composition of experimental diets (g/kg DM). Fatty acids
are presented as mole percentages (mole%). ............................................................ 146
Table 7. 3 Forward and reverse primer pairs (5ʹ – 3ʹ) used in the cloning of
eicosanoid metabolism genes in barramundi and real-time qPCR gene expression
analysis. .................................................................................................................... 148
Table 7. 4 Growth performance and feed utilisation of barramundi fed increasing
EPA analysed using polynomial contrasts. .............................................................. 151
Table 7. 5 Growth performance and feed utilisation of barramundi fed increasing
ARA analysed using polynomial contrasts. ............................................................. 152
Table 7. 6 Apparent digestibility (%) of nutrients and fatty acids in diets analysed by
one-way ANOVA. .................................................................................................... 155
Table 7. 7 Whole body and fatty acid composition of barramundi analysed by one-
way ANOVA (g/kg live-basis). Fatty acids are presented as mole percentages
(mole%). ................................................................................................................... 156
Table 7. 8 Retention efficiency of selected fatty acids in barramundi fed either
increasing EPA or ARA analysed by polynomial contrasts. .................................... 159
Table 7. 9 Summary of maintenance demand and utilisation efficiencies by
barramundi fed either increasing EPA or ARA. ....................................................... 161
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Table 8. 1. Performance parameters and chemical composition for initial and final
barramundi of varying sizes. Data are presented as mean ± SEM and composition
data are presented on a wet-weight basis. ................................................................ 174
Table 8. 2 Fatty acid density in the fish (mg/g/fish) after 21 days of fasting. Data
were fitted to logarithmic functions and presented as mean ± SEM. ....................... 175
Table 8. 3 Coefficient and exponent values derived from the power function (y=aXb)
of fatty acid loss over a wide range of fish sizes from ~10 g to ~440 g. Replication
was derived by manually bootstrapping each individual value and presented as mean
± SEM (n=18). .......................................................................................................... 180
Table 8. 4 Re-evaluation of the marginal efficiency of fatty acid utilisation in
barramundi. Data were transformed to LW exponent values determined from the
present study. Maintenance requirements and intake to gain ratio for each fatty acid
are presented ............................................................................................................. 182
Table 8. 5 Fatty acid demands in growing barramundi fed either 100% fish oil (FO)
or 100% poultry oil (PO) diets maintained at 30 °C. Calculations are based on the
predictive growth models and utilisation efficiencies from published studies for this
species ...................................................................................................................... 184
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List of figures Figure 1.1 Fish oil use over the past four decades. Major growth of the aquaculture
and the nutraceutical sectors in recent years has changed the supply and demand
dynamics of FO. Its use in industrial or non-human consumption application is now
limited (data adapted from http://www.IFFO.org). .................................................... 22
Figure 1.2 Chemical structure of long-chain polyunsaturated fatty acids (LC-PUFA)
n-3 (EPA, left) and n-6 (ARA, right) with twenty carbon chain length. The five
double (ethylenic) bonds of EPA are in the 5, 8, 11, 14 and 17 positions and the four
double bonds of ARA are in the 5, 8, 11 and 14 positions relative to the carboxyl
group (adapted from http://www.IUPAC.org). .......................................................... 26
Figure 1.3 The pathways of LC-PUFA biosynthesis. The Δ12 and Δ15 desaturase
enzymes are only found in photosynthetic plants, hence the essentiality of 18 carbon
precursors in vertebrates for LC-PUFA biosynthesis (Tocher, 2010). The grey
pathway is an alternative proposed by Tu et al. (2012) for barramundi that bypasses
the initial Δ6 desaturation rate limiting step. The fatty acids in red font are the
biologically important end products for vertebrates. Δ, desaturase enzymes; Elovl,
Elongation of very long carbon chains enzymes; CS, chain shortening by β-
oxidation. .................................................................................................................... 28
Figure 1.4. Typical fatty acid profiles of oils used in aquaculture and other terrestrial
animal nutrition. Data are adapted from Belcher et al. (2011); Glencross (2009);
Glencross and Rutherford (2011) and NUTTAB 2010 www.foodstandards.com.au. 31
Figure 3. 1 Whole-body retention of polyunsaturated fatty acids a) 18:2n-6 (y =
0.03x + 0.49, R2 = 0.13, P = 0.55) and b) 18:3n-3 (y = 0.65x + 0.10, R2 = 0.60, P =
0.13) and whole-body retention of long-chain polyunsaturated fatty acids c) ARA (y
= -1.08x + 1.03, R2 = 0.81, P < 0.05) , d) EPA (-0.04x + 0.52, R2 = 0.90, P < 0.05)
and e) DHA (y = 0.72x-0.44, R2 = 0.97, P < 0.05). ................................................... 63
Figure 3. 2 The linear relationship between the mass-independent intake relative to
mass-independent gain (marginal efficiency) of specific fatty acids by juvenile
barramundi. For comparative purposes the dotted line indicates the slope of 1.0 (y =
x). a) 18:2n-6 (y = 0.82x - 0.03, R2 = 0.75) b) 18:3n-3 (y = 1.04x - 0.01, R2 = 0.88)
c) ARA (y = 0.19x + 0.005, R2 = 0.43) d) EPA (y = 0.30x + 0.007, R2 = 0.95) e)
DHA (y = 0.27x + 0.01, R2 = 0.95). .......................................................................... 67
Figure 3. 3 Fillet (NQC) LC-PUFA deposition in juvenile barramundi expressed
relative to intake (y = -1.54x2 + 56.33x + 75.23, R2 = 0.99) as mg/100 g meat. By
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extrapolation the dotted line indicates that a formulation of ~11% total LC-PUFA
(EPA and DHA combined) would achieve a fillet composition of 500 mg/100 g. .... 68
Figure 5. 1 Hepatic gene expression of selected lipid metabolism genes in juvenile
barramundi (Lates calcarifer) after eight weeks of feeding. Relative expression is
calculated for each gene using cycle threshold values, normalised to control genes
(Elongation factor 1a and Luciferase). Values are shown as log-2 fold change relative
to the initial fish. Letters above error bars indicate significant differences between the
treatments. Analysed by two-way factorial ANOVA, df 1,1,1,8, post-hoc Tukey's
HSD. ......................................................................................................................... 112
Figure 6. 1 (A-D) Mass-balance computations of fatty acid retention and β-oxidation
in juvenile barramundi, mean (±SEM) n=3. Saturated fatty acid (a; SFA) retention
F=82.5***, β-oxidation F=5.2*; Monounsaturated fatty acid (b; MUFA) retention
F=44.5***, β-oxidation F=7.0*; Polyunsaturated fatty acid (c; PUFA) retention
F=10.8*, β-oxidation F=1.4, P=0.32; Long-chain polyunsaturated fatty acid (d; LC-
PUFA) retention F=6.1*, β-oxidation F=241.5***. Significant differences are
indicated by letters (*, P<0.05; **, P<0.01; ***, P<0.001), one - way ANOVA df
2,6., post-hoc Tukey’s HSD. .................................................................................... 132
Figure 6. 2 (A-F) Expression of lipid metabolism genes in the liver of juvenile
barramundi. All data are normalised to EF1α and Luc reference genes, log 2
transformed and expressed relative to the initial fish (WK 0), mean (±SEM) n=6. The
FO CTRL (control) groups are indicated by light bars and the FO FREE groups are
indicated by dark bars. Two-way repeated measures ANOVA, df of residuals 10,30.
Lc ACYL, Diet F=15.5**, Week F=2.3, P=0.09, Diet:Week F=1.0, P=0.39; Lc
CPT1a, Diet F=0.2, P=0.69, Week F=0.6, P=0.65, Diet:Week F=06, P=0.62; Lc
FAS, Diet F=48.5***, Week F=1.4, P=0.28, Diet:Week F=0.8, P=0.50; Lc SCD,
Diet F=126.6***, Week F=0.6, P=0.62, Diet:Week F=0.6, P=0.62; Lc ELOVL5,
Diet F=0.2, P=0.66, Week F=0.4, P=0.78, Diet:Week F=0.2, P=0.92; Lc FADS2,
Diet F=75.4***, Week F=0.7, P=0.59, Diet:Week F=0.2, P=0.91. Please refer to
Table 6.3 for individual gene details. ....................................................................... 136
19 | P a g e
Figure 7. 1 Specific fatty acid retention efficiency by barramundi fed increasing EPA
(a-e) or ARA (f-j). The control (FO CTRL) fed fish are represented in each Figure
with a triangle (∆). Bars indicate standard error means (n=3). ................................ 158
Figure 7. 2 Marginal utilisation efficiency assessments of 20:5n-3 (a) and 20:4n-6 (b)
gain with varying intake by juvenile barramundi. Efficiency functions are described
by the linear regression for 20:5n-3 gain y = 0.621x + 0.0003, R2 = 0.975 and 20:4n-6
gain y = 0.919x - 0.011, R2 = 0.965). ........................................................................ 160
Figure 7. 3 Eicosanoid pathway and mitochondrial fatty acid oxidation gene
expression in the liver (a) and kidney (b) of juvenile barramundi fed increasing EPA.
Gene expression is normalised to the EF1α and Luc reference genes and expressed
relative to the control fish. Data were analysed by students t-test with significant
differences defined as P<0.05. ................................................................................. 163
Figure 7. 4 Eicosanoid pathway and mitochondrial fatty acid oxidation gene
expression in the liver (a) and kidney (b) of juvenile barramundi fed increasing ARA.
Gene expression is normalised to the EF1α and Luc reference genes and expressed
relative to the control fish. Data were analysed by students t-test with significant
differences defined as P<0.05. ................................................................................. 164
Figure 8. 1 Energy density of barramundi of varying live-weight before
(●=4.221(±0.010)x0.088(±0.001), R2 = 0.845) and after
(○=3.359(±0.011)x0.118(±0.001), R2 = 0.844) fasting for 21 days. ........................ 176
Figure 8. 2 Lipid density of the barramundi of varying live-weight before
(●=0.807(±0.019)ln(x) + 2.312(±0.002), R2 = 0.711) and after (○=0.981(±0.014)ln(x)
– 0.083(±0.003), R2 = 0.744) fasting for 21 days. .................................................... 177
Figure 8. 3 Fatty acid density (mg/g lipid) in barramundi of varying live-weight after
fasting for 21 days. Values were fitted to a logarithmic curve and equations are
presented in Table 8.2. ............................................................................................. 178
Figure 8. 4 Fatty acid (A and B) loss in fasted barramundi of varying live-weight.
Data are (n=3) mean ± SEM. Values were fitted to a power function and equations
are presented in Table 8.3. ........................................................................................ 183
20 | P a g e
List of acronyms ADC Apparent digestibility coefficient ANF Anti-nutritional factor ANOVA Analysis of variance ARA Arachidonic acid cDNA Complementary deoxyribonucleic acid COX Cyclooxygenase (prostaglandin G/H synthase) DHA Docosahexaenoic acid DNA Deoxyribonucleic acid DPA Docosapentaenoic acid DP:DE Digestible protein to digestible energy ratio EFA Essential fatty acid EPA Eicosapentaenoic acid FAO Food and agricultural organisation FI:FO Fish in : Fish out ratio FM Fish meal FO Fish oil IUPAC International Union of Physical and Applied Chemists KO Krill oil LC-PUFA Long chain - polyunsaturated fatty acid LOX Lipoxygenase (arachidonate 5-lipoxygenase) MUFA Monounsaturated fatty acid NL Neutral lipid OLA Oleic acid PCR Polymerase chain reaction PF Poultry fat PL Polar lipid PO Palm oil PtdCho Phosphatidylcholine PtdEtn Phosphatidylethanolamine PtdIns Phosphatidylinositol PtdSer Phosphatidylserine PUFA Polyunsaturated fatty acid qRT-PCR Quantitative real time reverse transcription - polymerase chain reaction RNA Ribonucleic acid SEM Standard error of the mean SFA Saturated fatty acid SGR Specific growth rate SL Soybean lecithin SO Soybean oil STA Stearidonic acid TAG Triacylglycerol TGC Thermal growth coefficient WBFABM Whole-body fatty acid balance method
21 | P a g e
General Introduction
1.1 Introduction
The world supply of wild capture seafood is unable to meet the needs of the growing
human population. Aquaculture continues to be a rapidly expanding industry
potentially able to address this problem. However, aquaculture in itself is only part of
the solution. Over the last three decades world aquaculture production has grown at
an average rate of 8.8% per annum and for the first time in 2010 exceeded 60 million
tonnes of edible product (FAO, 2012). Commercially produced diets are feeding this
rapid expansion which ironically has an insatiable appetite for wild fishery resources
already stretched to their limits. This resource is primarily based on small pelagic
forage fish (eg. sardines, herrings, anchoveta, mackerel) targeted specifically for
rendering into fish meal (FM) and fish oil (FO) which can be used in dietary
formulations for compound feeds. There is strong debate over the fish-in to fish-out
ratio (FI:FO) of aquaculture (Naylor et al., 2009; Tacon and Metian, 2008) and also
the transformation of directly edible fishery products into higher value species
(Naylor et al., 2000), hence the urgency to find and use alternative ingredients for
aquaculture.
Fish and crustaceans grown on commercially produced feeds consume a large
proportion of the world’s available FM and FO resources. It has become clear that if
aquaculture is to continue to grow at such levels then alternative plant and animal
resources must be utilised as feed ingredients. Each year the world supply of FO is
mostly consumed by the aquaculture industry while the nutraceutical industry
acquires more FO than ever before compounding the issue (Figure 1.1). As FM and
FO are finite resources their price and availability relative to alternative ingredients
will influence their eventual use in aquafeeds. However, for the use of alternative
ingredients to be a success, a clear understanding of the farmed species nutritional
requirements must be known (Hardy, 2010). The changing supply and demand
dynamics for FM and FO resources have led to record high prices putting further
emphasis on feed companies to reduce costs and become more sustainable.
Replacement of FM and FO in aquafeeds has been a research priority for
several decades. The use of many terrestrial plant and animal by-products is now
widely accepted as nutritionally important cost effective ingredients, however,
precaution is necessary in dietary formulation for a range of reasons including but
not limited to:
22 | P a g e
The health effects on certain farmed aquatic animals caused by anti-
nutritional factors (ANF) present in plant ingredients (Gatlin et al.,
2007).
Maintaining high levels of feed utilisation and production efficiencies
of farmed aquatic animals (Naylor et al., 2009)
The functionality of alternative ingredients in terms of their storage
and processing characteristics (Glencross et al., 2007)
Cost benefit, economic viability and supply risks associated with
transport and availability of ingredients to feed producers compared to
FM and FO (Gatlin et al., 2007)
Maintaining appropriate n-3/n-6 ratios for specific species to maintain
metabolic homeostasis (Brown and Hart, 2011)
Figure 1.1 Fish oil use over the past four decades. Major growth of the
aquaculture and the nutraceutical sectors in recent years has changed the
supply and demand dynamics of FO. Its use in industrial or non-human
consumption application is now limited (data adapted from
http://www.IFFO.org).
The quality of the diets provided to aquatic animals can contribute to
improved production efficiency leading to shorter grow out periods and potentially
reduced environmental effects, critical to the aquaculture industry (Turchini et al.,
2009). However, the quality of the diets is defined by their ingredients and
0
200
400
600
800
1000
1200
1400
1600
1800
1970 1975 1980 1985 1990 1995 2000 2005 2010
Nutraceutical
Industrial
Aquaculture
23 | P a g e
alternatives such as those derived from the sources mentioned above are generally
considered to be inferior to FM and FO and for a range of reasons (Glencross et al.,
2007). Certain nutrients present in FM and FO support ideal growth and health of
most fish and crustaceans and some of these are considered essential for normal
metabolic processes to occur (Gatlin et al., 2007).
The essentiality of a nutrient is often difficult to determine in any animal and
this is particularly true of fish. However, many research groups have demonstrated to
varying degrees how essential certain nutrients are to a range of different species
under different biotic and abiotic conditions. This information is of critical
importance to the aquafeed industry as they increase the use of non-marine based
ingredients. For example, most plant protein lacks an amino acid profile similar to
that of FM, often being deficient in the first limiting amino acids (eg. lysine and
methionine). If used in complete replacement to FM an obvious deficiency would
soon manifest in most fish culture systems. Despite these limitations,
supplementation with crystalline amino acids and blending different plant proteins
has allowed a gradual decrease in the total FM used for many species (Gatlin et al.,
2007).
Similarly, the lipid component of the diet is composed of essential fatty acids
(EFA) and non-essential fatty acids necessary for normal metabolic processes. For
example the complete replacement of FO with alternative oils from plant or animal
sources could lead to a deficiency as the essential fatty acids for that species may be
missing or in a disproportionate combination. Many researchers have investigated the
options for FO replacement in a range of species for two main reasons, the first being
the clearly limited supply of FO discussed earlier and secondly the fact that the lipids
remain the least well understood of all nutrients due to their complexity (Sargent et
al., 2002). More recently, research groups have approached the question of FM and
FO replacement simultaneously in an effort to gain an understanding of the
interactions of both ingredients (Glencross et al., 2016; Panserat et al., 2009; Refstie
et al., 2001; Torstensen et al., 2011).
The barramundi (Asian seabass: Lates calcarifer) is typically cultured in the
Indo-pacific region in relatively small tonnages compared to other species, however,
it is a culturally important and profitable industry that has potential to expand. In
light of the perceived image of global aquaculture as a net consumer of seafood
products, it is important that research continues into the search for and use of
alternative ingredients. As overall fish consumption increases, the demand on our
24 | P a g e
oceans will be far exceeded by what it can produce and the sustainable production of
fish for human consumption is expected to meet that need. Research in this area will
continue to improve dietary formulations and efficient use of available ingredients.
25 | P a g e
1.2 The role of lipids in fish nutrition
1.2.1 Lipid classes in fish tissues
With increasing efforts to replace FO from fish diets there is added emphasis on
understanding the fate of ingested lipid sources as this dietary nutrient is not only an
important energy source but it also has a bioactive role in many physiological
functions. Neutral lipid principally in the form of triacylglycerides (TAG) is the most
abundant form of ‘energy’ lipid in fish tissues. Its chemical structure is represented
by three fatty acids esterified to different positions on a glycerol backbone with
saturated (SFA) and monounsaturated fatty acids (MUFA) generally esterified to the
stereospecific numbering position one and three (sn-1,3) and polyunsaturated fatty
acids (PUFA) esterified to the sn-2 position (Tocher, 2003). Phosphoglycerides are
important polar lipids forming the bilayer of cell membranes. Structurally they have
two fatty acids esterified to phosphatidic acid (L-glycerol 3-phosphate). The major
phosphoglycerides of animal, including fish, tissues are phosphatidylcholine
(PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and
phosphatidylinositol (PtdIns) formed by esterification of the phosphatidic acid group
to different bases such as choline, ethanolamine, serine, and inositol respectively
(Tocher, 2003).
The relative proportion of each major fatty acid is generally related to its
origins in the environment. Northern hemisphere forage fish typically contain higher
levels of 20:1n-9 and 20:1n-11 long-chain monounsaturated fatty acids, directly
related to their diet of marine zooplankton rich in wax esters whereas Southern
hemisphere forage fish contain typically higher levels of long-chain polyunsaturated
fatty acids (LC-PUFA) (Sargent and Henderson, 1995). There are other lipid classes
such as wax esters (neutral lipid; NL), sphingolipids (polar lipid; PL) and sterols that
are important in vertebrate physiology, however, discussion about these is beyond
the scope of this review. In the context of feeding fish increasing amounts of
vegetable oils it has been demonstrated that different fatty acids have markedly
different fates. In Atlantic salmon (Salmo salar) the shorter chain SFA such as 10:0
is rapidly catabolised for energetic requirements, whereas the longer chain MUFA
18:1n-9 is deposited as TAG (Denstadli et al., 2011).
1.2.2 Fatty acid biochemical structure
Fatty acids are defined by the International Union of Physical and Applied Chemists
(IUPAC) as aliphatic monocarboxylic acids that can be hydrolysed from naturally
26 | P a g e
occurring lipids (http://www.IUPAC.org). Systematic chemical names are given to
each fatty acid and these can be symbolically delta (Δ) classified as X:YΔa, b, c
where X is the number of carbons in the chain, Y is the level of double (or ethylenic)
bonds and Δ a, b, c represent the position of each double bond relative to the
carboxyl group of the fatty acid (Rigaudy and Klesney, 1979). A different lipid
numbering system is widely used and accepted whereby the number of carbon atoms
and double bonds are separated by a colon followed by the position of the first
double bond relative to the methyl terminus represented as n minus x (Glencross,
2009; Tocher, 2003; Turchini et al., 2009). Trivial common names are also applied to
fatty acids and they can be further abbreviated. Therefore in the order summarised
above 5,8,11,14-Icosatetraenoic acid is known also as 20:4Δ5,8,11,14, 20:4n-6,
arachidonic acid or ARA (Figure 1.2).
Figure 1.2 Chemical structure of long-chain polyunsaturated fatty acids (LC-PUFA) n-3 (EPA, left) and n-6 (ARA, right) with twenty carbon chain length. The five double (ethylenic) bonds of EPA are in the 5, 8, 11, 14 and 17 positions and the four double bonds of ARA are in the 5, 8, 11 and 14 positions relative to the carboxyl group (adapted from http://www.IUPAC.org).
1.2.3 Minimum and optimum lipid nutrition
Defining the role of lipids is a challenging area of research because of the complex
interaction between cellular function and various lipid types (Dowhan et al., 2008).
27 | P a g e
Dietary lipids are well known to affect the performance and overall nutritional
quality of fish and this can easily be demonstrated in vivo (Sargent et al., 2002). The
lipid requirements in fish differs among species and life stages, whereby faster
growing juvenile fish have higher essential fatty acid (EFA) demands for neural
tissue development and other physiological processes (Glencross, 2009), while in
some species the requirement for EFA appears to be proportional to the total dietary
lipid (Glencross et al., 2002).
A recent study by Turchini et al. (2011a) demonstrated that the minimum and
optimum supply of EFA in salmonid fish is very different. These authors
demonstrated that in fact salmonid fish can satisfy their EFA requirement provided
the appropriate amount of precursor fatty acid is present, in this case 18:3n-3
(linolenic acid; Fig. 1.3). In their study the fish fed on a linseed oil based diet grew at
the same rate as those fed FO, highlighting the moral dilemma between providing a
sustainably cultured product with the increasing consumer demand for the
consumption of LC-PUFA. On the other hand, many studies have shown that marine
fish are not able to meet their EFA requirements through endogenous biosynthesis
from dietary precursors as they lack at least one of the enzymes required (Sargent et
al., 2002). There are few exceptions to this fact; however, marine species of the
Siganidae family are almost exclusively herbivorous and are able to desaturate and
elongate dietary precursors to meet their EFA needs (Monroig et al., 2012). Some
research groups now argue that trophic level is more important in defining a fishes
capacity to synthesise polyunsaturated fatty acids (PUFA) (Li et al., 2010).
The physiological role of lipids in fish becomes increasingly apparent when
the minimum or maximum limits are approached for an individual species. A range
of studies on cultured marine species including Atlantic cod (Gadus morhua)
(Morais et al., 2001), barramundi (Williams et al., 2003), white seabass (Atractoscion
nobilis) (Lopez et al., 2009), Japanese seabass (Lateolabrax japonicas) (Ai et al.,
2004), European seabass (Dicentrarchus labrax) (Peres and Oliva-Teles, 1999) and
gilthead seabream (Sparus aurata L.) (Santinha et al., 1999) have shown that optimal
lipid levels are between 10-20% and growth is energy dependant. In addition, there is
little or no evidence of a protein sparing effect by lipids in marine species, differing
markedly from salmonids (Hillestad and Johnsen, 1994). Studies have also drawn
attention to increased body fat as a result of increased dietary lipid in fish as
28 | P a g e
Figure 1.3 The pathways of LC-PUFA biosynthesis. The Δ12 and Δ15
desaturase enzymes are only found in photosynthetic plants, hence the
essentiality of 18 carbon precursors in vertebrates for LC-PUFA biosynthesis
(Tocher, 2010). The grey pathway is an alternative proposed by Tu et al. (2012)
for barramundi that bypasses the initial Δ6 desaturation rate limiting step. The
fatty acids in red font are the biologically important end products for
vertebrates. Δ, desaturase enzymes; Elovl, Elongation of very long carbon
chains enzymes; CS, chain shortening by β-oxidation.
having downstream consequences for seafood processors and human health benefits
(Hillestad and Johnsen, 1994; Peres and Oliva-Teles, 1999; Williams et al., 2003).
Therefore, the significance of dietary lipids cannot be overlooked particularly
when dietary formulations are encroaching on the limits for each species. In
barramundi, it has been well documented that the digestible protein to digestible
energy ratio (DP:DE) decreases as the fish grow; however, they are not capable of
Elovl5 Δ5
Δ6 Δ12
Δ9
18:0
18:1n-9 18:2n-9
20:2n-9 20:3n-9
22:6n-3
CS Δ6 Elovl5
Δ5 Elovl5/2
Elovl2 Δ6
18:3n-3 18:4n-3
20:4n-3 20:5n-3
22:5n-3 24:5n-3
24:6n-3
20:3n-3
Elovl5
Δ6/8
Δ15
22:5n-6
CS Δ6 Elovl5
Δ5 Elovl5/2
Elovl2 Δ6
18:2n-6 18:3n-6
20:3n-6 20:4n-6
22:4n-6 24:4n-6
24:5n-6
20:2n-6
Elovl5
Δ6/8
29 | P a g e
utilising high dietary lipid as a primary energy source when protein is not adequately
provided (Williams et al., 2003). Likewise, when levels of lipid are too low
barramundi begin to exhibit symptoms of a nutrient deficiency (Catacutan and
Coloso, 1995).
1.2.4 Alternative oils
Along with the many advantages of using alternative lipid sources in aquafeeds there
are some important issues that arise. Most notable are the direct health impacts on the
cultured species when an imbalance or deficiency exists and then on the final product
quality for human consumption. For further information on the final product quality
aspects of fish nutrition refer to Turchini et al. (2009).
The range of lipids being explored for potential use in animal nutrition is
expanding. Traditionally, terrestrial oils with abundant n-3 and n-6 PUFA derived
from oil seeds have been extensively researched in a wide range of vertebrate models
largely due to their availability and price (Table 1.1).
Table 1. 1 Current world production (MMT) and value (USD/tonne) data of major and emerging food grade oils.
Major lipid sources Production Value
Fish oil1 1 2650
Soybean oil 43179 1076
Canola oil2 23798 1160
Olive oil3 2869 3960
Palm oil 53827 770
Rice bran oil ~0.5 N/A
Sunflower oil 13748 1460
Linseed oil 0.5 1671
Data are from USDA 2013; www.indexmundi.com; NUTTAB 2010 www.foodstandards.com.au. 1Crude fish oil, production data 2009, value based on FOB vessel in drums 2Also rapeseed oil 3Extra virgin
Generally, the plant oils contain a mix of SFA, MUFA and PUFA as either
16:0 (palmitic acid), 18:0 (stearic acid), 18:1n-9 (oleic acid; OLA), 18:2n-6 (linoleic
acid) and 18:3n-3 (linolenic acid) and they are characteristically devoid of any LC-
PUFA (Fig. 1.4). In addition to these, less common are plant oils that contain high
30 | P a g e
levels of 18:4n-3 (stearidonic acid; STA). STA in the human diet allows the
bypassing of the first rate limiting step of eicosapentaenoic acid (EPA) biosynthesis,
however, its commercial availability is limited (James et al., 2003) and its use in fish
diets has so far proven to be of limited value (Alhazzaa et al., 2011a; Tocher et al.,
2006b). More recently other products rich in LC-PUFA, including single cell
heterotrophs such as Schizochytrium sp. (Miller et al., 2007), Crypthecodinium sp
(Harel et al., 2002), Phaeodactylum tricornutum (Atalah et al., 2007) have been used
in FO replacement studies in fish. Furthermore, DuPont Applied Biosciences have
genetically modified a yeast species (Yarrowia lipolytica) to contain high levels of
EPA in its lipid content (Fig. 1.4) that is yet to be evaluated in fish (Belcher et al.,
2011; Gillies et al., 2012).
In addition to these existing alternatives, genetically modified oil seeds are a
promising alternative to meet the growing LC-PUFA demands and several plant
varieties now exist or are under development (Alonso and Maroto, 2000; Nichols et
al., 2010; Robert, 2006). Researchers have been able to develop high- EPA and high-
docosahexaenoic acid (DHA) producing cultivars of model species such as Nicotiana
benthemiana (Petrie and Singh, 2011) and Arabidopsis thaliana (Petrie et al., 2012)
by gene transfer from algae, effectively extending the natural biosynthetic pathways
found in plants. Commercially relevant production of LC-PUFA rich oil from
transgenic Camelina sativa was recently made available (Mansour et al., 2014; Ruiz-
Lopez et al., 2014) and evaluated in mice and fish without detrimental effects on
performance (Betancor et al., 2015; Tejera et al., 2016). These evolving technologies
are set to greatly improve the sustainability of aquaculture yet many challenges still
exist including approvals and consumer acceptance.
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Figure 1.4. Typical fatty acid profiles of oils used in aquaculture and other terrestrial animal nutrition. Data are adapted from Belcher et al. (2011); Glencross (2009); Glencross and Rutherford (2011) and NUTTAB 2010 www.foodstandards.com.au.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Anch
ovy
Cod
Live
r
Squi
d
Krill
ARA
Aqua
gro
EPA
yeas
t
DHA
Aqua
gro
Bovi
ne
Ovi
ne
Avia
n
Porc
ine
Cano
la
Lins
eed
Oliv
e
Palm
Rice
bra
n
Soyb
ean
Sunf
low
er
OLA LOA LNA ARA EPA DHA OTHER
Marine oils Algal & yeast oils
Animal oils Plant oils
32 | P a g e
1.3 Barramundi aquaculture nutrition
1.3.1 Macro nutrient requirements of barramundi
Macro nutrient requirements in barramundi have been determined over a range of
size classes and environmental conditions. Past studies have reached different
conclusions about the dietary strategies to improve production efficiency of
barramundi. Williams et al. (2003) reported that there was a tendency to spare
protein for growth particularly in juvenile barramundi whereas Catacutan and Coloso
(1995) found no protein sparing effect. In agreement, these authors also reported that
the most appropriate protein to energy ratio was in the range of 25 -27 mg/kJ.
However, they reported vastly different optimal nutrient specifications with 42.5%
and 10% (Catacutan and Coloso, 1995) and 60.3% and 18% (Williams et al., 2003)
protein and lipid respectively. In addition, Glencross and Bermudes (2012)
demonstrated that optimal DP:DE ratio decreased with increasing size of juvenile
barramundi. They also demonstrated an increased DP:DE requirement at higher
temperatures caused by decreased utilisation efficiency highlighting the importance
of different dietary strategies to improve production.
An early study into the use of dietary carbohydrate in barramundi found that
provided that dietary lipid was high then a high level of carbohydrate supported the
best performance (Catacutan and Coloso, 1997). However, in this study the diets
were not isoenergetic, nutrient dense nor were they formulated on a digestible basis.
Moreover, the pellets were produced using cold-screw press technology that would
not permit the complete gelatinisation of starch therefore reducing the energy
digestibility of the diets (Glencross et al., 2011a). Recent studies have shown that
barramundi actually have a poor ability to digest starch (Glencross et al., 2012).
These authors found that among a range of cereals commonly used in aquafeed,
wheat starch is one of the least digestible with a digestible value of only 9.6 kJ/g.
Studies in other carnivorous fish such as rainbow trout have demonstrated that the
value of carbohydrate is limited as a growth nutrient; however, it does have value as
an energy source (Saravanan et al., 2012). A similar response was seen in
barramundi, suggesting that they have a preference for metabolizable energy in the
form of protein or lipid and they derive little value from carbohydrate in terms of
growth (Glencross et al., 2014a).
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1.3.2 Alternative oils for barramundi
The replacement of FO with alternative oil sources continues to be a high priority for
aquafeed production worldwide. The barramundi is an obligate carnivorous fish
central to an expanding aquaculture industry in the Indo-Pacific region with a
reported LC-PUFA requirement in juvenile fish of around 1.2% (Williams et al.,
2006). However, variable responses of barramundi to FO replacement studies have
led to questions being raised about the upper limits of FO substitution in this species
(Glencross and Rutherford, 2011; Morton et al., 2014; Raso and Anderson, 2003; Tu
et al., 2013). Poultry fat (PF) is produced as a by-product of the chicken processing
industry and is characterised by its high 18:1n-9 and 18:2n-6 content (Turchini et al.,
2009). Poultry fat is commonly used to replace FO in fish diets providing an
excellent source of energy, however, it is characterised by a lack of n-3 LC-PUFA
(Turchini et al., 2009).
Depletion of LC-PUFA in the diet of barramundi can potentially lead to
reduced productivity and the onset of essential fatty acid deficiency symptoms
(Catacutan and Coloso, 1995; Glencross and Rutherford, 2011; Williams et al.,
2006). Moreover, a lack of dietary LC-PUFA will also likely be reflected in the flesh,
diminishing the human nutritional value of the product (Turchini et al., 2009). It is
well established that the regular consumption of food rich in n-3 LC-PUFA is a
fundamental part of a balanced diet and the Food and Agricultural Organisation
(FAO) recommend the consumption of 250 to 2000 mg/d (EPA and DHA) for adults
(FAO, 2010).
Highly variable or disproportionate retention of lipid or indeed specific fatty
acids may indicate metabolic changes as a direct result of the fed diet. Thomassen et
al. (2012) found that Atlantic salmon consuming a diet containing rapeseed oil with
supplemental EPA (20:5n-3) oil had significantly higher DPA (22:5n-3) retention via
elongation pathways. Moreover, these fish selectively retained DHA (22:6n-3)
efficiently and in absolute terms the proportion of DHA was significantly improved
when compared to those fish fed only rapeseed oil with no supplemental EPA.
Similarly, a dramatic reduction of LC-PUFA retention was demonstrated in both
barramundi and Atlantic salmon as dietary LC-PUFA increased (Glencross and
Rutherford, 2011; Glencross et al., 2014b). In contrast, Atlantic cod retained more
LC-PUFA as intake increased (Hansen et al., 2008).
Efficient feed utilisation has a determinant effect on costs and outputs in
aquaculture systems. In economics, a future return on an investment is estimated
34 | P a g e
based on financial inputs and is termed the marginal efficiency of capital (Kalecki,
1937). Similarly, this concept can be applied in aquaculture nutrition in order to
better understand the relationship between dietary inputs and fish outputs over time.
It differs from deposition or retention in that it is not just a mass-balance model but
rather is a bioenergetic approach based on the weight independent relationships
between the intake and gain of a specific nutrient. The exact fate of ingested nutrients
is difficult to measure, however, calculation of the marginal (partial) efficiency can
provide a clearer understanding of the discrete contributions of a dietary nutrient.
The slope coefficient of the linear relationship is termed the efficiency of utilisation
for production (kpf), protein (kp) and lipid (kf) and this can be used to estimate the
response over a range of nutrient intake levels independent of mass (NRC, 2011).
Moreover, the slope of the regression can be further extrapolated until recovered
energy for growth is equal to zero thus providing an estimate of nutrient maintenance
requirements (NRC, 2011).
A number of studies have used this bioenergetic approach in determining the
marginal efficiencies and estimating maintenance requirements of energy, lipid and
protein in a variety of fish species. The marginal efficiencies of protein (kp) and lipid
(kf) of a range of species including Atlantic salmon, rainbow trout (Oncorhynchus
mykiss), European seabass, gilthead sea bream, white grouper (Epinephelus aeneus)
and yellow-tail kingfish (Seriola lalandi) generally range between kp 0.53 - 0.64 and
kf 0.72 - 0.91 (Booth et al., 2010; Bureau et al., 2006; Helland et al., 2010; Lupatsch
et al., 2003). In barramundi, Glencross and Bermudes (2010) showed that over a
range of temperatures from 25 to 32 °C the partial efficiency of energy (kpf) was
relatively consistent at 0.56 and protein (kp) was relatively consistent at 0.51. Despite
the apparent importance of EFA the energetic efficiencies and maintenance
requirements of these nutrients do not appear to have been investigated in fish using
a bioenergetics approach.
1.3.3 Different fatty acid class use by barramundi
In the context of feeding fish alternative oils, many studies have focused on the
essential fatty acids and their bioactive long-chain derivatives, EPA and DHA.
However, it is suggested that SFA and MUFA are preferred substrates for β-
oxidation in fish, sparing the more essential fatty acids for their functional and
biological roles (review by Henderson 1996). As alternative oils are being
increasingly used in aquafeeds it is important to understand the effect of increasing
dietary levels of SFA and MUFA, which are generally abundant in such oils, on lipid
35 | P a g e
metabolism in fish. Carnivorous fish such as barramundi, rely heavily on β-oxidation
of lipid for their energetic requirements. Past studies have consistently shown
positive responses to the replacement of FO with a range of alternative oils such as
soybean, canola, rapeseed, Echium, usually included as blends (Alhazzaa et al.,
2011a; Raso and Anderson, 2003; Williams et al., 2006).
Atlantic salmon also performed well when fed diets with either low n-3
PUFA and high MUFA and in addition MUFA were found to be good substrates for
β-oxidation (Menoyo et al., 2003). In the same species, β-oxidation was found to be
dependent on energy demand and that when fatty acids were provided in excess to
synthetic demands they were increasingly β-oxidised for energy production
(Stubhaug et al., 2007; Torstensen et al., 2004). Denstadli et al. (2011) found that the
medium chain length decanoic acid (10:0) was rapidly oxidised for energy
production whereas OLA was primarily deposited in the intramuscular fat in Atlantic
salmon. In other species such as Atlantic cod reduced lipid deposition in the liver
was likely caused by selective β-oxidation of 16:0 and 18:0 SFA present in PO and
rapeseed oil (Jobling et al., 2008). In agreement, polka dot grouper (Cromileptes
altivelis) more readily oxidised lipid in diets containing SFA (coconut fat) for energy
rather than MUFA (olive oil); however, this was at the expense of efficient growth
(Smith et al., 2005). Further to this, the authors of a recent study on European sea
bass speculated that a single fatty acid (eg. OLA) might be more efficient at sparing
LC-PUFA from β-oxidation than oil blends (Eroldogan et al., 2013).
Using an in-vivo whole-body fatty acid balance approach for determining
fatty acid metabolism in rainbow trout, Turchini and Francis (2009) found that the
most heavily oxidised fatty acids were also the most dominant. However, in the fish
oil fed trout the most predominant dietary fatty acid (16:0) was not readily oxidised
for energy production, but rather was preferentially elongated to 18:0 or desaturated
to 16:1n-7. The same research group also found that the rate of β-oxidation of LC-
PUFA was reduced when Murray cod (Maccullochella peelii peelii) were fed diets
containing MUFA and SFA, which are generally abundant in alternative oils
(Turchini et al., 2011b). Moreover, a recent study on hybrid striped bass (White Bass
Morone chrysops × Striped Bass M. Saxatilis) concluded that diets containing SFA-
rich lipid and to a lesser extent MUFA-rich lipid were able to conserve tissue fatty
acid profiles possibly due to preferential β-oxidation (Trushenski et al., 2015).
36 | P a g e
1.3.4 Different lipid class use by barramundi
The phospholipids form the structural bilayer of cell membranes providing integrity
and fluidity (Hazel and Williams, 1990; Tocher et al., 2008). Also central to their
biological importance is their association with apoproteins (forming lipoproteins)
that assist in the extracellular transport of lipids thus improving parameters such as
growth, survival and health throughout the organism (Tocher et al., 2008). However,
the total lipid content in fish is mostly composed of neutral lipid in the form of TAG
(Glencross, 2009).
There is evidence to suggest that most larval and early juvenile fish have a
dietary requirement for intact phospholipids as endogenous biosynthesis is not
sufficient (Coutteau et al., 1997). Coutteau et al. (1997) reported that the
phospholipid requirement of fish and crustaceans varied depending on the life stage
and history. Freshwater fish generally have lower dietary requirements, of around 2%
whereas marine fish generally have higher requirements up to 7%. Early studies
found that in both rainbow trout and Atlantic salmon the phospholipid requirement of
first swim-up sized fish (<0.2 g) was 4% supplied in the form of soybean lecithin
(Poston, 1990a; b). However, larger salmon (~7.5 g initial) showed no improvement
in terms of growth suggesting that endogenous synthesis of phospholipid is sufficient
to support the requirement of the fish and that high dietary levels had a negative
effect on survival (Poston, 1990a). It should also be noted that the latter study, and
possibly others, refer to a requirements based on the use of phospholipid containing
ingredients rather than the precise phospholipid content which is often unclear.
There are very few studies on the effect of dietary phospholipids in juvenile
fish greater than 5 g as it is generally accepted that they don’t have a requirement
based on the historical evidence presented for Atlantic salmon (Poston, 1990a).
Recently, the influence of dietary phospholipid from either krill oil or soybean
lecithin was investigated in Atlantic salmon from first feeding up to smolt (0 to 70 g
range) (Taylor et al., 2015). These authors demonstrated a range of improvements
among the parameters tested and concluded that Atlantic salmon have a dietary
requirement for intact phospholipid particularly in early development. Therefore,
with the continual reduction of FM and FO in commercial feeds and the complex
biochemistry of the phospholipids particularly in juvenile fish, further investigation
is warranted.
In barramundi, some notable effects on the phospholipid composition of
tissues have been identified, which may suggest that juvenile barramundi have a
37 | P a g e
requirement for intact phospholipids (Alhazzaa et al., 2011b; Tu et al., 2013). It
appears that when dietary PL are not sufficient then very selective retention of tissue
phospholipids occurs in barramundi and other species, until depletion, this being a
mechanism to prevent the onset of phospholipid deficiency and secondary
pathologies as a result (Skalli and Robin, 2004; Tocher et al., 2008; Tu et al., 2013).
1.3.5 Essential fatty acids in barramundi
Nutrient deficiency in any species can be difficult to determine accurately due to the
difficulties in feeding a deficient diet. Early studies have specifically looked at fatty
acid deficiency in fish (Castell et al., 1972) while others studying nutrient
requirements have reached conclusions about deficiency signs and symptoms
(Catacutan and Coloso, 1995). There is a vast amount of literature on lipids and their
constituent fatty acids in fish diets and a focal point has been on their complexity,
uniqueness and biological importance (Sargent et al., 2002). The barramundi are
reported to have limited de novo capability to synthesise LC-PUFA (Alhazzaa et al.,
2011a; Mohd-Yusof et al., 2010; Tu et al., 2013). Moreover, the requirement of total
lipid and LC-PUFA is thought to be low based on the compositional status of wild
caught specimens compared to that of cultured barramundi (Nichols et al., 1998;
Nichols et al., 2014).
Clinical signs of nutrient deficiency become an important indicator of fish
health and productivity. If fish are fed a diet that is deficient in a particular nutrient
that cannot be synthesised endogenously, then their physical condition will begin
deteriorating once all body reserves become depleted. Further, secondary
pathological conditions may take hold as the animal becomes compromised and
without treatment premature death often follows. To avoid this situation arising,
commercially produced feeds need to ensure an adequate supply of all essential
nutrients, often achieved by the addition of FM and FO. However, global supply of
these resources is under increasing environmental and economic pressure. Therefore
it is important to determine critical inclusion levels of all essential nutrients in dietary
formulations for cultured species.
Several studies have induced EFA deficiency in a range of fish species. One
of the earliest was that of Castell et al. (1972) who documented the feeding of
rainbow trout over a prolonged time-course. These authors described a range of
deficiency symptoms such as; poor growth, fin erosion, changing pigmentation,
swollen livers and hearts and fainting or shock syndrome. A later study by Ruyter et
al. (2000) observed that Atlantic salmon exhibited poor growth, increased mortality
38 | P a g e
and altered blood chemistry and liver condition after one month of feeding an EFA
deficient diet. In other species such as channel catfish (Ictalurus punctatus) (Satoh et
al., 1989), red drum (Sciaenops ocellatus) (Lochmann and Gatlin, 1993), turbot
(Psetta maxima) (Bell et al., 1985) and gilthead sea bream (Ibeas et al., 1996), signs
of EFA deficiency include a consistent range of symptoms, with the most prominent
being reduced growth performance and survival.
The precursors to LC-PUFA, 18:2n-6 and 18:3n-3 are considered essential or
at least conditionally essential in all vertebrates, including fish, as they lack the
desaturase enzymes required for their synthesis (Cunnane, 2003; Tocher, 2003).
Typically, freshwater and some diadromous species, are able to synthesise sufficient
LC-PUFA from these precursor fatty acids while in most marine fish species the
pathway of fatty acid biosynthesis is incomplete (Tocher, 2003). However, there are
distinct differences that exist between the marine fish in their capacity to
biosynthesise fatty acids. Recently, the presence of an alternative delta-4 pathway to
LC-PUFA was identified in the herbivorous marine fish Siganus canaliculatus (Li et
al., 2010). Cell lines of the marine fish turbot were used to demonstrate that
elongation activity was limiting whereas in gilthead sea bream delta-5 desaturase
activity was limiting the conversion of precursor fatty acids to LC-PUFA (Ghioni et
al., 1999; Tocher and Ghioni, 1999). The barramundi also lacks delta-5 desaturase
activity and is known to have a desaturase with delta -6/delta-8 dual activity in vitro
(Mohd-Yusof et al., 2010; Tu et al., 2013). Therefore, this evidence supports that in
vivo this species should succumb to EFA deficiency due to this inability to produce
the reputedly required LC-PUFA including EPA, DHA and ARA.
The lipid, and to a lesser degree the fatty acid requirements were examined in
barramundi by a number of studies, yet there is still no clear documentation of the
onset and progression of deficiency symptoms. Early studies demonstrated that
performance of larval barramundi was improved by incorporating preformed LC-
PUFA into enriched live prey (Dhert et al., 1990; Rimmer et al., 1994). Other
juvenile barramundi studies have reported signs of EFA deficiency such as abnormal
reddening of the fins and reduced growth performance associated with low levels of
n-3 LC-PUFA (Buranapanidgit and Boonyaratpalin, 1988; Catacutan and Coloso,
1995). More recently, Williams et al. (2006) demonstrated improvements in
barramundi productivity with increasing n-3 LC-PUFA at either 20 or 29 °C.
Williams et al. (2006) described a ‘fainting attack’ from a small number of their fish
and this effect has also previously been observed in rainbow trout fed EFA deficient
39 | P a g e
diets (Castell et al., 1972). Glencross and Rutherford (2011) showed for the first time
that barramundi performance was clearly affected by the presence or absence of
certain EFA. These authors also reported symptoms such as reddening of the fins and
the opercular region, however, this was attributed to increasing DHA in the absence
of an equivalent increase of EPA.
1.3.6 EPA, ARA and eicosanoid metabolism in barramundi
Eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6) are
important for many metabolic and physiological functions, and are precursor
molecules for the production of eicosanoid hormones that play a role in the
inflammatory response, immune function and regulation as well as ionic regulation
and reproduction in fish and mammals (Rowley et al., 1995). In humans, the
incidence of diseases involving inflammatory processes can be related to the
production of these eicosanoids, for example prostaglandin E2 (PGE2) and
leukotriene B4 (LTB4) that are derived from high cellular concentrations of ARA
(Wall et al., 2010). The cyclooxygenase (COX; prostaglandin G/H synthase) and
lipoxygenase (LOX; arachidonate 5-lipoxygenase) enzymes interact and compete for
the EPA and ARA substrates and as such the eicosanoids produced are determined
by their availability (Calder, 2012; Tocher, 2003).
The COX enzyme catalyses 20-carbon chain fatty acids (ARA or EPA)
through bis-dioxygenation and subsequent reduction to produce 2 - and 3 - series
prostaglandins (PGG2/3 and PGH2/3) that are substrates for the synthesis of
biologically active prostaglandins, prostacyclins and thromboxanes (Calder, 2012;
Rouzer and Marnett, 2009; Rowley et al., 1995). While the LOX enzyme catalyses
the same substrates to yield biologically active metabolites of hydroperoxy-
eicosatetraenoic acid (HPETE), such as leukotrienes and lipoxins (Matsumoto et al.,
1988; Rowley et al., 1995). Unlike most vertebrates, the COX enzyme system in
teleost fish is complicated by an evolutionary duplication event that led to alternative
chromosomal regions of COX genes, often identified as ‘a and b’ isoforms of either
the COX-1 or -2 series, but rarely both (Ishikawa and Herschman, 2007; Ishikawa et
al., 2007). There is also an accepted paradigm that COX-1 is constitutively expressed
whereas COX-2 is inducible, however, this is now widely viewed as an
oversimplified view (Breder et al., 1995; Olsen et al., 2012; Rouzer and Marnett,
2009).
40 | P a g e
In many fish species, EPA and ARA are required for important metabolic and
physiological functions, and the dietary requirements are well understood (Bell and
Sargent, 2003; Izquierdo, 1996; Tocher, 2015). The consumption of diets rich in oils
that contain eicosanoid inhibiting or regulating substrates (Eg. EPA, 20:4n-3, 18:4n-
3) can have significant downstream modulatory effects on the fish (Ghioni et al.,
2002). However, without discounting the importance of ARA, there is great potential
for this fatty acid to affect growth, stress response, immune response and survival,
particularly at early life stages (Atalah et al., 2011; Bell and Sargent, 2003; Castell et
al., 1994; Montero et al., 2015b; Yuan et al., 2015).
Information on the EPA and ARA requirements in the barramundi or Asian
seabass are scarce In the only study available thus far, a single diet with a higher
inclusion of ARA in the absence of EPA showed negative effects on fish health,
including disproportionate tissue LC-PUFA retention and pathophysiological effects
such as subcutaneous haemorrhaging and disrupted ionic regulation (Glencross and
Rutherford, 2011). There is evidence to suggest that wild barramundi (either from
fresh or salt water) can contain ARA levels more than 5-fold higher than EPA in
their muscle tissue (Nichols et al., 2014). The increasing use of alternative oils in
farmed fish, often containing high levels of omega 6 fatty acids which can act as
ARA precursors (e.g. soybean oil, sunflower oil), and simultaneous reduction of
dietary fish oil, may impact the final ARA/EPA ratio in fish tissues, with possible
resulting issues associated with modified lipid metabolism (Brown and Hart, 2011).
1.3.7 Allometric scaling effects of LC-PUFA
A range of different approaches have been used to predict or determine growth as
well as feed requirements based on the dynamic flow of nutrients in aquatic systems
(Bar et al., 2007; Cho and Bureau, 1998; Glencross, 2008; Lupatsch and Kissil,
1998; Machiels and Henken, 1986). Predictive models started out as relatively simple
approaches such as the ubiquitous specific growth rate and thermal growth
coefficient calculations; however, progressive extensions of these models now exist
that consider the many biological properties of fish (Birkett and Lange, 2007; Dumas
et al., 2010). In addition, mass-balance models have also been developed and used in
understanding specific nutrient and metabolite flows in a range of model species
(Cunnane and Anderson, 1997; Turchini et al., 2006; Turchini et al., 2007) as well as
a whole ecosystem approach (Sawyer et al., 2016).
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There is a growing body of evidence regarding the essential fatty acid requirements
of many species generally determined by various forms of in vivo feeding
assessments (NRC, 2011). The efficient utilisation of fatty acids within an organism
depends on a number of factors and there are many complex interactions among
specific fatty acids potentially affecting their utilisation efficiency (Glencross, 2009;
Tocher, 2015). Despite the numerous studies to date, relatively little is known about
the maintenance requirements and utilisation efficiencies associated with specific
fatty acids and how these may be used in nutrient modelling. An obvious step in the
further development of these models is the incorporation of empirically derived
utilisation efficiency values. Maintenance requirements of protein, lipid and energy
typically described by linear equations of intake to gain ratios can give an insight
into the partitioning of production and maintenance costs (Bureau et al., 2006; NRC,
2011; Pirozzi et al., 2010). Similarly, it should be possible to determine estimates of
productivity for specific fatty acids using derived body weight scaling exponent
values to provide a size independent response (Bar et al., 2007; Glencross and
Bermudes, 2011; White, 2011).
1.4 Thesis structure
The thesis presented herein is an adaptation of a series of published manuscripts. The
manuscripts have been peer reviewed and published in high standing journals
relevant to the field of aquaculture nutrition. The following (second) chapter
summarises the materials and methodology utilised during experimentation. This
section is divided to cover key areas including experimental diet production,
experimental methodologies and experimental analyses. The second chapter also
includes an ethical statement encompassing all research chapters using live animals.
Chapter three to chapter eight are the six experimental research chapters that have
been adapted from published and submitted manuscripts. Each provides the reader
with a concise aims and methods section followed by detailed results and discussion.
The ninth chapter is a general summary followed by a general conclusion.
42 | P a g e
General methodology
2.1 Diet production
The following methods were used during the experiments conducted as part of this
thesis.
2.1.1 Ingredient preparation
This method applies to all of the experiments with the exception of Chapter 8 as
there were no diets produced. The dry ingredients were passed separately through a
hammermill (Mikro Pulverizer, type 1 SH, New Jersey, USA) such that the
maximum particle size was less than 750 μm. All ingredients were then thoroughly
mixed using an upright commercial mixer (Bakermix, Model 60 A-G, NSW,
Australia). Fish meal was defatted prior to use in experimental Chapter 4, Chapter 5,
Chapter 6 and Chapter 7. This was achieved by manually mixing hexane and fish
meal (2:1) in a large drum. The mix was left to soak for 3h before draining the excess
hexane and repeating the process a second time. The fish meal was then oven dried
overnight at 60 °C to a constant dry matter.
2.1.2 Diet preparation by screw press technology
This method applies to Chapter 4, Chapter 5 and Chapter 6. A single basal diet was
formulated and prepared without the addition of dietary lipids. The basal diet was
then separated into smaller batches and aliquots of lipid (8.2% diet) were added to
form the treatment diets. Fresh water was added at approximately 30% of dry mash
weight and mixed to form a consistent dough, then the dough was subsequently
screw pressed through a 4 mm die Dolly Pasta Extruder (La-Monferrina,
Sant’Ambrogio di Torino, Italy). The pellets were dried overnight at 60 °C to a
constant dry matter. Pellets were then stored at -20 °C until required.
2.1.3 Diet preparation by extrusion technology
This method applies to Chapter 3 and Chapter 7. The pellets were produced using a
laboratory-scale twin-screw extruder with intermeshing, co-rotating screws (MPF24,
Baker Perkins, Peterborough, United Kingdom). The following parameters were
used, however, these are only examples and vary depending on the formulation
across experiments.
The dry mash was delivered into the barrel at a feed rate of around 400 g/min.
Barrel temperatures were set for each of the four zones from drive to die at 60, 80,
100 and 110 °C respectively. The following methodology was based on Glencross et
43 | P a g e
al. (2016). The extruder barrel was a smooth-walled, open clam design with twin
screws measuring 24 x 600 mm (diameter x length). The screws were configured
with a series of feed screws (FS), forward paddles (FP) and lead screws (LS)
arranged to defined barrel diameters (D). The configuration was 16D FS, 2D FP, 1D
FS, 2D FP, 1D LS, 1D FP, 2D LS: to the die. Feeds were extruded through a 2 mm Ø
die with the machine running at c. 326 rpm. Water was peristaltically pumped
(Baoding Longer Precision Pump Co., Ltd, Hebei, China) into the barrel at 24
mL/min. Pellets obtained a 1.5-fold increase in diameter by expansion were cut off at
lengths of ~4 mm using a variable speed 4-blade cutter and dried overnight at 60 °C
to a constant dry matter.
2.1.4 Vacuum infusion of lipid
Prior to vacuum infusion the lipid sources were warmed at 60 °C to ensure a
homogeneous blend of the lipids as liquefied oils before the prescribed allocation of
dried pellets was placed into a large mixing bowl and oil slowly added while mixing
to ensure even coating. A perspex lid was then placed on the bowl with a rubber seal
and vacuum pump was then connected through a small fitting. Suction was applied to
achieve a maximum of 100 kPa pressure or until all visible signs of air escaping the
pellets had ceased at which point the suction was released and the oil infused as the
chamber re-equilibrated to atmospheric pressure. Pellets were then stored at -20 °C
until required.
2.2 Experimental methodology
2.2 Ethical statement
Ethical clearance was approved for each of the experimental procedures by the
CSIRO Animal Ethics Committee (Chapter 3.0 A09/2011; Chapter 4.0 A12/2013;
Chapter 5.0 A11/2013; Chapter 6.0 A10/2013; Chapter 7.0 A05/2014; Chapter 8.0
A3/2015). All manipulations with animals were conducted in adherence of the
ethical conditions. Wherever possible the use of animals was minimised and those
remaining after experimentation were returned to a stock holding facility.
2.3 Barramundi husbandry
For each experiment, juvenile barramundi (Lates calcarifer) were sourced from the
Betta Barra fish hatchery (Atherton, QLD, Australia), on-grown in a 10,000L tank
and fed a commercial diet (Marine Float; Ridley Aquafeed, Narangba, QLD,
Australia). Prior to commencement of the experiment the selected fish were
44 | P a g e
transferred to a series of experimental tanks with flow-through heated seawater at a
flow rate of about 3 L/min being supplied to each of the tanks. There were 20
individually weighed fish per tank used in Chapter 3, 26 individually weighed fish
per tank used in Chapter 4, Chapter 5 and Chapter 6, 30 individually weighed fish
per tank used in Chapter 7 and 15 individually weighed fish per tank used in Chapter
8. Photoperiod was constant at 12L:12D. Three experimental systems were used
during experimentation; 300 L aquaria were used for Chapter 4, Chapter 5 and
Chapter 6; 600 L aquaria were used for Chapter 3, and Chapter 8; and 1000 L aquaria
were used for Chapter 7. In each case, the experimental diets were randomly
distributed amongst the tanks with each treatment having three replicate tanks.
Depending on the experimental design, the fish were managed according to one of
the following protocols.
1) The diets were fed once daily to apparent satiety as determined over three separate
feeding events, for eight weeks. Any uneaten feed was collected shortly after and a
correction factor was applied (Helland et al., 1996). Briefly, the correction factor was
calculated as the proportion of soluble losses after immersion in water for 1 h
(Chapter 3, Chapter 4, Chapter 5 and Chapter 6).
2) The fish were restrictively fed, once daily, a sub-satietal (approximately 80%)
pair-feeding regime in order to avoid the potentially confounding issue of
unregulated feed intake (Chapter 7) (Glencross et al., 2003a).
3) At the beginning of the experiment the tanks held six treatment size classes of
fish, which were randomly distributed among the tanks with each treatment having
three replicate tanks. The fish were then starved for 21 days (Chapter 8).
2.4 Sample collection techniques
For each experiment six fish of similar size from the original stock were euthanized
by an overdose of AQUI-S™ (Lower Hutt, New Zealand) at the beginning of the
experiment and stored at -20 ºC until analysis. Upon termination of the experiment, a
further six fish were sampled from each tank. A sample of whole blood was removed
from the caudal vein of three fish using 1 mL pre-heparinised syringes and an 18 G
needle. Blood from the three fish was pooled in a single Vacutainer™ tube and then
centrifuged at 10,000 rpm for 5 min to settle the erythrocytes. The plasma was then
drawn off and transferred to a 1.5 mL Eppendorf™ tube and frozen at -80 °C before
being sent for chemical analysis at the West Australian Animal Health Laboratories
(South Perth, Western Australia). From the same three fish and three from the initial
stock, a sample of liver tissue for molecular analysis was removed upon termination
45 | P a g e
or at each fortnightly sampling event. We anticipated relatively minor changes in
gene expression and therefore we only included the control and extreme dietary
treatments for analysis. Samples were placed into 1.5 mL screw-top vials and kept on
dry ice before being transferred to a -80 °C freezer until analysis. All sampling
procedures occurred 24 h after feeding in the post absorptive phase (Wade et al.,
2014). If interim samples were required individual fish (n=3 per tank) were collected
in the same fashion after each fortnightly period and upon termination of the
experiment. Any remaining fish returned to their respective tank after a short
recovery.
2.5 Abnormalities and behaviour assessment
This method only applies to Chapter 6. The physical condition of all the individual
fish was recorded at each fortnightly sampling event. Any fish that had symptoms
such as erosion of the fins, reddening of the fins and extremities, gross lesions and
physical deformities were recorded. For the assessment, fish were considered either
normal or abnormal based on the presence or absence of at least one symptom. A
percentage score was given to each tank and each tank was used as a replicate within
each treatment. The behaviour of each tank of fish was assessed by an operator
holding a hand over the corner of a tank to simulate commencement of a feeding
event. Fish activity was scored as being either cryptic (0), ambivalent to the hand (1)
or actively searching for food (2). This assessment was carried out prior to feeding
on the same day of each week by the same operator to maintain consistency and the
results were averaged across time to give a repeated measures response to each tank.
Each tank was used as a replicate within each treatment (Glencross and Rutherford,
2011).
2.6.1 Digestibility analysis – settlement
Yttrium oxide was used in each diet formulation at 1g/kg as an inert marker for
digestibility analysis. Two different methods of faecal collection were used during
experimentation. The settlement method applies to Chapter 4, Chapter 5 and Chapter
6. Prior to the termination of the growth assay, faeces were collected using the
established settlement protocol described below (Blyth et al., 2014). Briefly, a
collection chamber was filled with water and frozen then attached to the evacuation
line of a swirl separator and left overnight. The following morning, the collection
chamber was removed and the chilled faeces were captured in a plastic sample
container and stored at -20 °C until analysis.
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2.6.2 Digestibility analysis - abdominal stripping
This method applies to Chapter 7. Upon termination of the growth assay, faeces were
collected using established abdominal stripping protocols (Blyth et al., 2014).
Briefly, the fish were netted from their tanks and anesthetised, then gentle abdominal
pressure was applied to the distal intestine to extract the faeces. Care was taken by
the operator to avoid contamination of the sample with foreign material and hands
were rinsed after each stripping. The faecal sample was placed into a small plastic
vial on ice before being stored in a freezer -20 °C until analysis.
2.7 Experimental analyses
2.7.1 Chemical analysis
These methods apply to all experiments. Prior to analysis the diets were each ground
to a fine powder using a bench grinder (KnifeTec™ 1095, FOSS, Denmark). The
initial and final fish were processed using the following method. The whole fish were
passed through a commercial meat mincer (MGT – 012, Taiwan) twice to obtain a
homogeneous mixture. A sample was taken for dry matter analysis and another
sample was freeze-dried along with the faecal samples until no further loss of
moisture was observed (Alpha 1-4, Martin Christ, Germany). Dry matter was
calculated by gravimetric analysis following oven drying at 105ºC for 24 h. Crude
protein was calculated after the determination of total nitrogen by organic elemental
analysis (CHNS-O Flash 2000, Thermo Scientific, USA), based on N x 6.25. Total
lipid content was determined gravimetrically following extraction of the lipids using
chloroform:methanol (2:1) following Folch et al. (1957). Gross ash content was
determined gravimetrically following loss of mass after combustion of a sample in a
muffle furnace at 550 °C for 24 h. Gross energy was determined by adiabatic bomb
calorimetry (Parr 6200 Calorimeter, USA). Total yttrium concentration in the diets
and faeces was determined after nitric acid digestion in a laboratory microwave
digester (Ethos One, Milestone, Italy) using inductively coupled plasma-mass
spectrophotometry (ICP-MS) (ELAN DRC II, Perkin Elmer, USA).
2.8.1 Biochemical analysis – Lipid transesterification
These methods apply to all experiments. Fatty acid composition was determined
following the methods of Christie (2003). Lipids were esterified by an acid-catalysed
methylation and 0.3 mg of an internal standard was added to each sample (21:0
Supelco, PA, USA). The fatty acids were identified relative to the internal standard
47 | P a g e
following separation by gas chromatography (GC). An Agilent Technologies 6890N
GC system (Agilent Technologies, California, USA) fitted with a DB-23 (60 m x
0.25 mm x 0.15 μm, cat 122-2361 Agilent Technologies, California) capillary
column and flame ionisation detection was used. The temperature program was 50–
175 ºC at 25 ºC /min then 175–230 ºC at 2.5 ºC /min. The injector and detector
temperatures were set at 250 ºC and 320 ºC, respectively. The column head pressure
was set to constant pressure mode at 170 kPa using hydrogen as the carrier gas. The
peaks were identified by comparing retention times to the internal standard and
further referenced against known standards (37 Comp. FAME mix, Supelco, PA,
USA). The resulting peaks were then corrected by the theoretical relative FID
response factors (Ackman, 2002) and quantified relative to the internal standard.
Lipid extracts were applied to silica Sep-Pak® (Waters, Massachusetts, USA)
columns and separated into neutral (non-phosphorus) lipid and polar (phosphorus)
lipid following (Juaneda and Rocquelin, 1985). Briefly, Sep-Pak® columns were
pre-conditioned with 4 mL of hexane before a 30 mg sample of lipid dissolved in
chloroform was applied to each column. The neutral lipid was first eluted with 20
mL of chloroform and followed by 5 mL of chloroform:methanol (49:1). The polar
lipid was then eluted with 30 mL of methanol. The proportion of either neutral or
polar lipid was quantified gravimetrically and then an aliquot was further esterified
and separated into fatty acids following the protocol above.
2.8.2 Biochemical analysis – Plasma chemistry
These methods only apply to Chapter 5 and Chapter 6. Plasma samples were sent to
the West Australian Animal Health Laboratories (South Perth, Western Australia) for
enzyme and chemistry assessment. The assays were run on an Olympus AU400
automated chemistry analyser (Olympus Optical Co. Ltd). Each of the assays used
was a standard kit developed for the auto-analyser. The tests performed included
alanine aminotransferase (ALT, EC 2.6.1.2) (Olympus kit Cat. No. OSR6107),
creatine kinase (CK, EC 2.7.3.2) (Olympus kit Cat. No. OSR6179), glutamate
dehydrogenase (GLDH, EC 1.4.1.2) (Randox kit Cat. No. GL441), total protein
(Olympus kit Cat. No. OSR6132), creatinine (Olympus kit Cat. No. OSR6178),
alkaline phosphatase (Olympus kit Cat. No. OSR6004), glucose (Olympus kit Cat.
No. OSR6121), haemoglobin (Randox kit Cat. No. HG1539) and haptoglobin
(Randox kit Cat. No. HP3886). Trace elements were determined after mixed acid
digestion using inductively coupled plasma mass spectrometry (ICP-MS).
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2.9.1 Molecular analysis - Cloning of putative prostaglandin G/H synthase
(COX) and arachidonate 5-lipoxygenase (LOX) genes
These methods only apply to Chapter 7. Sequences of the prostaglandin G/H
synthase and arachidonate 5-lipoxygenase (COX1a, COX1b, COX2 and ALOX-5)
genes from several teleost species were identified in the Genbank database. Highly
conserved regions from protein alignments across species were used to design pairs
of degenerate primers that were subsequently synthesised by Sigma-Aldrich (Table
3). PCR primers specific to each target gene were designed using PerlPrimer v.1.1.17
(Marshall, 2004). Pooled barramundi liver cDNA (1000 ng) was amplified in
reactions, with each degenerate primer pair (F and R; 10 μM) using platinum TAQ
mix (Thermofisher). Polymerase chain reaction (PCR) conditions including an initial
denaturation step at 90 °C for 2 min followed by 35 cycles of 94 °C/10 s, 50 °C/30 s
(50% ramp speed) and 72 °C/30 s with a final extension step of 72 °C/5 min were
used. The amplification products were separated by size using electrophoresis on a
1% agarose gel and then excised and extracted using the QIA quick gel extraction kit
(QIAGEN). The target was then ligated using the pGEM-T Easy vector system
(Promega). Ligation reactions were then transformed onto One Shot TOP10
chemically competent Escherichia coli cells (Thermofisher) which were then
cultured overnight on LB ampicillin plates (100 μg/mL). Positive clones were
selected by PCR amplification with primers flanking the multiple cloning site (M13F
and M13R), using an initial denaturation step at 94 °C/5 min followed by 35 cycles
of 94 °C/20 s, 55 °C/30 S, 72 °C/1.5 min with a final extension step of 72 °C/5 min.
Randomly selected positive clones were prepared for sequencing with a QIAprep
Miniprep kit (QIAGEN) and then the plasmid insert was sequenced using the
BigDye® Terminator V3.1 sequencing kit (Applied Biosystems) with an ABI 3130
Automated Capillary DNA Sequencer (Applied Biosystems).
2.9.2 Molecular analysis - Sequencing analysis
These methods only apply to Chapter 7. Multiple alignments of the target genes (Lc
COX-1a, Lc COX-1b, Lc COX-2 and Lc ALOX-5) were made using the CLC
Genomics Workbench software (CLC Bio, Aarhus, Denmark). The ORF and amino
sequence alignment and analysis were conducted using the Create Alignment tool,
and confirmed as the target gene sequence using the BLASTX algorithm
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence analysis of the previously
unreported Lates calcarifer COX and LOX genes identified expressed sequence tags
49 | P a g e
(EST) of between 752 and 999 base pairs, open reading frame protein alignments
showing similarity with other teleost fish are presented in Appendix -Supplementary
Figures 1 a-d.
2.9.3 Molecular analysis - RNA extraction and normalisation
These methods apply to Chapter 5, Chapter 6 and Chapter 7. Total RNA was
extracted from the liver samples using TRIzol reagent (Invitrogen) according to the
manufacturer’s instructions. RNA was precipitated using equal volumes of
precipitation solution (1.2 M sodium chloride and 0.8 M disodium citrate) and
isopropyl alcohol (Green and Sambrook, 2012). To eliminate any residual traces of
DNA, total RNA was DNase digested with the Turbo DNA free kit (Applied
Biosystems). To verify that RNA was not contaminated, an aliquot of DNase
digested RNA from each sample was pooled and later PCR amplified as a negative
control. A NanoDrop spectrophotometer (NanoDrop Technologies) was used to asses
RNA quantity and a Bioanalyser (Agilent Technologies) using RNA nanochips
(Agilent #5067-1511) was used to asses RNA quality. All RNA samples were
normalised to 200 ng/μl.
2.9.4 Molecular analysis - Quantitative real time reverse transcription
polymerase chain reaction (qRT-PCR)
These methods apply to Chapter 5, Chapter 6 and Chapter 7. Reverse transcription
was performed on 1 μg of total RNA using Superscript III (Invitrogen) with 25 μM
oligo (dT)20 and 25 μM random hexamers (Resuehr and Spiess, 2003). Expression of
a range of genes involved in fatty acid metabolism was analysed by real-time PCR as
described below. Real-time PCR primers specific to each target gene were designed
using PerlPrimer v.1.1.17 (Marshall, 2004). Real-time PCR amplification reactions
were carried out using 2X SYBR Green PCR Master Mix (Applied Biosystems), 0.2
μM of Real-Time PCR primers specific to each gene and the equivalent of 7.5 ng of
reverse-transcribed RNA. Of the genes examined in Chapter 5 and Chapter 6, fatty
acid elongation 5, fatty acid desaturation 6 and elongation factor 1α (GQ214180.1,
GQ214179.1, GU188685) are contained within the public nucleotide database. The
remaining genes were previously identified and reported following next generation
sequencing of barramundi liver tissue and BLAST similarity searches (Wade et al.,
2014). The raw sequence read data are made available online through the CSIRO
Data Access Portal (http://doi.org/10.4225/08/55E799BA0F73E). Amplification
cycle conditions were 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles of 15 s
50 | P a g e
at 95 °C and 40 s at 60 °C. After amplification, a melt curve analysis was routinely
performed to verify the specificity of the target gene. Reactions were setup using the
epMotion 5070 robot (Eppendorf) and run in triplicate on a Viia7 real-time PCR
system (Applied Biosystems). Changes in expression levels of each gene were
determined by normalising the cycle threshold values for each gene to elongation
factor 1 alpha (EF1α) and Luciferase reference genes. The EF1α and Luciferase
genes are routinely used as a reference in this species (De Santis et al., 2011; Wade
et al., 2014).
2.10 Calculations
The following equations have been used for the calculation of results during
experimentation.
1) Differences in the ratio of dry matter, protein, lipid and energy to yttrium in the
diet and faeces were calculated to determine the apparent digestibility coefficients
(ADC) using the formula:
Where Y diet and Y faeces represent the yttrium content in both the diet and faeces,
respectively and Parameter diet and Parameter faeces represent the nutritional parameter
(dry matter, protein, lipid and energy) in the diet and faeces, respectively (Maynard
and Loosli, 1979).
2) Nutrient deposition efficiencies were calculated as the ratio of the nutrient or
specific fatty acid gained relative to their respective consumption during the study
period using the formula:
Where Nf and Ni are the final and initial nutrient composition (g/fish) of the fish on a
wet basis, respectively, and Nc is the amount of the nutrient consumed (g/fish) during
the study period (Maynard and Loosli, 1979).
3) Maintenance demands and utilisation efficiencies were then determined from the
regression of marginal fatty acid intake against marginal fatty acid gain on a
transformed live-weight basis following (Glencross, 2008) using the formula:
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Where Intake FA or Gain FA is the specific fatty acid consumed or gained (g/fish) on a
weight specific (geometric mean live-weight g/fish) basis then transformed to a fatty
acid specific exponent. The duration of the study period in days is defined as d.
4) The assessment of somatic losses was based on the formula previously reported by
Glencross and Bermudes (2011):
Where the Wi and Wf are the initial and final weights of the fish respectively. Ei and
Ef are the initial and final energy content of the whole fish on a live-weight basis
respectively. The time of the assessment is denoted as t. The determination of lipid
and fatty acid loss was calculated in the same way by substituting the appropriate Ei
and Ef values with the corresponding values for either lipid or fatty acids.
5) The computation of apparent in vivo fatty acid elongation, desaturation and β-
oxidation was performed using the whole-body fatty acid balance method
(WBFABM) following Turchini et al. (2007). Briefly, this involved determination of
the appearance/disappearance of specific fatty acids by mass balance. The resulting
values of net appearance/disappearance were then transformed to a molecular weight
basis per gram of body weight per day (nmol/g fish/d). Subsequent back calculations
along the known fatty acid bioconversion pathways were used to determine the fate
of specific fatty acids
2.11 Statistical interpretation
All data are expressed as mean ± standard error of the mean (SEM) or mean with
pooled standard error mean unless otherwise specified. All data were checked for
normal distribution and homogeneity of variance by qualitative assessment of
residual and normal Q-Q plots. Growth performance data were analysed using a
range of statistical tests including polynomial contrasts, repeated-measures analysis,
linear regression, two-way factorial ANOVA, one-way ANOVA, or paired t-test,
Holm adjusted. Levels of significance were compared using Tukey’s HSD a
posteriori test.
Energy, lipid and fatty acid losses were examined relative to the geometric
mean weight in grams of the initial and final fish from each treatment size class. All
relationships were examined using a power function (y=aXb) or a logarithmic
52 | P a g e
function (y=b ln(x)+a). Microsoft Excel (Microsoft Office 2007) was used to
generate the equations and figures. A bootstrapping approach was used to generate
replications of coefficient and exponent values of energy, lipid and fatty acid loss in
order to analyse the data statistically.
The RStudio package v.0.98.501was used for all statistical analyses (R Core Team,
2012). Any percentage data were arcsine transformed prior to analysis. Significance
among the treatments defined as P < 0.05.
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Experimental Chapters
3.0 Marginal efficiencies of long chain-polyunsaturated fatty acid
use by barramundi (Lates calcarifer) when fed diets with varying
blends of fish oil and poultry fat
Adapted from
Salini, M.J., Irvin, S.J., Bourne, N., Blyth, D., Cheers, S., Habilay, N., Glencross,
B.D., 2015. Marginal efficiencies of long chain-polyunsaturated fatty acid use by
barramundi (Lates calcarifer) when fed diets with varying blends of fish oil and
poultry fat. Aquaculture. 449, 48-57
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3.1 Aims
It was hypothesised that barramundi would reach a critical limit of FO substitution
when absolute levels of dietary LC-PUFA dropped below estimated requirement of
around 1.2% (Williams et al., 2006). Therefore a series of diets were developed to
examine the effects of diluting fish oil with poultry fat on the growth and feed
utilisation performance of juvenile barramundi and to determine the consequences of
this on the fillet fatty acid profiles. This study also aimed to develop a strategy for
fish oil replacement with defined impacts on meat n-3 LC-PUFA levels.
3.2 Materials and Methods
The methodology used in the present experiment are detailed in the section 2.0
General methodology. Ethical clearance was approved for the experimental
procedures by the CSIRO Animal Ethics Committee (Application A09/2011). The
chemical composition of the main dietary ingredients is presented in Table 3.1. The
formulation and chemical composition of the five diets are presented in Table 3.2.
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Table 3. 1 Chemical composition of key ingredients. All data are g/kg DM unless otherwise stated. Fatty acid data are expressed as a percentage of total fatty acids (%). FM WF LM WG PM FO PF Dry matter 933 868 903 921 953 998 997 Crude protein 706 112 432 816 671 9 7 Lipid 101 18 76 97 164 949 996 Ash 139 6 28 4 137 1 0 Gross energy (MJ/kg) 20.0 16.5 18.9 21.3 21.0 39.2 38.7 14:0 5.2 0.2 0.2 0.1 1.5 8.3 1.1 16:0 24.1 20.3 12.1 20.2 27.3 19.0 23.0 18:0 7.2 1.5 5.5 1.2 8.6 3.7 6.1 16:1n-7 5.3 0.4 0.1 0.1 6.4 10.3 5.6 18:1n-9 13.2 14.4 0.0 12.4 41.3 10.1 42.8 18:2n-6 1.5 56.5 41.3 59.4 8.9 1.6 16.2 18:3n-3 0.8 3.5 4.9 2.8 0.7 0.7 2.0 20:4n-6 0.1 0.0 0.0 0.0 0.0 1.1 0.0 20:5n-3 9.0 0.4 1.5 0.6 0.0 17.3 0.6 22:5n-3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 22:6n-3 17.3 0.0 0.0 0.0 0.0 13.6 0.0 SFA 39.1 22.4 18.5 22.1 37.8 33.3 30.1 MUFA 24.2 16.6 33.5 14.7 51.3 24.9 51.0 PUFA 3.5 60.0 46.3 62.2 9.5 5.2 18.2 LC-PUFA 30.3 0.4 1.5 0.6 0.2 32.0 0.6 n-3 28.7 4.0 6.4 3.4 0.7 35.3 2.6 n-6 5.2 56.5 41.3 59.4 9.1 2.7 16.2
FM fish meal, WF wheat flour, LM lupin meal, WG wheat gluten, PM poultry meal, FO fish oil, PF poultry fat
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Table 3. 2 Experimental diet formulation and composition. All data are g/kg DM unless otherwise stated. Fatty acid data are expressed as a percentage of total fatty acids (%).
F100:
P0 F60: P40
F30: P70
F15: P85
F0: P100
Formulation Fish meal a 150 150 150 150 150 Fish oil b 85 51 25 12 0 Wheat flour c 119 119 119 119 119 Wheat gluten d 85 85 85 85 85 Lupin meal e 100 100 100 100 100 Poultry meal f 455 455 455 455 455 Poultry fat g 0 34 59 72 85 Premix h 5 5 5 5 5 Yttrium oxide i 1 1 1 1 1 Composition Dry matter 908.2 921.7 977.6 977.6 981.3 Crude protein 518.8 536.9 543.8 531.3 539.5 Crude lipid 149.7 151.2 157.0 159.4 161.9 Ash 92.9 90.3 91.3 94.9 93.8 Gross energy (MJ/kg) 21.9 22.4 22.6 22.7 22.6 14:0 4.7 3.4 2.4 1.9 1.4 16:0 21.1 22.0 22.7 22.8 22.9 18:0 5.6 6.1 6.5 6.6 6.7 16:1n-7 7.7 6.8 6.0 5.7 5.4 18:1n-9 24.0 30.0 34.8 36.7 38.9 18:2n-6 8.9 11.6 13.9 14.8 15.6 18:3n-3 1.1 1.4 1.5 1.6 1.7 20:4n-6 0.9 0.8 0.7 0.6 0.5 20:5n-3 8.8 5.9 3.3 2.3 1.2 22:5n-3 1.2 0.9 0.0 0.0 0.0 22:6n-3 7.6 5.4 3.5 2.7 1.8 SFA 33.7 27.1 25.6 25.4 24.9 MUFA 36.9 46.8 50.8 52.3 54.3 PUFA 11.7 14.0 16.0 16.8 17.4 LC-PUFA 17.3 12.1 7.5 5.5 3.5 n-3 19.4 13.7 9.0 7.0 4.7 n-6 10.0 12.4 14.5 15.3 16.2
a Fish meal; Ridley aquafeeds, Narangba, QLD, Australia b Fish (anchovy) oil; Ridley aquafeeds, Narangba, QLD, Australia c Plain wheat flour; Manildra Group, Rocklea, QLD, Australia d Wheat gluten; Manildra Group, Rocklea, QLD, Australia e Lupinus angustifolius cv. Coromup; Coorow Seeds, WA, Australia. f Poultry meal; Ridley aquafeeds, Narangba, QLD, Australia g Poultry fat; Ridley aquafeeds, Narangba, QLD, Australia h Vitamin and mineral premix includes (IU kg-1 or g/kg of premix): vitamin A, 2.5MIU; vitamin D3, 0.25 MIU; vitamin E, 16.7 g; vitamin K3, 1.7 g; vitamin B1, 2.5 g; vitamin B2, 4.2 g; vitamin B3, 25 g; vitamin B5, 8.3; vitamin B6, 2.0 g; vitamin B9, 0.8; vitamin B12, 0.005 g; biotin, 0.17 g; vitamin C, 75 g; choline, 166.7 g; inositol, 58.3 g; ethoxyquin, 20.8 g; copper, 2.5 g; ferrous iron, 10.0 g; magnesium, 16.6 g; manganese, 15.0 g; zinc, 25.0 g i Yttrium oxide; Stanford Materials, Aliso Viejo, California, United States
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3.3 Results
3.3.1. Growth performance and feed utilisation
During the 82 d growth period, the fish responded to the experimental diets, growing
consistent with the predicted model growth (Glencross and Bermudes, 2012).
Survival was 100% in all treatments. No significant differences were observed
among the treatment diets in terms of growth performance (Table 3.3). During the
growing period, there was greater than 2.5-fold increase in weight among the groups
of fish with final fish weights ranging between 545 to 553 g. Similarly, there were no
significant differences in feeding parameters with FCR values ranging from 1.12 to
1.15 (Table 3.3). The diet dry matter, protein, lipid and energy digestibility were also
unaffected by the modified lipid profile of the treatment diets (Table 3.3). The
digestibility of individual fatty acids was not significantly affected by treatment
(Table 3.3).
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Table 3. 3 Growth, feed utilisation and nutrient deposition parameters.
F100:
P0 F60: P40
F30: P70
F15: P85
F0: P100
P SEM
Regression R2, P#
Fish performance Initial (g/fish) 208.8 211.2 208.6 207.1 207.5 1.13 0.04, 0.48 Week-12 (g/fish) 545.0 546.4 553.6 546.6 549.7 4.20 0.01, 0.70 Gain (g/fish) 336.2 335.1 345.0 339.5 342.2 4.08 0.03, 0.57 Growth rate (g/d) 4.1 4.0 4.2 4.1 4.1 0.05 0.03, 0.55 Feed Intake (g/fish) 384.4 386.0 394.6 380.6 382.6 4.54 0.00, 0.93 FCR (feed/gain) 1.14 1.15 1.14 1.12 1.12 0.01 0.11, 0.22 Survival (%) 100.0 100.0 100.0 100.0 100.0 0.00 0.50, 0.10 Diet digestibility (%) Dry matter 57.5 55.3 53.9 56.7 59.1 4.00 0.00, 0.94 Protein 76.2 74.2 74.5 76.2 77.6 2.20 0.00, 0.84 Lipid 93.7 91.7 93.5 92.9 92.4 0.65 0.02, 0.67 Energy 71.7 69.4 68.4 70.6 70.4 2.41 0.00, 0.85 16:0 88.6 86.8 91.1 91.6 89.7 1.17 0.06, 0.45 18:0 81.7 80.4 86.6 88.1 84.5 1.74 0.11, 0.30 18:1n-9 95.0 94.4 96.3 96.4 96.7 0.48 0.22, 0.12 18:2n-6 93.6 93.4 95.5 95.9 97.2 0.78 0.29, 0.07 18:3n-3 96.5 95.3 96.4 97.4 97.8 0.66 0.07, 0.42 20:4n-6 95.0 100.0 97.4 100.0 93.2 1.32 0.00, 0.91 20:5n-3 99.2 98.5 99.4 95.3 97.0 0.82 0.13, 0.25 22:5n-3 100.0 100.0 100.0 100.0 100.0 0.00 0.45, 0.14 22:6n-3 98.0 96.1 96.0 94.9 94.8 0.99 0.14, 0.23 SFA 90.9 89.7 93.0 92.0 90.9 0.86 0.01, 0.72 MUFA 91.5 90.7 93.9 93.1 93.2 0.69 0.14, 0.23 PUFA 94.8 94.1 95.7 96.2 97.2 0.67 0.18, 0.17 LC-PUFA 98.6 97.7 97.6 95.6 95.3 0.89 0.18, 0.17 n-3 98.6 97.3 97.6 95.9 96.4 0.74 0.13, 0.25 n-6 93.9 93.9 95.5 96.1 97.0 0.75 0.25, 0.10 Nutrient deposition (%) Protein 33.5 31.0 33.7 34.2 35.2 0.01 0.04, 0.47 Lipid 64.2 82.1 73.9 87.2 81.5 0.03 0.31, * Energy 36.7 42.1 40.6 42.3 40.1 0.01 0.12, 0.22
P SEM, Pooled standard error of the mean; # Linear regression of all replicates with 1,13 df P < 0.05 *
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When compared to the initial samples the dry matter, lipid and gross energy
content of the carcass of all treatments were numerically higher upon termination of
the experiment (Table 3.4). However, there were no significant differences in the
final whole body composition of the fish fed the treatment diets. In terms of the fatty
acid composition, some significant effects among the dietary treatments were
observed (Table 3.4). The fatty acid composition of the whole body was in most
cases a reflection of the dietary treatments and changed accordingly. DPA was the
only exception to this as it was not detected in the diets containing less than 30% FO,
while whole body DPA levels were maintained at levels similar to that of the initial
fish (R2 = 0.76, P < 0.001). There was a significant difference in whole body ARA
(R2 = 0.47, P < 0.05) and this fatty acid appears to be well conserved compared to the
initial fish. Both EPA and DHA whole body proportions were significantly reduced
by the dietary treatments (R2 = 0.90, P < 0.001 and R2 = 0.76, P < 0.001,
respectively).There was no significant difference in SFA composition despite the
altered dietary profiles.
There were no significant differences in protein or energy retention efficiency
in the whole body among the treatment groups (Table 3.3). However, there was a
slight increase in lipid retention efficiency values with increasing PF. Whole body
retention efficiency of linoleic acid was not significantly affected by increasing
intake and the values ranged between 55% to 86% (y = 0.03x – 0.49, R2 = 0.13, P =
0.55, Figure 3.1a). Similarly, the retention efficiency of 18:3n-3 was not significantly
affected by increasing intake with the retention efficiency values ranging between
45% and 69% (y = 0.65x – 0.10, R2 = 0.65, P = 0.13, Figure 3.1b). There was
disproportionate retention of LC-PUFA’s including arachidonic acid (ARA), EPA
and DHA which are presented in Figures 3.1c-e. The ARA retention efficiency in the
whole body decreased sharply from 74% to 47% with increasing intake while the
dietary intake values of ARA were very low (y = -1.08x + 1.03, R2 = 0.81, P < 0.05).
Similarly, the retention efficiency of EPA decreased from 49% at the lowest dietary
inclusion to 32% at the highest inclusion (y = -0.04x + 0.52, R2 = 0.90, P < 0.05).
The whole body retention efficiency of DHA was 73% at the lowest dietary inclusion
levels then showed a curvilinear decline to around 37% (y = 0.72x-0.44, R2 =0.97, P <
0.05).
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Table 3. 4 Whole body chemical composition of the experimental fish on a live-weight basis. All data are g/kg unless otherwise stated. Fatty acid data are expressed as a percentage of total fatty acids (%).
Initial F100:
P0 F60: P40
F30: P70
F15: P85
F0: P100
P SEM
Regression R2, P#
Dry matter 28.9 33.2 35.7 35.5 35.4 35.2 0.01 0.24, 0.06 Crude protein 20.9 20.3 22.1 21.0 20.6 21.1 0.01 0.58, 0.10 Crude lipid 5.3 9.1 10.6 10.1 11.5 11 0.01 0.58, 0.10 Gross energy (MJ/kg) 6.3 8.1 8.9 8.9 9.1 8.7 0.18 0.15, 0.16 14:0 2.7 4.3 3.6 2.4 2.0 1.6 0.29 0.94, *** 16:0 22.9 23.8 26.1 23.6 23.2 23.6 0.32 0.02, 0.65 18:0 7.3 7.0 7.9 7.3 7.3 7.4 0.10 0.08, 0.36 16:1n-7 5.7 7.5 6.0 4.2 5.7 3.0 0.76 0.01, 0.71 18:1n-9 37.6 32.6 39.6 39.3 40.2 42.5 1.05 0.77, *** 18:2n-6 10.9 8.2 10.1 11.7 12.1 13.2 0.52 0.96, *** 18:3n-3 1.1 0.8 0.9 1.2 1.2 1.4 0.06 0.71, *** 20:4n-6 0.6 0.7 0.5 0.6 0.5 0.5 0.03 0.47, * 20:5n-3 1.7 3.9 2.3 1.9 1.3 0.8 0.36 0.90, *** 22:5n-3 1.0 1.3 0.9 0.9 0.7 0.6 0.09 0.76, *** 22:6n-3 3.8 4.2 2.7 2.7 2.2 1.9 0.33 0.76, *** SFA 34.1 36.6 38.9 34.3 33.3 33.3 0.59 0.30, 0.06 MUFA 44.8 41.8 41.9 44.9 47.2 46.6 0.74 0.76, *** PUFA 13.0 10.2 11.7 13.7 13.9 15.2 0.53 0.91, *** LC-PUFA 7.8 10.7 7.0 6.8 5.4 4.6 0.80 0.80, *** n-3 8.2 11.3 7.7 7.5 6.0 5.2 0.81 0.82, *** n-6 12.5 9.6 11.0 13.1 13.2 14.7 0.53 0.91, ***
P SEM, Pooled standard error of the mean; # Linear regression of all replicates with 1,13 df P < 0.05 *; P < 0.01 **; P < 0.001 ***
63 | P a g e
(a)
(c)
(e)
(d)
(b)
Figure 3. 1 Whole-body retention of polyunsaturated fatty acids a) 18:2n-6 (y = 0.03x + 0.49, R2 = 0.13, P = 0.55) and b) 18:3n-3 (y = 0.65x + 0.10, R2 = 0.60, P = 0.13) and whole-body retention of long-chain polyunsaturated fatty acids c) ARA (y = -1.08x + 1.03, R2 = 0.81, P < 0.05) , d) EPA (-0.04x + 0.52, R2 = 0.90, P < 0.05) and e) DHA (y = 0.72x-0.44, R2 = 0.97, P < 0.05).
64 | P a g e
3.3.2. Nutrient marginal efficiencies
The marginal efficiencies of nutrient utilisation were calculated using a bioenergetic
approach to determine the discrete effects on nutrient utilisation and maintenance
requirements for juvenile barramundi (Figures 3.2 a-e). The marginal efficiency of
18:2n-6 (k18:2n-6) was determined as 82% (y = 0.82x – 0.03, R2 = 0.75) and the
maintenance requirement estimated at 0.068 g/kg0.9/d. Similarly, the marginal
efficiency of 18:3n-3 (k18:3n-3) was determined as 104% (y = 1.04x – 0.01, R2 = 0.88)
and the maintenance requirement was estimated at 0.012 g/kg0.9/d. The marginal
efficiencies of the LC-PUFA’s were contrasting to those of the PUFA’s. The
marginal efficiency of ARA (kARA) was low at 19% (y = 0.19x + 0.005, R2 = 0.43)
and the regression suggests there was no maintenance requirement for this fatty acid.
Similarly, the marginal efficiencies of EPA and DHA (kEPA and DHA) were also low at
30% (y = 0.30x + 0.0, R2 = 0.95) and 27% (y = 0.27x + 0.01, R2 = 0.95), respectively
and the regression model suggested that there was also no maintenance requirement
for these LC-PUFA’s. A summary of the marginal efficiencies and the intake to gain
ratio of key fatty acids are presented in Table 3.5.
3.3.3. Fillet quality assessment
The consequence of changing dietary lipids in juvenile barramundi was evaluated in
terms of fillet quality using a standard sample of flesh (NQC). There were no
significant differences in macro nutrient composition of the NQC samples (Table
3.6). Overall, the dietary fatty acid profiles were closely reflected in the flesh with
the exception of DPA which was barely present in the diets F100:P0 and F60:P40
and not detected in the other diets. Consequently, some significant differences in
NQC fatty acid composition were observed (Table 3.6). Myristic acid (14:0) was the
only saturate affected showing a reduced concentration with increasing FO
substitution (R2 = 0.80, P < 0.001). Oleic acid (OLA; 18:1n-9) was significantly
increased with FO substitution (R2 = 0.50, P < 0.01). Both 18:2n-6 and 18:3n-3
composition increased with increasing FO substitution (R2 = 0.65, P < 0.001 and R2
= 0.58, P < 0.01, respectively). ARA composition in the fillet did not change;
however, n-3 LC-PUFA (EPA and DHA combined) deposition in the flesh showed
an increasing curvilinear response relative with increasing intake (Figure 3.3).
65 | P a g e
Table 3. 5 Summary of marginal efficiencies of specific fatty acids by juvenile barramundi when fed diets with varying blends of fish oil and poultry fat. Fatty acid Efficiency constant k R2 Intake:gain ratio 18:2n-6; Linoleic acid 0.82 0.75 1.2:1 18:3n-3; Linolenic acid 1.04 0.88 1.0:1 20:4n-6; Arachidonic acid 0.19 0.43 5.3:1 20:5n-3; Eicosapentaenoic acid 0.30 0.95 3.3:1 22:6n-3; Docosahexaenoic acid 0.27 0.95 3.7:1
66 | P a g e
Table 3. 6 Fillet (NQC) composition on a live-weight basis. All data are g/kg unless otherwise stated. Fatty acid profiles of NQC samples from each treatment are expressed as mg/100g meat.
F100:
P0 F60: P40
F30: P70
F15: P85
F0: P100
P SEM
Regression R2, P#
Dry matter 33.2 35.7 35.5 35.4 35.2 0.01 0.20, 0.10 Crude protein 26.5 28.5 28.3 28.3 27.4 0.01 0.08, 0.30 Crude lipid 4.9 5.2 5.6 6.3 5.4 0.01 0.15, 0.16 Gross energy (MJ/kg) 8.2 8.8 8.9 9.1 8.8 0.13 0.24, 0.06 14:0 4.0 2.9 2.1 1.8 1.5 0.24 0.80, *** 16:0 22.9 23.0 22.4 22.8 23.1 0.13 0.11, 0.22 18:0 7.1 7.4 7.2 7.3 7.4 0.09 0.14, 0.17 16:1n-7 7.1 6.2 5.6 5.4 5.1 0.18 0.15, 0.15 18:1n-9 30.9 34.4 37.8 39.3 40.8 0.96 0.50, ** 18:2n-6 8.4 10.3 11.9 12.6 13.2 0.46 0.65, *** 18:3n-3 0.9 1.1 1.2 1.3 1.2 0.04 0.58, ** 20:4n-6 1.0 0.9 0.8 0.7 0.7 0.03 0.15, 0.15 20:5n-3 5.2 3.7 2.4 1.7 1.0 0.41 0.84, *** 22:5n-3 1.7 1.4 1.1 0.9 0.7 0.10 0.62, *** 22:6n-3 6.2 5.2 4.0 3.4 2.8 0.36 0.61, *** SFA 35.0 34.1 32.4 32.5 32.7 0.35 0.02, 0.57 MUFA 39.5 41.9 44.8 45.9 47.1 0.75 0.38, * PUFA 10.6 12.5 14.1 14.7 15.1 0.45 0.56, ** LC-PUFA 14.2 11.2 8.5 6.7 5.2 0.90 0.71, *** n-3 15.1 12.2 9.4 7.6 5.8 0.93 0.67, *** n-6 9.6 11.5 13.3 13.9 14.5 0.48 0.64, ***
P SEM, Pooled standard error of the mean; # Linear regression of all replicates with 1,13 df P < 0.05 *; P < 0.01 **; P < 0.001 ***
67 | P a g e
Figure 3. 2 The linear relationship between the mass-independent intake relative to mass-independent gain (marginal efficiency) of specific fatty acids by juvenile barramundi. For comparative purposes the dotted line indicates the slope of 1.0 (y = x). a) 18:2n-6 (y = 0.82x - 0.03, R2 = 0.75) b) 18:3n-3 (y = 1.04x - 0.01, R2 = 0.88) c) ARA (y = 0.19x + 0.005, R2 = 0.43) d) EPA (y = 0.30x + 0.007, R2 = 0.95) e) DHA (y = 0.27x + 0.01, R2 = 0.95).
68 | P a g e
Figure 3. 3 Fillet (NQC) LC-PUFA deposition in juvenile barramundi expressed relative to intake (y = -1.54x2 + 56.33x + 75.23, R2 = 0.99) as mg/100 g meat. By extrapolation the dotted line indicates that a formulation of ~11% total LC-PUFA (EPA and DHA combined) would achieve a fillet composition of 500 mg/100 g.
3.4 Discussion
The benefits of regular consumption of seafood rich in n-3 LC-PUFA are well
known. These fatty acids are implicated in a range of physiological and metabolic
processes and many studies have demonstrated their positive benefit in the
prevention and management of cardio-vascular disease and inflammation (Calder,
2012). The FAO’s guideline to consume a dose of 250 mg/d EPA and DHA is an
achievable yet rarely met target due to the production and subsequent consumption
of the predominant vegetable oils including soybean, palm and canola (FAO, 2010).
This study investigated the potential effects of fish oil substitution with poultry fat in
diets fed to juvenile barramundi and provides empirical support for the development
of a strategy to manage the LC-PUFA content in the fillet. However, in addition to
this objective the study also provides some unique insights into fatty acid metabolism
in this species based on understanding the marginal utilisation of these nutrients and
comparing that with what we know about similar parameters for utilisation of other
nutrients.
This study demonstrated that the growth and feed utilisation of barramundi
was not significantly affected by the complete replacement of fish oil with poultry
fat. Until recently, variable responses to fish oil replacement and fatty acid
69 | P a g e
requirement studies have led to further questions being raised about lipid metabolism
in this species (Catacutan and Coloso, 1995; Glencross and Rutherford, 2011;
Morton et al., 2014; Raso and Anderson, 2003; Williams et al., 2006). The fish in
this study achieved a minimum 2.5-fold increase in weight over the twelve week
study period. Experimental duration is of particular interest in the realm of fatty acid
nutrition as the increasing use of alternative oils has a definitive influence on flesh
quality (Robin et al., 2003). The NRC (2011) suggest that 300% or a 3-fold increase
in weight should be respected and a range of other studies suggest different methods
of assessment (Glencross et al., 2003b; Jobling, 2004; Morton et al., 2014; Robin et
al., 2003). Clearly the fish in this study achieved sufficient biological turnover as
evidenced by the whole fish and flesh fatty acid profiles mirroring the diets similar to
that of other studies (Glencross et al., 2014b; Tu et al., 2013; Turchini et al., 2011b).
Moreover, past studies have demonstrated that fatty acid profiles of fish can be
manipulated in as little as two weeks (Castell et al., 1994; Skonberg et al., 1994).
Poultry fat (PF) is a commonly utilised alternative lipid source for aquafeeds
in Australia, having been widely used for over a decade, despite containing little or
no n-3 LC-PUFA. In this study, FO substitution with PF covered a range of LC-
PUFA inclusion from 22.0 to 4.8 g/kg, theoretically surpassing the reported
minimum requirement of 12 g/kg LC-PUFA (Williams et al., 2006). It is noteworthy
that this species is a catadromous, obligate carnivore and the reduced levels of LC-
PUFA fed in this study should probably have induced a range or EFA deficiency
symptoms similar to those previously reported (Catacutan and Coloso, 1995;
Glencross and Rutherford, 2011). Indeed, when other marine carnivorous fish were
fed diets with high levels of FO substitution, this led to growth retardation
(Glencross et al., 2003b; Izquierdo et al., 2005; Montero et al., 2005). We suggest
that the absence of problems seen in the present study may be reflective of the use of
larger fish which had previously been raised on high LC-PUFA diets and therefore
their underpinning n-3 LC-PUFA requirements in the demanding early juvenile
growth phase had already been met and that there was limited subsequent turnover.
Alternatively, it may also be that the actual requirement for EFA by this species is
much lower than previously thought. In this study, the digestibility of lipid and
individual fatty acids were not significantly affected. Further suggesting that despite
the contrasting differences in fatty acid profiles of PF and FO, the diets were digested
by barramundi equally efficiently.
70 | P a g e
The LC-PUFA content of the F0:P100 diet was only 3.5% of the total lipid
(equivalent to 4.8 g/kg diet) with this residual LC-PUFA coming from the fish meal
appearing sufficient to maintain biological functions in the fish. The inclusion of
15% fish meal in the present study is based on the findings of Glencross et al.
(2011b) as the use of a fully purified diet containing no fish meal was shown to be a
risky approach and lacks practical application (Tu et al., 2013). In light of this, the
results of this study demonstrate that in terms of growth performance, 100% of the
FO can be effectively replaced by poultry fat in growing barramundi, however,
discrete effects on deposition and marginal efficiencies of specific fatty acids were
apparent.
Bioenergetic models have come a long way to understanding how different
nutrients are deposited in the body of an animal and that macro nutrients such as
protein and lipid have different energetic efficiencies (Bureau et al., 2006). The
individual fatty acids have vastly different metabolic fates and hence are likely to
have different energetic efficiencies. For example, the n-3 and n-6 series
eicosapolyenoic fatty acids are implicated in a competing nature for the synthesis of
the autocrine hormones (Tocher, 2003). Arguably they may also have different
allometric relationships, as do protein and lipid, and this remains to be explored
(Glencross and Bermudes, 2011). In the present study we have relied on the
determined exponent of total lipid utilisation of LW0.90 to define the energetic
relationships of the different fatty acids, though we acknowledge that this is still an
assumption.
The present study demonstrated that the marginal efficiencies of LC-PUFA
(kDHA, kEPA and kARA) were 0.27, 0.30 and 0.19, respectively and the PUFA (k18:3n-3
and k18:2n-6) were significantly higher at 1.04 and 0.82, respectively. At first glance,
the examination of the retention and marginal efficiencies of LC-PUFA in this study
gives further evidence to the hypothesis that barramundi are unable to elongate and
desaturate precursor FA to form essential LC-PUFA (Mohd-Yusof et al., 2010). This
is evidenced by the direct accumulation of those fatty acids (18:2n-6 and 18:3n-3) in
a manner directly reflective of their intake levels, indicating no loss of these nutrients
from what was consumed.
Clearly in the case of DHA, EPA and ARA, retention efficiency in the whole
body decreased as dietary intake of those nutrients increased (Figure 1c-e). In
agreement, the marginal efficiencies of these nutrients were low compared to other
the shorter-chain and more saturated FA. These responses are likely due to the
71 | P a g e
physiological requirements of these nutrients in growing barramundi and suggest that
they were catabolised into other forms or used for energy. The strong conservation of
DHA in the whole body at low dietary intakes may be indicative of a priority for
retaining this nutrient (essentiality) as the organism attempts to retain what it has to
sustain necessary functions, but from our data it is implied that these necessary
functions require a transformation of the nutrient into another metabolite or energy
(Tocher, 2003). However, by extrapolating the marginal efficiency of the DHA
regression it appears as if there is no maintenance requirement for LC-PUFA in the
barramundi. This is contrasting to that identified for many other nutrients (and
energy), each of which have clear maintenance requirement levels.
This observation may be partly explained by the extrapolation method in
itself which has been found to slightly underestimate maintenance requirements
compared to a factorial approach (Bureau et al., 2006). However, it is also likely that
the dietary formulations in this study all supplied LC-PUFA in excess or at least
beyond the point of sensitivity for this species. Given that the barramundi is clearly
very effective at selective LC-PUFA retention it probably has a very low requirement
for these specific fatty acids. As discussed earlier, we are also assuming that the
relationships of the different fatty acids to animal live-weight is consistent with that
of total lipid (LW0.90), though this needs to be further validated and this, and other
further evidence may still be required to draw conclusions on the use of this method
to estimate baseline metabolic requirements of individual LC-PUFA.
Recent fatty acid studies have demonstrated similar metabolic responses for a
range of species. In Atlantic salmon (Glencross et al., 2014b; Torstensen et al.,
2004), barramundi (Glencross and Rutherford, 2011) and gilthead seabream
(Montero et al., 2001) there was a curvilinear decline in the retention efficiency of
DHA in response to increasing DHA level in the diet. In contrast, DHA and EPA
retention efficiency in the Atlantic cod (Gadus morhua) increased in response to
increasing dietary supply (Hansen et al., 2008) . In the latter example, the authors
argued that DHA was probably used as a substrate for energy production at lower
dietary intake. Many studies have demonstrated that despite up regulation of genes
involved in FA synthesis pathways, most fish species are unable to completely
compensate for a lack of dietary LC-PUFA (Alhazzaa et al., 2011b; Betancor et al.,
2014; Francis et al., 2007; Geay et al., 2010; Tu et al., 2013). Interestingly in this
study, the reduced retention of EPA may be attributable in part, to the apparent
concomitant appearance of DPA via elongation of C20 to C22. This is a likely
72 | P a g e
scenario as the barramundi attempts to synthesise DHA from dietary precursor fatty
acids in response to the treatment diets. However, to achieve this goal it requires a
Δ5 desaturation enzyme that probably does not exist in the species hence the building
up of DPA (Mohd-Yusof et al., 2010). Another possible explanation for the
accumulation of DPA may be that the animal is β-oxidising available DHA to EPA
for eicosanoid production and DPA is an intermediate in this process.
Moreover, the high retention and low marginal efficiency of ARA and EPA
could be related to the requirement for eicosanoid production under inadequate or
stressful dietary conditions. The eicosanoids particularly the n-6 series are implicated
in a range of physiological roles such as ion transfer and osmoregulation (Castell et
al., 1994) and the n-3 series eicosanoids have an anti-inflammatory role (Wall et al.,
2010). In addition, the majority of eicosanoid products are derived from ARA in fish
adding to the idea that ARA may be implicated in a range of biological processes in
barramundi (Henderson, 1996; Tocher, 2003). Few studies have investigated ARA
metabolism in barramundi, however, current evidence suggests that ARA may be
implicated in reduced growth and increased sub-clinical signs of essential fatty acid
deficiency (Glencross and Rutherford, 2011). In agreement, the results of the present
study emphasized the relative importance of ARA and future work in this area is
warranted.
Much awareness is now placed on the consumption of seafood, rich in LC-
PUFA, in an attempt to ameliorate a range of largely preventable diseases (Calder,
2012; Wall et al., 2010). Many nutritional feeding studies have focused on the
replacement of FO with alternative oils and also how this translates to the flesh
(Sales and Glencross, 2011; Turchini et al., 2009). Past studies have demonstrated
that fish fillet adiposity can be altered by the use of common vegetable oils (Bell et
al., 2002); however, the same response in this study was not found with poultry fat.
Fillet lipid levels remained constant among the treatment groups and the fillet fatty
acid profiles were largely reflective of the diets. The fatty acids deposited in the flesh
of barramundi were clearly proportional to their respective diet with few exceptions
which is typical of most fish (Turchini et al., 2009).
Given the limited ability of barramundi to desaturate and elongate precursor
FA to EPA and then DHA it was not surprising that these FA were strongly related to
the dietary composition. However, the relationship was best described as curvilinear
and the fillet concentration of LC-PUFA plateaued as the dietary concentration
exceeded 6% (Figure 3). There was a linear decrease in the fillet EPA concentration
73 | P a g e
with increasing replacement of the fish oil that appeared to be related to a slight
increase in DPA relative to the diet. The accumulation of DPA in the fillet is likely to
be a result of chain length modification in the liver followed by transport to the
adipose tissue for storage or eventual secondary processing, however, the effect was
minimal. Similarly, the fillet DHA concentration also decreased linearly with
increasing fish oil replacement, however, it was selectively retained in the meat at
levels higher than the dietary supply. Similarly, studies have shown that fillet DHA
levels in Atlantic salmon (Bell et al., 2001; Bell et al., 2002), European seabass
(Mourente and Bell, 2006), gilthead sea bream (Izquierdo et al., 2005) and Murray
cod (Turchini et al., 2011b) can be elevated relative to the diet. Possible mechanisms
underpinning this selective retention among species may be attributable to the high
specificity of fatty acyl transferases for DHA (Bell et al., 2001) and reduced
catabolism due to the complex peroxisomal β-oxidation required for the DHA
molecule (Tocher, 2003). Furthermore, this study showed that OLA and palmitic
acid (16:0) dominated within the flesh and these FA are known to be heavily
oxidised as energy substrates, potentially promoting more efficient accumulation of
other FA (Codabaccus et al., 2012; Turchini et al., 2011b).
By extrapolation, the results of this study allow the estimation of fillet FA
composition of barramundi (Figure 3). These observations are of practical use for
predicting the outcome of feeding diets with reduced levels of FO to barramundi and
promoting the most efficient use of the FO resource. It is estimated that feeding
barramundi with approximately 11% LC-PUFA would result in a fillet concentration
of 500 mg/100 g. The FAO recommend a minimum daily consumption of 250 mg of
n-3 LC-PUFA (FAO, 2010). In recognition of the FAO guideline, a 350 g fillet
portion would theoretically meet the weekly needs of adult consumers, aiming to
maintain a healthy and balanced diet.
74 | P a g e
4.0 Effect of dietary saturated and monounsaturated fatty acids in
juvenile barramundi Lates calcarifer
Adapted from
Salini, M.J., Turchini, G.M., Glencross, B.G., 2015. Effect of dietary saturated and
monounsaturated fatty acids in juvenile barramundi Lates calcarifer. Aquac. Nutr.
doi: 10.1111/anu.12389
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76 | P a g e
77 | P a g e
4.1 Aims
Consistent responses of a range of fish species indicate that SFA and MUFA
provided in surplus are heavily oxidised as substrates for energy, effectively sparing
LC-PUFA for biological needs and deposition. Likewise, it is unfavourable to
provide excess n-3 LC-PUFA, typical of FO, as these FA can also be readily β-
oxidised (Torstensen et al., 2000; Turchini et al., 2013). Recent studies have shown
that barramundi are capable of utilising poultry oil, characterised by high MUFA
content, however, to what extent SFA are preferentially utilised or able to ‘spare’
LC-PUFA remains unclear (Salini et al., 2015a). The proposed experiment examined
the effect of diets containing a 2:1 or 1:2 ratio of SFA to MUFA in the lipid as it was
hypothesised that these fatty acid classes would be metabolised differently. The
digestibility of the diets was also measured and mass-balance computations used to
estimate the fatty acid metabolism in vivo.
4.2 Materials and methods
The methodology used in the present experiment is detailed in the section 2.0
General methodology. Ethical clearance was approved for the experimental
procedures by the CSIRO Animal Ethics Committee (Application A12/2013). The
chemical composition of the main dietary ingredients is presented in Table 4.1. The
formulation and chemical composition of the three diets are presented in Table 4.2.
78 |
Pa
ge
Tab
le 4
. 1 C
hem
ical
com
posi
tion
of k
ey in
gred
ient
s, al
l val
ues a
re p
rese
nted
as g
/kg
DM
unl
ess o
ther
wis
e st
ated
.
DF
Fish
m
eal
Poul
try
mea
l So
y is
olat
e
Whe
at
glut
en
Whe
at
flour
C
asei
n
Whe
at
star
ch
Fish
oi
l O
live
oil
Palm
O
il
Palm
Fl
ake
Com
posi
tion
Dry
mat
ter (
g/kg
) 98
4 95
8 95
8 92
7 83
9 92
4 83
6 99
2 98
7 10
0 99
Prot
ein
789
641
895
823
112
870
5 4
4 3
4
Ash
16
3 13
8 46
1
6 11
3
1 N
D
ND
N
D
Li
pid
46
151
57
121
22
5 N
D
956
973
963
986
C
HO
# 1
70
2 55
86
0 11
3 99
2 39
23
34
10
Gro
ss e
nerg
y (M
J/kg
) 18
.9
20.4
21
.8
21.2
15
.3
21.9
14
.5
39.3
39
.5
39.5
39
.3
Fatty
aci
ds (%
) ^
16:0
25
.8
25.4
17
.8
- -
- -
22.9
9.
9 51
.9
46.2
18:0
8.
7 8.
6 4.
5 -
- -
- 5.
1 3.
0 4.
6 51
.3
18
:1
15.5
43
.8
25.1
-
- -
- 18
.6
73.8
34
.6
0.4
18
:2n-
6 1.
7 11
.0
46.5
-
- -
- 2.
0 11
.0
6.7
ND
18:3
n-3
0.8
1.0
5.1
- -
- -
1.0
1.0
ND
N
D
20
:4n-
6 2.
5 0.
6 N
D
- -
- -
1.5
ND
N
D
ND
20:5
n-3
9.0
0.5
ND
-
- -
- 11
.3
ND
N
D
ND
22:5
n-3
2.2
ND
N
D
- -
- -
2.1
ND
N
D
ND
22:6
n-3
19.9
N
D
ND
-
- -
- 14
.2
ND
N
D
ND
SFA
39
.8
36.1
22
.6
- -
- -
36.4
13
.4
58.6
99
.6
M
UFA
22
.2
50.8
25
.8
- -
- -
29.1
74
.6
34.7
0.
4
C18
PUFA
3.
4 12
.0
51.6
-
- -
- 4.
9 12
.0
6.7
ND
LC-P
UFA
34
.5
1.2
ND
-
- -
- 29
.7
ND
N
D
ND
n-3
32
.7
1.6
5.1
- -
- -
30.5
1.
0 N
D
ND
n-6
5.
2 11
.6
46.5
-
- -
- 4.
1 11
.0
6.7
ND
# C
HO
, car
bohy
drat
e ca
lcul
ated
by
diff
eren
ce (e
g. C
HO
= 1
000
– (p
rote
in +
lipi
d +
ash)
^
All
fatty
aci
ds a
re p
rese
nted
as a
per
cent
age
of th
e to
tal f
atty
aci
ds. Q
uant
itativ
e da
ta c
an b
e ob
tain
ed b
y m
ultip
lyin
g th
e to
tal F
A (m
g/g
lipid
) by
spec
ific
fatty
aci
ds (%
). 1
8:1,
sum
of 1
8:1n
-7, 1
8:1n
-9 c
is, 1
8:1n
-9 tr
ans;
satu
rate
d fa
tty a
cids
(SFA
), su
m o
f 12:
0, 1
4:0,
16:
0, 1
8:0,
20:
, 22:
0, 2
4:0;
m
onou
nsat
urat
ed fa
tty a
cids
(MU
FA),
sum
of1
4:1n
-5, 1
6:1n
-7, 1
8:1n
-7, 1
8:1n
-9 (c
is a
nd tr
ans)
, 20:
1n-7
, 20:
1n-9
, 22:
1n-9
, 24:
1n-9
; pol
yuns
atur
ated
fa
tty a
cids
(PU
FA),
sum
18:
2n-6
(cis
and
tran
s), 1
8:3n
-6, 1
8:3n
-3, 1
8:4n
-3; l
ong
chai
n po
lyun
satu
rate
d fa
tty a
cids
(LC
-PU
FA),
sum
20:
2n-6
, 20:
3n-
6, 2
0:4n
-6, 2
2:4n
-6, 2
-:3n-
3, 2
0:5n
-3, 2
2:5n
-3, 2
2:6n
-3; n
-3 a
nd n
-6, s
um o
f om
ega
3 an
d 6
PU
FA a
nd L
C-P
UFA
.
79 | P a g e
Table 4. 2 Formulation and composition of experimental diets. All values are g/kg DM unless otherwise stated.
CTRL-D (Fish oil)
SFA-D (Palm oil)
MUFA-D (Olive oil)
Formulation DF Fish meal a 150 150 150 Poultry meal a 150 150 150 Soy protein isolate b 150 150 150 Wheat gluten b 150 150 150 Wheat flour b 109 109 109 Casein c 100 100 100 Pregelled wheat starch b 80 80 80 DL-Methionine c 10 10 10 Di-calcium phosphate 10 10 10 Pre-mix vitamins and minerals d 8 8 8 Yttrium oxide e 1 1 1 Fish oil a 82 41 41 Olive oil f 0 0 41 Palm Oil f 0 15 0 Palm Flake f 0 26 0 Composition as analysed Dry matter (g/kg) 940 952 979 Protein 598 601 585 Ash 64 63 60 Lipid 126 137 135 CHO # 204 192 217 Gross energy (MJ/kg DM) 21.5 21.6 22.2 Fatty acids (%) ^ Total FA (mg/g lipid) 801.7 734.7 717.4 16:0 22.9 32.0 18.4 18:0 5.5 16.1 4.8 18:1 22.7 20.6 42.6 18:2n-6 10.0 10.5 13.3 18:3n-3 1.3 1.0 1.3 20:4n-6 1.2 0.7 0.7 20:5n-3 8.1 4.1 4.0 22:5n-3 1.6 0.9 0.9 22:6n-3 10.6 5.4 5.4 SFA 34.5 51.8 26.4 MUFA 31.3 25.0 47.3 C18PUFA 12.7 12.1 15.3 LC-PUFA 21.5 11.0 10.9 n-3 22.9 12.0 12.2 n-6 11.3 11.1 14.0
# CHO, carbohydrate calculated by difference (eg. CHO = 1000 – (protein + lipid + ash). ^ refer to Table 4.1 for details. a Ridley aquafeeds, Narangba, QLD, Australia b Manildra Group, Rocklea, QLD, Australia c Bulk Powders, www.bulkpowders.com.au d Vitamin and mineral premix (IU kg-1 or g/kg of premix): vitamin A, 2.5MIU; vitamin D3, 0.25 MIU; vitamin E, 16.7 g; vitamin K3, 1.7 g; vitamin B1, 2.5 g;
80 | P a g e
vitamin B2, 4.2 g; vitamin B3, 25 g; vitamin B5, 8.3; vitamin B6, 2.0 g; vitamin B9, 0.8; vitamin B12, 0.005 g; biotin, 0.17 g; vitamin C, 75 g; choline, 166.7 g; inositol, 58.3 g; ethoxyquin, 20.8 g; copper, 2.5 g; ferrous iron, 10.0 g; magnesium, 16.6 g; manganese, 15.0 g; zinc, 25.0 g e Yttrium oxide; Stanford Materials, Aliso Viejo, California, United States f Sydney Essential Oil Co. (Sydney, NSW, Australia)
81 | P a g e
4.3 Results
4.3.1 Growth and feed utilisation
During the 56 d growth assay, the fish in all treatments responded readily to the
experimental diets and growth in the CTRL-D group was consistent with the
predicted model growth, achieving 106% of the modelled potential (Glencross and
Bermudes, 2012). Live-weight measurements were conducted after four weeks of
feeding and then again after eight weeks and although there was a tendency towards
lower growth in the SFA-D and MUFA-D fish at both time points, there were no
significant differences (Table 4.3). The same trend was observed in terms of daily
feed intake and growth rate with no significant differences and there was no
difference in FCR values (Table 4.3). There were no differences in terms of survival
with only one fish that died and was removed from the system over the term of the
experiment (Table 4.3).
The apparent digestibility of macro nutrients and specific fatty acids was
affected by the diets (Table 4.4). Both dry matter (DM) and lipid were significantly
less digestible in the SFA-D fed group of fish. There were no significant differences
in either protein or gross energy (GE) digestibility among the treatments (Table 4.4).
There were no significant differences in the digestibility of individual and total LC-
PUFA among the diets, each showing almost complete digestion in most cases.
Similarly, there were no significant differences in the digestibility of individual and
total PUFA and 18:1. However, the saturated fatty acids were significantly affected
with 18:0 and 16:0 being less digestible in the SFA-D fed fish compared to the other
treatments. The total SFA digestibility was also significantly reduced in the SFA-D
fed fish (Table 4.4).
82 | P a g e
Table 4. 3 Growth performance and feed utilisation of juvenile barramundi fed experimental diets for eight weeks. All data (n=3 per treatment) are presented as mean ± SEM.
CTRL-D SFA-D MUFA-D TEST ^ Week 0 weight (g) 46.9 ± 0.1 47.3 ± 0.2 47.1 ± <0.1 F=2.2, P=0.20 Week 4 weight (g) 139.2 ± 1.5 135.3 ± 7.8 136.0 ± 3.0 F=0.9, P=0.45 Week 8 weight (g) 238.3 ± 1.2 231.2 ± 9.0 230.9 ± 6.2 F=0.4, P=0.66 Feed intake (g/fish) 209.6 ± 2.7 202.4 ± 5.0 200.7 ± 4.9 F=0.6, P=0.58 Growth rate (g/fish/d) 3.4 ± <0.1 3.3 ± 0.2 3.3 ± 0.1 F=0.5, P=0.65 FCR 1.10 ± <0.1 1.12 ± <0.1 1.09 ± <0.1 F=2.0, P=0.21 Survival (%) 98.0 ± 0.1 100.0 ± 0.0 100.0 ± 0.0 F=1.0, P=0.42 Protein retention (%) 34.1 ± 1.1 29.8 ± 1.9 32.5 ± 0.9 F=2.5, P=0.16 Lipid retention (%) 48.6 ± 4.5 40.2 ± 1.7 45.9 ± 0.7 F=2.4, P=0.17 Energy retention (%) 37.8 ± 0.6a 33.5 ± 1.0b 34.4 ± 0.3b F=10.1*
Values <0.1 are reported as <0.1. ^ One-way ANOVA, DF 2,6, post-hoc Tukey’s HSD. P<0.05 *. Superscript letters indicate different levels of significance between treatment diets, percentage data were arcsine transformed prior to analysis.
83 | P a g e
Table 4. 4 Apparent digestibility coefficients of macro nutrients and specific fatty acids of experimental diets fed to juvenile barramundi. All data (n=3 per treatment) are reported as apparent digestibility percentage (%)
CTRL-D SFA-D MUFA-D TEST ^ Macro nutrient digestibility (%) Dry matter 64.3 ± 1.6ab 60.3 ± 1.5b 69.4 ± 0.2a F=9.6* Protein 91.1 ± 0.4 92.0 ± 1.2 92.1 ± 1.8 F=0.2, P=0.85 Lipid 91.0 ± 0.9a 73.7 ± 2.6b 92.9 ± 1.4a F=21.8** Gross energy 87.3 ± 0.4 82.4 ± 3.3 89.2 ± 2.0 F=1.6, P=0.31 Fatty acid digestibility (%) 16:0 82.1 ± 1.7a 59.2 ± 3.8b 86.6 ± 2.1a F=17.7* 18:0 77.5 ± 4.1a 42.4 ± 5.4b 82.1 ± 3.2a F=15.5* 18:1 92.4 ± 0.3 89.2 ± 1.5 94.3 ± 1.2 F=3.6, P=0.12 18:2n-6 96.0 ± 0.1 94.3 ± 0.8 96.3 ± 1.1 F=1.5, P=0.33 18:3n-3 98.6 ± 1.4 100.0 ± 0.0 97.7 ± 0.8 T=0.6, P=0.60 20:4n-6 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 N/A 20:5n-3 99.1 ± <0.1 100.0 ± 0.0 98.6 ± 0.5 T=0.9, P=0.45 22:5n-3 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 N/A 22:6n-3 98.5 ± 0.2 100.0 ± 0.0 97.3 ± 1.0 T=1.1, P=0.35 SFA 82.9 ± 2.0a 55.6 ± 4.2b 86.4 ± 2.2a F=18.3** MUFA 92.9 ± 0.5 90.0 ± 1.3 94.3 ± 1.2 F=2.9, P=0.17 C18PUFA 96.7 ± 0.1 95.0 ± 0.7 96.6 ± 1.0 F=1.2, P=0.38 LC-PUFA 98.9 ± 0.1 100.0 ± 0.0 98.1 ± 0.7 T=1.1, P=0.40 n-3 98.9 ± <0.1 100.0 ± 0.0 98.1 ± 0.7 T=1.2, P=0.36 n-6 96.4 ± 0.1 94.6 ± 0.8 96.5 ± 1.1 F=1.5, P=0.32
Values <0.1 are reported as <0.1. ^ P<0.05*, P<0.01**, P<0.001***; One-way ANOVA, DF 2,6, post-hoc Tukey’s HSD; T-test, DF 4, was used to test two variables; Superscript letters indicate different levels of significance between treatment diets, percentage data were arcsine transformed prior to analysis.
84 | P a g e
4.3.2 Biochemical analysis
The whole-body DM, protein and lipid composition on a wet weight basis was not
significantly affected by the diets; however, the GE was significantly lower in the
SFA-D fed fish (Table 4.5). Whole-body total fatty acids were not significantly
different; however, there were some significant differences between the specific fatty
acids. Among the LC-PUFA the whole-body 22:6n-3 and 22:5n-3 was significantly
higher in the CTRL-D fish compared to the MUFA-D fish. The whole-body 18:2n-6
was significantly highest in the MUFA-D and lowest in the CTRL-D (Table 4.5).
The whole-body 18:1 was significantly highest in the MUFA-D fed fish whereas
18:0 and 16:0 were lowest in the MUFA-D fed (Table 4.5). The total LC-PUFA and
the total n-3 fatty acids were significantly lowest in the MUFA-D fed fish (Table
4.5).
The liver fatty acid composition was affected in a similar fashion to that of
the whole-body where 22:6n-3, 22:5n-3, 20:5n-3 and 20:4n-6 had significantly
higher composition in the CTRL-D fed fish. However, there were no significant
differences among the MUFA-D or SFA-d diets (Table 4.5). There was no
significant difference in PUFA composition of the liver. The total LC-PUFA and n-3
composition were significantly higher in the CTRL-D fish and there was no
significant difference in n-6 composition of the liver (Table 4.5).
85 |
Pa
ge
Tab
le 4
. 5 W
hole
bod
y co
mpo
sitio
n (n
=3 p
er tr
eatm
ent,
g/kg
live
bas
is).
Who
le b
ody
and
liver
fatt
y ac
id d
ata
(n=3
per
trea
tmen
t, %
tota
l).
Initi
al
CTR
L-D
SF
A-D
M
UFA
-D
TEST
^
Who
le b
ody
com
posi
tion
Dry
mat
ter
265
± 0.
4 32
9 ±
0.5
318
± 0.
3 32
4 ±
0.3
F=2.
1, P
=0.2
1
Prot
ein
188
± 0.
5 20
6 ±
0.7
191
± 0.
8 20
0 ±
0.6
F=1.
2, P
=0.3
6
Lipi
d 51
± 0
.4
95 ±
0.6
91
± 0
.2
94 ±
0.2
F=
0.3,
P=0
.73
G
ross
ene
rgy
(MJ/
kg)
61 ±
0.2
84
± 0
.2a
77 ±
0.1
b 79
± <
0.1ab
F=
8.0*
W
hole
bod
y fa
tty a
cids
(%) #
Tota
l FA
(mg/
g lip
id)
681.
0 ±
35.1
61
6.6
± 19
.2
665.
7 ±
68.1
64
7.7
± 44
.7
F=0.
4, P
=0.6
9
16:0
26
.1 ±
0.2
26
.9 ±
0.5
a 28
.0 ±
0.2
a 22
.4 ±
0.1
b F
=74.
3***
18:0
7.
8 ±
0.2
7.4
± 0.
1b 9.
9 ±
0.2c
6.4
± <0
.1a
F=1
48.8
***
18
:1
37.9
± 0
.8
30.1
± 0
.5a
30.9
± 0
.2a
41.7
± 0
.1b
F=37
1.0*
**
18
:2n-
6 9.
7 ±
0.3
8.8
± 0.
2a 10
.2 ±
0.2
b 11
.0 ±
0.1
c F
=37.
4***
18:3
n-3
1.0
± 0.
1 1.
0 ±
<0.1
1.
0 ±
<0.1
1.
1 ±
<0.1
F
=3.3
, P=0
.11
20
:4n-
6 0.
4 ±
0.0
0.7
± <0
.1a
0.5
± <0
.1b
0.5
± <0
.1b
F=14
.7**
20:5
n-3
1.4
± 0.
1 3.
7 ±
0.3a
2.6
± 0.
1b 2.
2 ±
0.1b
F=1
3.1*
*
22:5
n-3
0.8
± 0.
0 1.
6 ±
0.1a
1.3
± 0.
1ab
1.2
± <0
.1b
F=5
.3*
22
:6n-
3 2.
1 ±
0.2
5.5
± 0.
6a 4.
3 ±
0.2ab
3.
8 ±
<0.1
b F
=6.1
*
SFA
38
.5 ±
0.2
39
.3 ±
0.7
a 41
.4 ±
0.4
a 31
.9 ±
<0.
1b F=
102.
4***
MU
FA
45.7
± 0
.5
37.7
± 0
.7a
36.6
± 0
.1a
46.6
± 0
.1b
F19
8.1*
**
C
18PU
FA
11.0
± 0
.1
11.7
± 0
.3a
13.2
± 0
.3b
13.9
± 0
.1b
F=26
.6**
LC-P
UFA
4.
7 ±
0.2
11.4
± 1
.1a
8.8
± 0.
4a 7.
6 ±
0.1b
F=7.
7*
n-
3
4.3
± 0.
0 11
.9 ±
1.2
a 9.
3 ±
0.4a
8.2
± 0.
1b F=
7.0*
n-6
11
.4 ±
0.1
11
.2 ±
0.2
a 12
.7 ±
0.2
b 13
.4 ±
0.1
b F
=38.
1***
Li
ver f
atty
aci
ds (%
) #
16
:0
27.8
± 0
.4
30.8
± 0
.4ab
33
.7 ±
0.7
b 27
.6 ±
1.3
a F=
12.9
**
18
:0
11.5
± 0
.2
10.6
± 0
.2a
13.9
± 0
.4b
9.8
± 0.
8a F
=17.
5**
18
:1
34.8
± 0
.3
31.1
± 0
.1a
33.3
± 0
.8a
39.8
± 0
.6b
F=5
9.3*
**
86 |
Pa
ge
18
:2n-
6 6.
4 ±0
.2
4.8
± 0.
2 4.
3 ±
0.6
6.9
± 1.
2 F=
3.0,
P=0
.12
18:3
n-3
0.6
± 0.
0 0.
5 ±
<0.1
0.
4 ±
0.1
0.6
± 0.
2 F=
1.5,
P=0
.29
20:4
n-6
0.7
± 0.
0 0.
8 ±
<0.1
a 0.
5 ±
<0.1
b 0.
5 ±
<0.1
b F
=37.
4***
20:5
n-3
1.7
± 0.
1 2.
7 ±
0.1a
1.3
± 0.
1b 1.
5 ±
0.3b
F=1
8.0*
*
22:5
n-3
1.1
± 0.
1 1.
4 ±
0.1a
0.7
± 0.
1b 0.
9 ±
0.1b
F=1
4.4*
*
22:6
n-3
4.4
± 0.
1 4.
8 ±
0.1a
2.7
± 0.
2b 2.
8 ±
0.3b
F=1
5.0*
*
SFA
42
.9 ±
0.3
45
.5 ±
0.5
ab
50.5
± 0
.9b
40.5
± 1
.8a
F=17
.4**
MU
FA
40.0
± 0
.3
37.9
± 0
.3a
38.2
± 0
.7a
44.8
± 0
.6b
F=48
.9**
*
C
18PU
FA
8.5
± 0.
1 6.
2 ±
0.2
5.5
± 0.
7 8.
4 ±
1.2
F=3.
4, P
=0.1
0
LC
-PU
FA
8.6
± 0.
1 10
.4 ±
0.1
a 5.
7 ±
0.4b
6.3
± 0.
7b F
=29.
6***
n-3
8.
6 ±
0.1
9.7
± 0.
1a 5.
6 ±
0.5b
6.1
± 0.
9b F
=17.
6**
n-
6
8.5
± 0.
2 6.
8 ±
0.2
5.7
± 0.
7 8.
6 ±
1.0
F=3
.9, P
=0.0
8 V
alue
s <0.
1 ar
e re
porte
d as
<0.
1.
^ P<
0.05
*, P
<0.0
1**,
P<0
.001
***;
One
-way
AN
OV
A, D
F 2,
6, p
ost-h
oc T
ukey
’s H
SD; S
uper
scrip
t let
ters
indi
cate
diff
eren
t lev
els o
f sig
nific
ance
be
twee
n tre
atm
ent d
iets
, per
cent
age
data
wer
e ar
csin
e tra
nsfo
rmed
prio
r to
anal
ysis
.. # R
efer
to T
able
1 fo
r det
ails
.
87 | P a g e
4.3.3 Mass-balance computations
There were no significant differences in protein or lipid retention, however, there
was significantly higher energy retention in the CTRL-D fish (Table 4.3). There was
significantly higher β-oxidation of specific n-3 series fatty acids (22:6n-3, 20:5n-3
and 18:4n-3) and n-6 series fatty acids (20:4n-6) in the CTRL-D diet compared to the
other treatment diets (Table 4.6). There was no recorded β-oxidation for any of the
dominant saturates (including 12:0, 14:0, 16:0 and 18:0); however, there was a
significant increase in β-oxidation of n-7 series FA (16:1n-7 and 18:1n-7) in the
CTRL-D diet compared to the other treatment diets (not shown). There was a
significant increase in the β-oxidation of 18:1 fatty acids in the MUFA-D diet
compared to the other treatment diets (Table 4.6).
There was significantly higher fatty acid de novo production of 12:0
(neogenesis) in the MUFA-D fed fish compared to the other treatment diets (Table
6). The recorded elongation activity of 14:0 was significantly higher in the CTRL-D
and MUFA-D fed fish while the elongation of 16:0 was highest in the CTRL-D and
SFA-D fed fish. Elongation of 20:5n-3 to 22:5n-3 was only recorded in the fish fed
the SFA-D and MUFA-D diets and there was no significant difference (Table 4.6).
The Δ-9 desaturation activity was significantly highest in the SFA-D fed fish
followed by the control fed fish and there was none recorded in the MUFA-D fed
fish (Table 4.6). There was no Δ-5 or Δ-6 desaturation activity detected in any of the
diet treatments (data not presented).
Relative to the total intake of specific fatty acids, there was a significantly
greater proportion of total LC-PUFA deposited in the SFA-D and MUFA-D fed fish;
however, there was no difference between these two groups (Table 4.7). Likewise,
there was proportionally less β-oxidation in the SFA-D and MUFA-D fish; however,
there was no difference between these two groups (Table 4.7). There was only a
minor proportion of the consumed LC-PUFA converted or excreted in the groups of
fish.
88 | P a g e
Table 4. 6 Whole-body fatty acid mass balance computations of β-oxidation, elongation and desaturation activity of juvenile barramundi fed experimental diets for eight weeks. All data (n=3 per treatment) are reported on a nmol/g fish/d basis.
ND, not detected, N/A, not analysed, values <0.1 are reported as <0.1. # Computations following Turchini et al (2007). Refer to Table 4.1 for details. ^ P<0.05*, P<0.01**, P<0.001***; One-way ANOVA, DF 2,6, post-hoc Tukey’s HSD; T-test, DF 4, was used to test two variables. Superscript letters indicate different levels of significance between treatment diets, percentage data were arcsine transformed prior to analysis.
CTRL-D SFA-D MUFA-D TEST ^ β-oxidation # 16:0 ND ND ND N/A 18:0 ND ND ND N/A 18:1 54.0 ± 2.8a 7.7 ± 3.9a 580.4 ± 48.6b F=127.6*** 18:2n-6 298.3 ± 16.8 312.5 ± 27.4 240.5 ± 14.5 F=3.5, P=0.09 18:3n-3 ND ND ND N/A 20:4n-6 64.3 ± 3.3a 25.8 ± 1.3 b 27.6 ± 0.7b F=108.0*** 20:5n-3 482.9 ± 23.9a 183.7 ± 12.8b 167.7 ± 4.8b F=125.0*** 22:5n-3 25.7 ± 9.9 ND ND N/A 22:6n-3 631.3 ± 50.9a 220.5 ± 17.3b 187.9 ± 3.7b F=63.0*** SFA ND ND ND N/A MUFA 348.4 ± 12.9b 38.5 ±13.8c 639.9 ± 60.9a F=69.8*** C18PUFA 558.1 ± 32.0a 382.9 ± 44.0b 492.7 ± 19.8a F=7.3* LC-PUFA 1623.6 ± 115.5a 574.1 ± 37.9b 508.6 ± 15.4b F=78.4*** n-3 1616.3 ± 116.4a 560.1 ± 38.3b 494.7 ± 15.1b F=78.2*** n-6 565.4 ± 31.1a 396.9 ± 43.6b 506.6 ± 20.1a F=7.1* Neogenesis # 12:0 188.3 ± 31.6a 166.0 ± 29.8a 312.3 ± 22.6b F=7.7* Elongation # 12:0 202.6 ± 28.8 184.9 ± 29.9 285.5 ± 22.4 F=3.1, P=0.12 14:0 350.8 ± 30.0b 241.4 ± 30.2a 363.7 ± 20.0b F=6.1* 16:0 216.8 ± 17.7b 235.9 ± 15.6b 107.2 ± 3.6a F=25.4** 20:5n-3 ND 13.9 ± 4.8 12.9 ± 1.1 T=0.5, P=0.55 Δ-9 desaturation # 18:0 98.0 ± 22.7b 288.3 ± 28.6a ND T=5.2**
89 | P a g e
Table 4. 7 Calculated summary of LC-PUFA flux in juvenile barramundi. All data are (n=3) reported as a percentage based on the total intake of each fatty acid (nmol/g fish/d).
CTRL-D SFA-D MUFA-D TEST ^ 22:6n-3# % Converted ND ND ND N/A % Oxidised 58.1 ± 5.1a 38.6 ± 3.5b 36.7 ± 0.4b F=10.9*
% Excreted 1.5 ± 0.1 ND 2.7 ± 0.6 T=2.0, P=0.11
% Deposited 40.4 ± 5.0 a 61.4 ± 3.5 b 60.6 ± 0.3 b F=11.4** 22:5n-3 % Converted ND ND ND N/A % Oxidised 20.5 ± 8.0 ND ND N/A % Excreted ND ND ND N/A % Deposited 79.5 ± 8.0 100 ± 0.0 100 ± 0.0 N/A
20:5n-3 % Converted ND 3.5 ± 1.2 3.6 ± 0.3 T=0.1, P=0.96
% Oxidised 63.4 ± 3.7 a 47.3 ± 3.9 b 47.3 ± 1.1 b F=8.7*
% Excreted 1.0 ± 0.1 ND 1.4 ± 0.2 T=1.5, P=0.21
% Deposited 35.6 ± 3.6 a 49.2 ± 2.7 b 47.6 ± 0.7 b F=7.8*
n-3 LC-PUFA % Converted ND 1.2 ± 0.4 1.2 ± 0.1 T=0.1, P=0.96
% Oxidised 47.3 ± 5.6 a 28.6 ± 2.5 b 28.0 ± 0.5 b F=9.7*
% Excreted 0.8 ± 0.1 ND 1.4 ± 0.3 T=1.8, P=0.14
% Deposited 51.8 ± 5.5 a 70.2 ± 2.1 b 69.4 ± 0.2 b F=9.2* ND, not detected, N/A, not analysed. ^ P<0.001*** P<0.01** P<0.05*; One-way ANOVA, DF 2,6, post-hoc Tukey’s HSD; T-test, DF 4, was used to test two variables; Superscript letters indicate different levels of significance between treatment diets, percentage data were arcsine transformed prior to analysis. #Assumed that no further conversion occurs (Turchini et al 2007).
90 | P a g e
4.4 Discussion
The present study demonstrated that the inclusion of a 2:1 or 1:2 ratio of either SFA
or MUFA, did not affect the growth performance of juvenile barramundi. The
simultaneous reduction of dietary LC-PUFA (~11% LC-PUFA in SFA- and MUFA-
D vs 21.5% LC-PUFA in CTRL-D), provided that they were still above reported
requirements of 1.2% LC-PUFA, also had no effect on performance (Williams et al.,
2006). Moreover, the feed intake and FCR values were unaffected. This is consistent
with a range of species showing that substitution of FO with lipid rich in either SFA
or MUFA does not affect fish growth performance (Turchini et al., 2009). The fish in
the present study more than tripled in size suggesting that trial duration or nutrient
turnover was not a confounding issue. The fish in the SFA-D and MUFA-D
treatments did show a numerical reduction in growth; however, this was not
confirmed statistically. It is uncertain whether longer trial duration would have
resulted in significant differences as recent studies with barramundi have
demonstrated that changes to the lipid profile of the diets can have rapid metabolic
effects (Salini et al., 2015c). Lipid and in particular the saturated fatty acids are
generally less digestible at lower environmental temperature and as a result less
energy is available for growth (Ng et al., 2004; Olsen and Ringø, 1998). The
barramundi is a tropical species adapted to high water temperature and potentially
better able to cope with dietary SFA and MUFA rich lipid.
The replacement of FO with MUFA rich lipid such as that from poultry oil
had no effect on growth or FCR in a range of species including rainbow trout
(Oncorhynchus mykiss) juveniles (Fonseca-Madrigal et al., 2005), post-smolt
Atlantic salmon (Salmo salar) (Bell et al., 2002) or large 1.5 kg Atlantic salmon
(Torstensen et al., 2000), juvenile red hybrid tilapia (Oreochromis sp.) (Bahurmiz
and Ng, 2007) humpback grouper (Cromileptes altivelis) (Shapawi et al., 2008) and
African catfish (Clarias gariepinus) (Ng et al., 2003). However, consistent among
these studies when FO was completely replaced by MUFA rich lipid was the
modified tissue FA profile resulting in reduced concentration of the beneficial n-3
LC-PUFA. The same effect was clearly noted in the present study. However,
proportional to the LC-PUFA intake, the results of most studies and those of the
present study conclude that MUFA rich lipid is an ideal energy source capable of
‘sparing’ the more valuable LC-PUFA from β-oxidation.
An important consideration when formulating with SFA rich palm oil (PO) is
the fraction used (Ng and Gibon, 2011). Past studies demonstrated that growth
91 | P a g e
performance (Ng et al., 2003; Shapawi et al., 2008) and digestibility of a range of
species was not affected by different PO fractions (Bahurmiz and Ng, 2007) while
the digestibility of palm products was significantly reduced compared to FO. Recent
studies have also concluded that other sources of lipid rich in SFA such as beef
tallow did not affect the growth of Atlantic salmon (Emery et al., 2014) or rainbow
trout (Trushenski et al., 2011). Consistent with these reports, the present study
demonstrated that the blend of palm products used did not compromise growth
performance in barramundi while there were notable reductions to the lipid and
specific fatty acid digestibility.
In the present study, the digestibility of total lipid was significantly reduced
in the SFA fed fish and this did not lead to a reduction in energy availability or any
changes in whole-body lipid composition or retention. The reduction in lipid
digestibility was evidently a result of the greatly reduced digestibility of the
saturates, including both 16:0 and 18:0 FA. This is in agreement with other studies
showing that the digestibility of the saturated fatty acids was reduced when a range
of species were fed SFA rich oil (Caballero et al., 2002; Ng et al., 2004; Torstensen
et al., 2000; Turchini et al., 2013). Moreover, in the present study the digestibility of
SFA decreased with increasing chain length, consistent with other studies (Caballero
et al., 2002; Johnsen et al., 2000; Ng et al., 2004). The reduced rate of lipolysis and
absorption of longer chain SFA, such as 18:0 is caused by the lower ability of these
FA to form lipid emulsions prior to digestion (Olsen et al., 1998).
In contrast to the present study, negative effects of the high level inclusion of
SFA (e.g. as occurs with high level use of PO) have been reported and that these
effects are arguably more pronounced in carnivorous and/or marine fish with higher
n-3 LC-PUFA requirements. Fountoulaki et al. (2009) found that gilthead sea bream
growth was negatively impacted by reduced digestibility of SFA rich lipid over an
extended growth trial of six months. Japanese seabass (Lateolabrax japonicas)
growth performance was significantly reduced with a high inclusion of SFA rich
lipid after only 50 days of growth and similarly reduced digestibility was inferred to
be the cause (Gao et al., 2012). In agreement, Turchini et al. (2013) found that over a
long duration (27 weeks) rainbow trout fed SFA rich lipid (at 75% replacement level)
showed depressed performance compared to that of a control diet.
An in vivo whole-body fatty acid balance method (WBFABM) was used in
the present study in order to understand the apparent fate of specific fatty acids
(Turchini et al., 2007). Theoretically, β-oxidation of specific fatty acids should be
92 | P a g e
recorded if they are provided in excess (Eroldogan et al., 2013; Stubhaug et al., 2007;
Turchini et al., 2013). However, in the present study there was no recorded β-
oxidation of any SFA despite the relatively high proportion of SFA in all three diets.
This effect is likely to be caused by the low lipid levels of all three diets, a strategy
that was intended to highlight the potential effects of the lipid classes. Based on the
β-oxidation results, it appears that MUFA are marginally better at sparing LC-PUFA
from β-oxidation (Table 4.6) which is in agreement with past studies (Codabaccus et
al., 2012; Turchini et al., 2011b). However, in the present study the final composition
of the fish suggests that the SFA-D fed fish were significantly more efficient at
depositing or ‘sparing’ LC-PUFA in the whole body (Table 4.5). To resolve this
discrepancy, it is necessary to look at the net intake and total intake budgets to clarify
the situation (Table 4.7). When expressed proportional to FA intake, the SFA-D and
MUFA-D fed fish consequently deposited almost exactly the same n-3 LC-PUFA
and the differences between the two diets were insignificant.
Previous studies have demonstrated that limited de novo FA production
(neogenesis) occurs when diets with adequate SFA were fed to Atlantic salmon
(Emery et al., 2014) or rainbow trout (Turchini et al., 2013). However, neogenesis
was evident in MUFA rich (canola oil) fed trout (Turchini et al., 2013). This is
broadly similar to the results obtained in the present study in that neogenesis in
barramundi was significantly higher in the MUFA fed fish. This may be partly
explained by the up-regulation of genes related to lipogenic activity in response to
vegetable oil observed in other species (Bell et al., 2001; Bell et al., 2002; Tocher et
al., 2002). In contrast, dietary PUFA clearly led to a suppression of the lipogenic
enzyme, fatty acid synthase (FAS) in the rat (Blake and Clarke, 1990). Moreover, in
rainbow trout hepatocytes, FAS expression was strongly inhibited by PUFA (18:3n-3
and 20:5n-3). These results and those of the present study confirm that the
energetically expensive process of initial synthesis of palmitic acid from acetyl- and
malonyl-CoA is avoided by the presence of dietary SFA thus allowing energy to be
utilised more efficiently for growth and other cellular processes.
Elongation and desaturation activity also occurred in the CTRL-D fed fish
suggesting that the total lipid content of the diet was limiting as intended based on
the optimal specification of at least 18% for growing barramundi (Glencross et al.,
2013). However, there was clearly adequate n-3 LC-PUFA in the diets based on the
known requirement data for barramundi of around 1.2% (Glencross and Rutherford,
2011; Williams et al., 2006). Consistent with Glencross and Rutherford (2011), there
93 | P a g e
was no elongation of 20:5n-3 to 22:5n-3 in the control fed fish. However, in the
SFA-D and MUFA-D fed fish, there was a slight increase in the elongation of
available 20:5n-3 to 22:5n-3 possibly in an attempt to achieve 22:6n-3 synthesis.
However, barramundi, like most marine fish are not equipped with the complete set
of enzymes required to endogenously synthesise sufficient LC-PUFA from precursor
FA (Mohd-Yusof et al., 2010). Moreover, a recent study also demonstrated a similar
increase in 22:5n-3 from 20:5n-3, lending further support to the elongation results
obtained in the present study (Salini et al., 2015a).
94 | P a g e
5.0 The effect of marine and non-marine phospholipid rich oils when
fed to juvenile barramundi (Lates calcarifer)
Adapted from
Salini, M.J., Wade, N.M., Bourne, N., Turchini, G.M., Glencross, B.D., 2016. The
effect of marine and non-marine phospholipid rich oils when fed to juvenile
barramundi (Lates calcarifer). Aquaculture. 455, 125-135
95 | P a g e
96 | P a g e
97 | P a g e
5.1 Aims
Most phospholipid requirement studies to date have used soybean lecithin containing
high levels of n-6 PUFA, while others have used egg lecithin or various other marine
sources such as fish roe lecithin (Cahu et al., 2009). Recent studies have clearly
demonstrated the potential of marine derived phospholipid sources to improve larval
and juvenile fish performance (Betancor et al., 2012; Taylor et al., 2015). To date,
information is scarce on the effect of phospholipid in juvenile barramundi diets.
Therefore, an experiment was designed to compare the metabolic effect of marine
and non-marine neutral lipid (NL) and polar (PL) sources using a two-by-two
factorial approach in juvenile barramundi. The biochemical and molecular
mechanisms underpinning the role of phospholipids was also investigated.
5.2 Materials and methods
The methodology used in the present experiment are detailed in the section 2.0
General methodology. Ethical clearance was approved for the experimental
procedures by the CSIRO Animal Ethics Committee (Application A11/2013). The
chemical composition of the main dietary ingredients is presented in Table 5.1. The
formulation and chemical composition of the four diets are presented in Table 5.2.
The neutral and polar lipid composition of the experimental diets are presented in
Table 5.3. Genes involved in fatty acid metabolism were analysed by real-time qPCR
as described and are presented in Table 5.4.
98 |
Pa
ge
Tab
le 5
. 1 C
hem
ical
com
posi
tion
of in
gred
ient
s use
d in
exp
erim
enta
l die
ts, a
ll va
lues
are
g/k
g D
M u
nles
s oth
erw
ise
stat
ed
Fish
m
eal#
Poul
try
mea
l So
y is
olat
e
Whe
at
glut
en
Whe
at
flour
C
asei
n
Whe
at
star
ch
Fish
oil
K
rill o
il So
ybea
n oi
l So
ybea
n le
cith
in
Com
posi
tion
Dry
mat
ter (
g/kg
) 98
.4
95.8
95
.8
92.7
83
.9
92.4
83
.6
99.2
99
.9
100.
0 98
.0
Pr
otei
n 78
.9
64.1
89
.5
82.3
11
.2
87.0
0.
5 0.
4 4.
5 1.
0 7.
5
Ash
16
.3
13.8
4.
6 0.
1 0.
6 1.
1 0.
3 0.
1 2.
9 N
D
9.8
Li
pid
4.6
15.1
5.
7 12
.1
2.2
0.5
ND
95
.6
92.6
94
.6
75.7
Car
bohy
drat
e 0.
1 7.
0 0.
2 5.
5 86
.0
11.3
99
.2
3.9
ND
4.
5 7.
1
Gro
ss e
nerg
y (m
J/kg
) 18
.9
20.4
21
.8
21.2
15
.3
21.9
14
.5
39.3
36
.3
39.5
29
.7
Fatty
aci
ds (m
g/g
lipid
)
16
:0
149.
0 16
1.7
NA
N
A
NA
N
A
NA
12
8.4
107.
3 93
.7
111.
5
18:0
50
.9
55.4
N
A
NA
N
A
NA
N
A
29.0
6.
3 35
.4
23.7
18:1
89
.8
277.
0 N
A
NA
N
A
NA
N
A
104.
0 90
.0
220.
7 53
.3
18
:2n-
6 10
.4
71.4
N
A
NA
N
A
NA
N
A
11.7
12
.2
430.
6 32
6.1
18
:3n-
3 4.
7 7.
1 N
A
NA
N
A
NA
N
A
5.8
7.1
49.7
41
.8
20
:4n-
6 16
.4
4.5
NA
N
A
NA
N
A
NA
9.
1 3.
2 N
D
ND
20:5
n-3
57.6
3.
7 N
A
NA
N
A
NA
N
A
70.3
14
4.5
ND
N
D
22
:5n-
3 13
.0
ND
N
A
NA
N
A
NA
N
A
12.4
0.
0 N
D
ND
22:6
n-3
152.
2 N
D
NA
N
A
NA
N
A
NA
10
5.3
94.8
N
D
ND
SFA
23
1.5
230.
1 N
A
NA
N
A
NA
N
A
205.
5 17
5.3
137.
1 13
5.8
M
UFA
12
9.1
322.
0 N
A
NA
N
A
NA
N
A
163.
2 13
5.5
221.
7 53
.3
C
18PU
FA
20.0
78
.5
NA
N
A
NA
N
A
NA
27
.5
39.6
48
3.6
367.
9
LC-P
UFA
24
4.4
8.2
NA
N
A
NA
N
A
NA
20
0.5
242.
6 N
D
ND
Tota
l n-3
22
2.7
3.7
NA
N
A
NA
N
A
NA
19
8.0
259.
7 N
D
ND
Tota
l n-6
36
.7
83.0
N
A
NA
N
A
NA
N
A
29.9
22
.5
483.
6 36
7.9
# Fi
sh m
eal w
as d
efat
ted
usin
g he
xane
. Ple
ase
see
met
hods
for d
etai
ls. N
A, N
ot a
naly
sed;
ND
, Not
det
ecte
d.^
18:1
, sum
of 1
8:1n
-7, 1
8:1n
-9 c
is, 1
8:1n
-9
trans
; sat
urat
ed fa
tty a
cids
(SFA
), su
m o
f 12:
0, 1
4:0,
16:
0, 1
8:0,
20:
, 22:
0, 2
4:0;
mon
ouns
atur
ated
fatty
aci
ds (M
UFA
), su
m o
f 14:
1n-5
, 16:
1n-7
,
99 |
Pa
ge
18:1
n-7,
18:
1n-9
(cis
and
tran
s), 2
0:1n
-7, 2
0:1n
-9, 2
2:1n
-9, 2
4:1n
-9; p
olyu
nsat
urat
ed fa
tty a
cids
, with
18
carb
on a
tom
s (C
18 P
UFA
), su
m 1
8:2n
-6 (c
is
and
trans
), 18
:3n-
6, 1
8:3n
-3, 1
8:4n
-3; l
ong
chai
n po
lyun
satu
rate
d fa
tty a
cids
, with
20
or m
ore
carb
on a
tom
s (LC
-PU
FA),
sum
20:
2n-6
, 20:
3n-6
, 20:
4n-
6, 2
2:4n
-6, 2
0:3n
-3, 2
0:5n
-3, 2
2:5n
-3, 2
2:6n
-3; n
-3, s
um o
f om
ega
3 C
18 P
UFA
and
LC
-PU
FA; n
-6, s
um o
f om
ega
6 C
18 P
UFA
and
LC
-PU
FA
100 | P a g e
Table 5. 2 Formulation and composition (as analysed) of experimental diets, all values are g/kg DM unless otherwise stated.
n-3 NL (Fish)
n-3 PL (Krill)
n-6 NL (Soybean)
n-6 PL (Lecithin)
Formulation Fish meal a 150 150 150 150 Poultry meal a 150 150 150 150 Soy protein isolate b 150 150 150 150 Wheat gluten b 150 150 150 150 Wheat flour b 109 109 109 109 Casein c 100 100 100 100 Pregelled wheat starch b 80 80 80 80 DL-Methionine 10 10 10 10 Di-calcium phosphate 10 10 10 10 Pre-mix vitamins d 8 8 8 8 Yttrium oxide e 1 1 1 1 Fish oil a 82 10 10 10 Krill Oil f 0 72 0 0 Soy oil g 0 0 72 0 Soy lecithin g 0 0 0 72 Composition Dry matter (g/kg) 940 933 959 952 Protein(g/kg DM) 598 613 597 593 Digestible protein (g/kg DM) 547 561 559 555 Ash (g/kg DM) 64 65 67 70 Lipid (g/kg DM) 122 124 122 118 Carbohydrate (g/kg DM) 212 198 214 219 Gross energy (mJ/kg) 21.5 21.1 21.8 21.0 Digestible energy (mJ/kg) 18.9 18.4 19.8 18.6
a Ridley Aquafeed, Narangba, QLD, Australia. Fish meal defatted (see methods 2.2.1). b Manildra Group, Rocklea, QLD, Australia. c Bulk Powders, Moorabbin, Victoria, Australia. d Vitamin and mineral premix (IU kg-1 or g/kg of premix): vitamin A, 2.5MIU; vitamin D3, 0.25 MIU; vitamin E, 16.7 g; vitamin K3, 1.7 g; vitamin B1, 2.5 g; vitamin B2, 4.2 g; vitamin B3, 25 g; vitamin B5, 8.3; vitamin B6, 2.0 g; vitamin B9, 0.8; vitamin B12, 0.005 g; biotin, 0.17 g; vitamin C, 75 g; choline, 166.7 g; inositol, 58.3 g; ethoxyquin, 20.8 g; copper, 2.5 g; ferrous iron, 10.0 g; magnesium, 16.6 g; manganese, 15.0 g; zinc, 25.0 g e Yttrium oxide; Stanford Materials, Aliso Viejo, California, United States f Sydney Essential Oil Co. (Sydney, NSW, Australia)
101 | P a g e
Table 5. 3 Neutral and polar lipid composition of experimental diets, all values are mg/g lipid unless otherwise stated.
Diets
n-3 NL (Fish)
n-3 PL (Krill)
n-6 NL (Soybean)
n-6 PL (Lecithin)
Lipid class (% total lipid) Neutral 91.5 67.5 89.7 51.1 Polar 8.5 32.5 10.3 48.9 Neutral lipid fatty acids ^ 16:0 190.9 186.7 137.7 207.6 18:0 45.9 34.0 44.0 52.3 18:1n-9 194.6 223.6 252.9 225.4 18:2n-6 48.0 69.7 351.5 183.5 18:3n-3 10.2 10.8 40.3 22.2 20:4n-6 10.9 5.6 0.0 6.2 20:5n-3 79.4 92.6 13.1 35.0 22:5n-3 14.4 5.0 3.6 7.3 22:6n-3 117.5 71.8 19.7 54.7 SFA 298.6 304.0 195.1 291.5 MUFA 284.0 293.1 274.7 278.4 C18PUFA 58.3 97.7 395.7 210.9 LC-PUFA 222.2 175.1 36.4 103.3 Total n-3 211.2 186.7 40.2 102.3 Total n-6 69.2 86.1 391.9 211.9 Total fatty acids 863.1 869.9 901.8 884.0 Polar lipid fatty acids ^ 16:0 139.5 147.3 121.4 118.7 18:0 44.1 18.4 35.9 27.7 18:1n-9 101.8 87.0 109.0 68.1 18:2n-6 121.4 55.3 150.9 317.6 18:3n-3 7.7 7.0 11.6 37.3 20:4n-6 10.1 ND 6.9 ND 20:5n-3 27.5 87.8 29.3 5.9 22:5n-3 7.4 4.4 4.9 ND 22:6n-3 60.0 68.2 41.6 12.1 SFA 204.8 188.9 166.1 146.4 MUFA 144.9 110.4 128.5 71.6 C18PUFA 129.1 71.6 162.5 354.9 LC-PUFA 105.0 160.4 82.6 18.0 Total n-3 94.9 169.7 75.7 18.0 Total n-6 139.2 62.3 169.4 354.9 Total fatty acids 583.8 531.2 539.7 591.0
n-3 NL, Fish oil; n-3 PL, Krill oil; n-6 NL, Soybean oil; n-6 PL, Soybean lecithin. ND, not detected. ^ Refer to Table 5.1 for details.
102
| Pa
ge
Tab
le 5
. 4 R
eal t
ime
quan
titat
ive
PCR
pri
mer
pai
rs fo
r fa
tty
acid
met
abol
ism
and
con
trol
gen
es
NA
, Not
ava
ilabl
e.
Targ
et n
ame
Abb
revi
atio
n EC
num
ber
Prim
er n
ame
Sequ
ence
Le
ngth
Fa
tty a
cid
synt
hase
Lc
FAS
EC
2.3
.1.8
5 FA
S qP
CR
.For
1 TG
AA
TCTC
AC
CA
CG
CTT
CA
G
20
FAS
qPC
R.R
ev1
AG
GC
AG
CA
ATA
GA
AC
CC
TCA
20
Ster
oyl C
oA d
esat
uras
e Lc
SC
D
EC 1
.14.
19.1
SC
D q
PCR
.For
1 C
CTG
GTA
CTT
CTG
GG
GTG
AA
20
SC
D q
PCR
.Rev
1 A
AG
GG
GA
ATG
TGTG
GTG
GTA
20
Car
nitin
e pa
lmito
yltra
nsfe
rase
Lc
CPT
1α
EC 2
.3.2
.21
CPT
1a q
PCR
.For
1 TG
ATG
GTT
ATG
GG
GTG
TCC
T 20
C
PT1a
qPC
R.R
ev1
CG
GC
TCTC
TTC
AA
CTT
TGC
T 20
ATP
citr
ate
lyas
e Lc
ACY
L EC
2.3
.3.8
Lc
al a
cyl F
1 C
AA
CA
CC
ATT
GTC
TGTG
CTC
20
Lc
al a
cyl R
1 G
AA
ATG
CTG
CTT
AA
CA
AA
GTC
C
21
Fa
tty a
cid
elon
gatio
n 5
Lc E
LOVL
5 EC
2.3
.1.n
8 Lc
al F
ads2
F1
ATC
CA
GTT
CTT
CTT
AA
CC
GT
20
Lcal
Fad
s2 R
1 G
GTT
TCTC
AA
ATG
TCA
ATC
CA
C
22
Fa
tty a
cid
desa
tura
se 6
Lc
FAD
S2
EC 1
.14.
19
Lcal
ELO
VL5
F1
TCA
TAC
TAC
CTT
CG
CTA
CTT
CTC
23
Lc
al E
LOV
L5 R
1 A
CA
AA
CC
AG
TGA
CTC
TCC
AG
20
Luci
fera
se
Luc
NA
Lu
c qP
CR
For
G
GTG
TTG
GG
CG
CG
TTA
TTTA
20
Lu
c qP
CR
Rev
C
GG
TAG
GC
TGC
GA
AA
TGC
18
Elon
gatio
n fa
ctor
1α
EF1α
N
A
Lcal
EF1
a F
AA
ATT
GG
CG
GTA
TTG
GA
AC
19
Lc
al E
F1a
R
GG
GA
GC
AA
AG
GTG
AC
GA
C
18
103 | P a g e
5.3 Results
5.3.1 Growth and feed utilisation
In the present study the two levels of omega status were defined as n-3 and n-6 and
the two levels of lipid class were defined as neutral lipid and phospholipid. There
was no difference in the initial weight of the fish among the treatments; however,
there were significant differences in the growth and feed utilisation parameters (as
final weight, feed intake and FCR) upon termination of the 56 d growth assay (Table
5.5). There were significant interaction terms indicating that the effectiveness of
phospholipid-rich ingredients was found to be dependent on the omega status for the
final weight, feed intake and FCR. The lowest growth performance and feed intake
was seen in the fish fed n-6 NL (soybean oil) and the FCR was reduced in the n-6 PL
fed fish (soybean lecithin). There were no differences in terms of survival with only
three fish removed from the system. There were no significant differences in either
protein or lipid retention (Table 5.5).
There were no differences in apparent digestibility of dry matter, protein or
gross energy (Table 5.6). There was a significant interaction effect indicating that the
digestibility of total lipid in the phospholipid-rich treatments was dependent on the
omega status with the lowest lipid digestibility in the n-6 PL (soybean lecithin)
treatment. There was a significant interaction in the digestibility of 16:0 which
showed that the highest digestibility in the n-6 NL treatment was dependent on the
lipid class. There was also a significant interaction noted for the digestibility of
18:2n-6, total C18PUFA and total n-6 showing that the digestibility of fatty acids
from phospholipid-rich ingredients was dependent on the omega status. In each case
the digestibility was lowest in the n-6 PL treatment (soybean lecithin). Similarly, the
digestibility of 18:3n-3 was significantly lower in the n-6 PL fed fish.
104
| Pa
ge
Tab
le 5
. 5 G
row
th a
nd fe
ed u
tilis
atio
n pa
ram
eter
s of j
uven
ile b
arra
mun
di fe
d ex
peri
men
tal d
iets
for
eigh
t wee
ks. D
ata
(n=3
) are
pre
sent
ed a
s m
ean
± SE
M.
n-3
NL;
Fis
h oi
l, n-
3 PL
; Kril
l oil,
n-6
NL;
Soy
bean
oil,
n-6
PL;
Soy
bean
leci
thin
. R
et. n
utrie
nt =
(nut
rient
fina
l - n
utrie
nt in
itial
/ nu
trien
t con
sum
ed)*
100.
‘
* ’ <
0.0
5, ‘
** ’
< 0.
01, ‘
***
’ <
0.00
1; su
pers
crip
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s am
ong
mea
ns.
† Tw
o-w
ay fa
ctor
ial A
NO
VA
, df 1
,1,1
,8, p
ost-h
oc T
ukey
's H
SD; p
ost-h
oc T
ukey
’s H
SD. N
S, n
ot si
gnifi
cant
P >
0.0
5.
D
iets
Test
†
n-
3 N
L
(Fis
h)
n-3
PL
(Kril
l) n-
6 N
L
(Soy
) n-
6 PL
(L
ecith
in)
Om
ega
Cla
ss
Inte
ract
ion
In
itial
wei
ght (
g)
46.9
± 0
.1
46.7
± 0
.2
47.1
± 0
.1
46.9
± 0
.2
NS
NS
NS
Fi
nal w
eigh
t (g)
23
8.3
± 1.
2 a
237.
1 ±
1.1
a 21
7.5
± 1.
6 b
233.
4 ±
3.8 a
**
* *
**
Fe
ed in
take
20
9.6
± 2.
7 b
211.
1 ±
2.6 b
18
9.4
± 0.
7 c
222.
2 ±
0.9 a
*
***
***
FC
R
1.10
± 0
.1 a
1.11
± 0
.1 a
1.11
± 0
.1 a
1.19
± 0
.1 b
*
* *
Su
rviv
al (%
) 98
.0
98.0
10
0.0
98.0
N
S N
S N
S
Ret
. Pro
tein
(%)
34.1
± 1
.1
34.1
± 1
.5
31.7
± 0
.9
28.4
± 0
.5
NS
NS
NS
R
et. l
ipid
(%)
48.6
± 4
.5
49.8
± 1
.4
49.2
± 1
.4
43.1
± 3
.6
NS
NS
NS
105
| Pa
ge
Tab
le 5
. 6 A
ppar
ent d
iges
tibili
ty (%
) par
amet
ers o
f the
die
ts fe
d to
juve
nile
bar
ram
undi
. Dat
a (n
=3) a
re p
rese
nted
as m
ean
± SE
M.
‘ * ’
< 0.
05, ‘
**
’ < 0
.01,
‘ **
* ’ <
0.0
01; s
uper
scrip
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s am
ong
mea
ns.
† Tw
o-w
ay fa
ctor
ial A
NO
VA
, df 1
,1,1
,8, p
ost-h
oc T
ukey
's H
SD; 1
8:3n
-3 a
naly
sed
by o
ne-w
ay A
NO
VA
df 3
,7, P
<0.0
1, p
ost-h
oc T
ukey
's H
SD.
NA
, not
ana
lyse
d; N
S, n
ot si
gnifi
cant
P >
0.0
5. ^
Ref
er to
Tab
le 5
.1 fo
r det
ails
.
Die
ts
Te
st †
n-
3 N
L
(Fis
h)
n-3
PL
(Kril
l) n-
6 N
L
(Soy
bean
) n-
6 PL
(L
ecith
in)
Om
ega
Cla
ss
Inte
ract
ion
Die
t
Dry
mat
ter
64.3
± 1
.6
67.4
± 1
.6
63.7
± 4
.1
66.6
± 2
.2
NS
NS
NS
Pr
otei
n 91
.1 ±
0.7
91
.2 ±
1.8
92
.6 ±
0.3
92
.8 ±
0.4
N
S N
S N
S
Lipi
d 90
.7 ±
0.9
ab
91.2
± 1
.2 ab
94
.2 ±
0.4
a 89
.3 ±
0.6
b N
S *
*
Ener
gy
87.3
± 1
.5
86.6
± 1
.3
89.3
± 0
.7
87.5
± 0
.8
NS
NS
NS
Fatty
aci
ds ^
16:0
81
.5 ±
1.9
a 84
.2 ±
1.0
ab
87.7
± 0
.8 b
84.4
± 0
.8 ab
*
NS
*
18:0
76
.7 ±
3.0
77
.5 ±
2.9
84
.6 ±
1.2
79
.5 ±
1.1
N
S N
S N
S
18:1
n-9
92.1
± 0
.8
90.4
± 2
.7
95.0
± 0
.3
90.5
± 0
.4
NS
NS
NS
18
:2n-
6 95
.8 ±
0.2
ab
94.9
± 1
.0 a
b 97
.3 ±
0.1
a
90.3
± 1
.6 b
N
S **
*
18
:3n-
3 98
.6 ±
1.2
a
100.
0 ±
0.0
98.3
± 0
.1 a
92.5
± 1
.4 b
N
A
NA
N
A
20
:4n-
6 10
0.0
± 0.
0 10
0.0
± 0.
0 10
0.0
± 0.
0 10
0.0
± 0.
0 N
A
NA
N
A
20
:5n-
3 99
.0 ±
0.4
97
.7 ±
0.2
10
0.0
± 0.
0 10
0.0
± 0.
0 N
A
NA
N
A
22
:5n-
3 10
0.0
± 0.
0 10
0.0
± 0.
0 10
0.0
± 0.
0 10
0.0
± 0.
0 N
A
NA
N
A
22
:6n-
3 98
.5 ±
0.2
96
.0 ±
0.2
92
.9 ±
0.6
98
.0 ±
2.0
N
S N
S N
S
SFA
82
.3 ±
1.8
85
.3 ±
1.1
87
.3 ±
1.0
84
.1 ±
0.6
N
S N
S N
S
MU
FA
92.6
± 0
.8
91.3
± 2
.3
94.7
± 0
.4
91.1
± 0
.4
NS
NS
NS
C
18PU
FA
96.5
± 0
.2 a
96.1
± 0
.7 a
97.5
± 0
.1 a
90.5
± 1
.5 b
*
**
*
LC-P
UFA
98
.9 ±
0.2
97
.1 ±
0.2
96
.2 ±
0.3
98
.9 ±
1.1
N
S N
S N
S
Tota
l n-3
98
.9 ±
0.2
97
.5 ±
0.1
97
.6 ±
0.1
95
.2 ±
1.5
N
S N
S N
S
Tota
l n-6
96
.3 ±
0.1
a 95
.2 ±
0.9
ab
97.2
± 0
.1 a
91.1
± 1
.4 b
NS
**
*
106 | P a g e
5.3.2 Biochemical analysis
As intended, the neutral and polar composition of the diets was reflective of the lipid
composition of the ingredients used (Table 5.3).There were no significant differences
in the neutral and polar lipid composition of the whole fish with all treatments in the
range of 89.4 to 94.0% neutral lipid (Table 5.7). Fatty acid analysis of the neutral and
polar fractions revealed several significant differences among the treatments.
Significant interaction terms were noted for many of the neutral lipid fatty acids.
Most notably, the effect of phospholipid-rich ingredients on LC-PUFA (specifically
22:6n-3, 22:5n-3 and 20:5n-3) composition in the whole fish were found to be
dependent on the omega status with the highest levels present in the n-3 treatments.
Of the polar lipid fatty acids, the18:2n-6 composition was significantly higher in the
n-6 treatments whereas the 16:0, 20:5n-3 and 22:6n-3 composition was highest in the
n-3 treatments.
There was several significant interaction terms noted on the apparent in-vivo
β-oxidation activity (Table 5.8). The phospholipid-rich ingredients were found to be
effective in modifying the β-oxidation of MUFA in the n-6 PL fed fish; however, this
was not the case for the n-3 PL fish. There was a significant interaction term noted in
the β-oxidation C18PUFA and Total n-6 with the highest activity in the phospholipid-
rich lipid being dependent on the omega status. There was also a significant
interaction for the β-oxidation of LC-PUFA and Total n-3 which was dependent on
the omega status with the highest activity in the n-3 NL treatments.
There was a significant interaction indicating that elongation activity of SFA
was highest in the n-6 NL treatment and lowest activity was in the n-3 NL treatment
(Table 5.8). There was no detectable elongation activity of the LC-PUFA or Total n-
3 in the n-3 NL fed fish; however, when analysed by one-way ANOVA there was
significantly greater activity in the n-3 PL fed fish compared to both of the n-6
treatments. There was also a significant interaction in the delta-9 (Steroyl CoA
desaturase) desaturation activity with greatest activity recorded in the n-6 NL and the
lowest activity in the n-3 NL treatment.
107
| Pa
ge
Tab
le 5
. 7 N
eutr
al a
nd p
olar
lipi
d co
mpo
sitio
n in
the
who
le b
ody
of ju
veni
le b
arra
mun
di fe
d ex
peri
men
tal d
iets
. All
valu
es a
re m
g/g
lipid
un
less
oth
erw
ise
stat
ed. D
ata
(n=3
) are
pre
sent
ed a
s mea
n ±
SEM
Die
ts
Te
st †
n-3
NL
(F
ish)
n-
3 PL
(K
rill)
n-6
NL
(S
oybe
an)
n-6
PL
(Lec
ithin
) O
meg
a C
lass
In
tera
ctio
n Li
pid
clas
s (%
tota
l lip
id)
Neu
tral
91.7
± 1
.1
91.4
± 1
.4
94.0
± 1
.5
89.4
± 0
.6
NS
NS
NS
Po
lar
8.3
± 1.
1 8.
6 ±
1.4
6.0
± 1.
5 10
.6 ±
0.6
N
S N
S N
S N
eutr
al li
pid
fatty
aci
ds ^
16
:0
227.
1 ±
2.1 a
22
9.6
± 1.
1 a
198.
0 ±
4.5 b
23
8.4
± 3.
9 a
* **
* **
*
18:0
61
.2 ±
0.6
ab
56.5
± 0
.7 a
64.6
± 1
.9 b
71
.3 ±
0.9
c
***
NS
**
18
:1n-
9 25
4.8
± 3.
3 a
257.
1 ±0
.6 a
286.
7 ±8
.0 b
24
4.1
± 1.
6 a
NS
**
***
18
:2n-
6 76
.4 ±
1.6
a
82.5
± 0
.8 a
264.
5 ±
8.9 b
20
2.0
± 0.
4 c
***
***
***
18
:3n-
3 9.
0 ±
0.3 a
10
.0 ±
0.1
a
27.0
± 1
.2 b
21.2
± 0
.2 c
***
**
***
20
:4n-
6 5.
6 ±
0.1
1.2
± 1.
2 N
D
ND
N
A
NA
N
A
20
:5n-
3 32
.1 ±
0.9
a 53
.2 ±
1.1
b 8.
7 ±
0.7
c 10
.5 ±
0.2
c **
* **
* **
*
22:5
n-3
13.4
± 0
.4 a
11.6
± 0
.2 b
5.9
± 0.
3 c
6.5
± 0.
1 c
***
NS
**
22
:6n-
3 54
.7 ±
2.4
a 47
.4 ±
1.4
b 18
.0 ±
1.1
c 21
.1 ±
0.7
c **
* N
S **
SFA
33
5.3
± 1.
8 a
331.
4 ±
0.8 a
28
6.7
± 7.
0 b
338.
3 ±
4.7 a
**
**
* **
*
MU
FA
322.
9 ±
3.9 a
31
5.3
± 0.
6 a
319.
1 ±
8.3 a
28
4.7
± 2.
0 b
**
**
*
C18
PUFA
97
.7 ±
1.8
a
109.
7 ±
0.8 a
29
7.4
± 10
.0 c
231.
3 ±
0.3 b
**
* **
* **
*
LC-P
UFA
10
5.8
± 3.
8 a
113.
3 ±
3.0 a
37
.5 ±
2.3
b
43.1
± 1
.0 b
**
* *
NS
To
tal n
-3
106.
4 ±
3.9 b
12
3.1
± 2.
7 a
32.5
± 2
.1 c
38.1
± 0
.9 c
***
**
NS
To
tal n
-6
97.0
± 1
.7 a
99.9
± 1
.9 a
302.
3 ±
10.2
c
236.
2 ±
0.3 b
**
* **
* **
*
Tota
l fat
ty a
cids
86
1.6
± 5.
7 a
869.
7 ±
3.8 a
94
0.7
± 25
.6 b
897.
3 ±
5.4 a
b **
N
S N
S Po
lar l
ipid
fatty
aci
ds ^
16
:0
171.
4 ±
5.7 a
16
3.6
± 0.
2 a
135.
8 ±
2.4 b
14
3.8
± 2.
8 b
**
NS
NS
18
:0
65.9
± 4
.5
62.0
± 1
.2
69.9
± 0
.6
61.7
± 3
.5
NS
NS
NS
108
| Pa
ge
18
:1n-
9 15
5.0
±1.0
14
5.2
± 3.
4 13
9.1
± 4.
5 14
3.3
± 8.
9 N
S N
S N
S
18:2
n-6
42.9
± 0
.9 a
39.5
± 1
.7 a
129.
7 ±
3.2 b
12
0.3
± 9.
9 b
***
NS
NS
18
:3n-
3 N
D
ND
N
D
9.8
± 1.
6 N
A
NA
N
A
20
:4n-
6 12
.2 ±
2.6
6.
9 ±
1.3
8.9
± 0.
1 5.
7 ±
0.5
NS
NS
NS
20
:5n-
3 26
.5 ±
3.2
ab
35.2
± 5
.4 a
11.1
± 0
.1 b
11
.4 ±
0.7
b
**
NS
NS
22
:5n-
3 11
.8 ±
1.6
10
.8 ±
1.8
10
.0 ±
0.3
7.
0 ±
0.1
NS
NS
NS
22
:6n-
3 66
.8 ±
13.
0 a
57.6
± 1
1.9 a
42
.1 ±
0.9
ab
24.5
± 1
.7 b
*
NS
NS
SF
A
254.
9 ±
6.4 a
24
4.9
± 3.
2 ab
205.
7 ±
3.0 c
22
1.2
± 5.
3 bc
**
NS
NS
M
UFA
18
9.9
± 6.
0 17
5.7
± 6.
3 15
6.5
± 4.
8 17
2.7
± 8.
3 N
S N
S N
S
C18
PUFA
48
.4 ±
3.6
a
43.2
± 4
.7 a
133.
3 ±
6.1 b
13
5.0
± 12
.2 b
**
* N
S N
S
LC-P
UFA
11
7.3
± 20
.4 a
110.
5 ±
20.4
a
83.9
± 1
.4 a
b 56
.0 ±
1.8
b
* N
S N
S
Tota
l n-3
10
6.7
± 16
.4 a
100.
4 ±
17.7
a
63.2
± 1
.3 a
b 42
.9 ±
1.1
b
* N
S N
S
Tota
l n-6
59
.0 ±
0.3
a
48.3
± 2
.0 a
154.
1 ±
6.0 b
14
8.1
± 11
.5 b
**
* N
S N
S
Tota
l fat
ty a
cids
61
0.6
± 1
7.2
569.
4 ±
6.1
579.
4 ±
12.5
58
4.8
± 13
.4
NS
NS
NS
‘ * ’
< 0.
05, ‘
**
’ < 0
.01,
‘ **
* ’ <
0.0
01; s
uper
scrip
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s am
ong
mea
ns.
† Tw
o-w
ay fa
ctor
ial A
NO
VA
, df 1
,1,1
,8, p
ost-h
oc T
ukey
's H
SD
NA
, not
ana
lyse
d; N
D, n
ot d
etec
ted;
NS,
not
sign
ifica
nt P
> 0
.05.
^
Ref
er to
Tab
le 5
.1 fo
r det
ails
.
109
| Pa
ge
Tab
le 5
. 8 W
hole
bod
y fa
tty
acid
bal
ance
cal
cula
tions
of β
-oxi
datio
n, e
long
atio
n an
d de
satu
ratio
n of
juve
nile
bar
ram
undi
fed
expe
rim
enta
l di
ets f
or e
ight
wee
ks. A
ll va
lues
are
pre
sent
ed a
s nm
ol/g
fish
/d. D
ata
(n=3
) are
pre
sent
ed a
s mea
n ±
SEM
.
SCD,
Ste
royl
CoA
des
atur
ase;
FA
DS2
, Fat
ty a
cid
desa
tura
se 6
. FA
DS1
(Δ-5
Des
.) an
d ch
ain
shor
teni
ng w
ere
not d
etec
ted
or re
porte
d.
‘ * ’
< 0.
05, ‘
**
’ < 0
.01,
‘ **
* ’ <
0.0
01; s
uper
scrip
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s am
ong
mea
ns.
† Tw
o-w
ay fa
ctor
ial A
NO
VA
, df 1
,1,1
,8, p
ost-h
oc T
ukey
's H
SD; E
long
atio
n LC
-PU
FA a
nd to
tal n
-3 w
ere
anal
ysed
by
one-
way
AN
OV
A d
f 3,7
, pos
t-ho
c Tu
key'
s HSD
. NA
, not
ana
lyse
d; N
D, n
ot d
etec
ted;
NS,
not
sign
ifica
nt P
> 0
.05.
^
Ref
er to
Tab
le 5
.1 fo
r det
ails
.
Die
ts
Te
st †
n-
3 N
L
(Fis
h)
n-3
PL
(Kril
l) n-
6 N
L
(Soy
bean
) n-
6 PL
(L
ecith
in)
Om
ega
Cla
ss
Inte
ract
ion
β-O
xida
tion
^
SFA
33
9.7
± 18
.2
ND
N
D
45.7
± 0
.3
NA
N
A
NA
MU
FA
554.
1 ±
43.9
b 12
0.4
± 5.
7c 8.
1 ±
6.1c
1338
.2 ±
113
.2a
***
***
***
C
18PU
FA
661.
6 ±
34.9
b 41
1.9
± 22
.9bc
46
.5 ±
1.5
c 33
76.6
± 2
08.4
a **
* **
* **
*
LC-P
UFA
16
95.1
± 1
02.7
a 10
49.5
± 5
3.3b
171.
5 ±
5.9c
136.
9 ±
22.8
c **
* **
* **
*
Tota
l n-3
16
88.5
± 1
03.5
a 11
01.8
± 5
6.7b
125.
2 ±
5.8c
116.
7 ±
20.9
c **
* **
**
Tota
l n-6
66
8.1
± 34
.3b
359.
5 ±
19.9
bc
92.8
± 1
.8c
3396
.9 ±
209
.2a
***
***
***
Elon
gatio
n ^
SF
A
73.0
± 1
0.1d
3154
.3 ±
297
.9b
4894
.0 ±
205
.1a
1301
.4 ±
438
.2c
***
NS
***
M
UFA
N
D
ND
17
.8 ±
3.5
N
D
NA
N
A
NA
C18
PUFA
N
D
ND
N
D
ND
N
A
NA
N
A
LC
-PU
FA
ND
11
8.5
± 7.
1a 55
.4 ±
1.0
b 65
.1 ±
2.9
b N
A
NA
N
A
To
tal n
-3
ND
11
8.5
± 7.
1a 55
.4 ±
1.0
b 65
.1 ±
2.9
b N
A
NA
N
A
To
tal n
-6
ND
N
D
ND
N
D
NA
N
A
NA
D
esat
urat
ion
SC
D (Δ
-9 D
es.)
2.4
± 2.
4c 46
6.7
± 60
.9b
1138
.0 ±
41.
2a 19
.2 ±
14.
0c **
**
* **
*
FAD
S2 (Δ
-6 D
es.)
ND
N
D
29.1
± 1
.2
40.1
± 1
.6
NA
N
A
NA
110 | P a g e
There was a significant interaction in the plasma GLDH enzyme with an
elevated level present in the n-6 PL treatment which was dependant on the omega
status (Table 5.9). Similarly, there was a significant interaction noted on plasma
creatinine levels with an elevated level in the n-6 PL fish which was dependent on
the omega status. The plasma cholesterol level was significantly elevated in the n-3
PL treatment and lowest in the n-6 NL and n-6 PL treatments. Circulating protein
levels in the plasma were significantly higher in the phospholipid class treatments.
Among the haematological parameters, Hb was significantly elevated in the n-3 NL
fish and haptoglobin was significantly elevated in the n-6 PL fish (Table 5.9).
5.3.3 Gene expression
Several significant differences were observed in the expression of genes related to
fatty acid metabolism (Figure 5.1). There was a significant interaction in the relative
gene expression of Lc ACYL, Lc FAS and Lc FADS2 with increased expression in the
n-6 PL treatment that was dependent on the lipid class. The expression of these genes
was at least 1.5-fold higher in the n-6 PL compared to the n-3 PL treatments. There
was also a significant interaction effect in the modification of Lc CPT1α expression
by the phospholipid-rich ingredients which was dependent on the omega status. The
expression of Lc CPT1α was down regulated by approximately 1.5-fold in the n-3 PL
compared to the control. Similarly, the expression of Lc SCD was significantly up
regulated in the n-6 treatments compared to the n-3 treatments and there was no
interaction effect observed. The expression of Lc ELOVL5 in all treatments was
down regulated compared to the initial fish levels and there were no significant
differences among the treatments.
111
| Pa
ge
Tab
le 5
. 9 P
lasm
a ch
emis
try
of b
arra
mun
di fe
d ex
peri
men
tal d
iets
for
eigh
t wee
ks. D
ata
(n=3
) are
pre
sent
ed a
s mea
n ±
SEM
.
CK
, cre
atin
e ki
nase
; ALT
, ala
nine
am
inot
rans
fera
se; G
LDH
, glu
tam
ate
dehy
drog
enas
e.
‘ * ’
< 0.
05, ‘
**
’ < 0
.01,
‘ **
* ’ <
0.0
01; s
uper
scrip
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s am
ong
mea
ns.
† Tw
o-w
ay fa
ctor
ial A
NO
VA
, df 1
,1,1
,8, p
ost-h
oc T
ukey
's H
SD
NS,
not
sign
ifica
nt P
> 0
.05.
D
iets
Test
†
n-
3 N
L
(Fis
h)
n-3
PL
(Kril
l) n-
6 N
L
(Soy
bean
) n-
6 PL
(L
ecith
in)
Om
ega
Cla
ss
Inte
ract
ion
CK
(U/L
) 74
86.3
± 3
156.
8 33
94.0
± 1
048.
1 36
67.7
± 6
31.9
35
91.0
± 1
076.
3 N
S N
S N
S A
LT (U
/L)
8.7
± 4.
3 4.
3 ±
1.5
5.7
± 4.
3 9.
0 ±
3.9
NS
NS
NS
GLD
H (U
/L)
10.0
± 2
.6 a
9.3
± 6.
5 a
11.3
± 3
.0 a
24.7
± 8
.3 b
***
***
***
Ure
a (m
mol
/L)
2.9
± 0.
6 2.
2 ±
0.4
3.1
± 0.
5 3.
0 ±
0.3
NS
NS
NS
Cre
atin
ine
(um
ol/L
) 22
.7 ±
0.3
a 23
.3 ±
1.1
a 21
.7 ±
1.0
a 28
.0 ±
1.2
b *
***
**
Ca
(mm
ol/L
) 3.
2 ±
0.1
3.0
± 0.
2 3.
3 ±
0.2
3.3
± 0.
1 N
S N
S N
S M
g (m
mol
/L)
1.8
± 0.
2 1.
4 ±
0.6
1.6
± 0.
2 1.
7 ±
0.3
NS
NS
NS
Phos
phat
e (m
mol
/L)
3.6
± 0.
2 3.
3 ±
0.2
3.7
± 0.
4 4.
0 ±
0.3
NS
NS
NS
Cho
lest
erol
(mm
ol/L
) 5.
6 ±
0.3
ab
6.8
± 0.
2 a
4.4
± 0.
6 b
4.4
± 0.
3 b
***
* *
Tota
l Pro
tein
(g/L
) 48
.1 ±
1.2
a 49
.9 ±
2.8
ab
48.1
± 0
.9 a
54.3
± 0
.8 b
NS
**
NS
Alb
umin
(g/L
) 13
.6 ±
0.5
13
.8 ±
0.9
14
.1 ±
0.4
16
.0 ±
0.4
N
S N
S N
S Fe
(um
ol/L
) 21
.5 ±
0.7
21
.7 ±
1.0
19
.6 ±
2.6
27
.1 ±
2.6
N
S N
S N
S H
b (m
g/m
l) 0.
26 ±
0.5
b 0.
10 ±
0.1
a 0.
07 ±
0.7
a 0.
14 ±
0.9
ab
NS
NS
**
Hap
togl
obin
(mg/
ml)
1.3
± 0.
1 a
1.3
± 0.
1 a
1.3
± 0.
1 ab
1.
4 ±
0.1
b **
N
S N
S
112 | P a g e
Figure 5. 1 Hepatic gene expression of selected lipid metabolism genes in juvenile barramundi (Lates calcarifer) after eight weeks of feeding. Relative expression is calculated for each gene using cycle threshold values, normalised to control genes (Elongation factor 1a and Luciferase). Values are shown as log-2 fold change relative to the initial fish. Letters above error bars indicate significant differences between the treatments. Analysed by two-way factorial ANOVA, df 1,1,1,8, post-hoc Tukey's HSD.
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
Rela
tive
fold
cha
nge
Lc ACYL
N-3 NL N-3 PL N-6 NL N-6 PL
Omega P=0.06Class P=0.72Interaction P<0.05
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0 Lc CPT1α
N-3 NL N-3 PL N-6 NL N-6 PL
abab
b
a
Omega P=0.98Class P=0.08Interaction P<0.01
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5 Lc ELOVL5
N-3 NL N-3 PL N-6 NL N-6 PL
Omega P=0.38Class P=0.88Interaction P=0.16
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5 Lc FADS2
N-3 NL N-3 PL N-6 NL N-6 PL
a
b
c
bcOmega P<0.001Class P=0.07Interaction P<0.01
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5 Lc FAS
N-3 NL N-3 PL N-6 NL N-6 PL
ab
a
c
bc
Omega P<0.001Class P=0.72Interaction P<0.01
-2.0
-1.0
0.0
1.0
2.0
3.0
4.0
Rela
tive
fold
cha
nge
Lc SCD
N-3 NL N-3 PL N-6 NL N-6 PL
a
abab
b
Omega P<0.05Class P=0.11Interaction P=0.73
a
abab
b
113 | P a g e
5.4 Discussion
It is well established that larval and early juvenile fish have a dietary requirement for
intact phospholipids that can lead to long term improvements in many growth
performance parameters (Coutteau et al., 1997; Tocher et al., 2008). Historically
most phospholipid studies were conducted with commercial phospholipid
preparations of commonly available emulsifying agents such as lecithin from
soybeans or corn (Tocher et al., 2008). However, in some cases the use of these
products has potentially led to unclear requirement data as the composition of polar
lipid and polar lipid classes is seldom reported and there are other potential
interacting effects of the ingredients themselves. The aims of the present study were
to provide an up to date assessment of the physiological and metabolic effect of
commercially available preparations of phospholipid-rich ingredients such as krill oil
and soybean lecithin against neutral lipid-rich ingredients such as fish oil and
soybean oil in juvenile barramundi.
Based on the performance data of the present study, it is clear that for
barramundi in the range of ~47 to ~238 g, a response to the lipid ingredients was
evident. It should be noted that each of the diets were prepared with a minimum (1%
diet) inclusion of FO in order to prevent the onset of essential fatty acid deficiency
(Salini et al., 2015c). The growth, feed intake and FCR of the n-3 NL and n-3 PL fed
fish were nearly identical. Despite the n-6 PL fed fish being statistically the same
weight as the two n-3 diets they consumed significantly more feed which resulted in
a higher FCR. Many studies have clearly demonstrated that growth potential is
driven by the demand for energy (derived mostly from protein and lipid) in this
species (Glencross et al., 2013). However, the diets in the present study were
equivalent in digestible energy and the n-6 PL fish consumed more feed to maintain
growth, comparable to the control (n-3 NL) fish suggesting that the difference in
FCR is attributable to the lipid ingredients. Therefore this suggests that the lecithin
was poorly utilised by the fish as also supported by numerically lower lipid retention
in this treatment. There may be other features of the soybean lecithin that contributed
to the results observed, however, investigation of these was beyond the objectives of
the present study.
A recent study on dietary phospholipids in Atlantic salmon over a range of
sizes (from first feeding to ~60 g) found that at 2.6% phospholipid in the form of
soybean lecithin reduced growth performance and FCR (Taylor et al., 2015).
However, the most profound negative effect of soybean lecithin on growth rate
114 | P a g e
(SGR) was in early phase (first feeding to 2.5 g) rather than the latter stages of
growth studied. It should be clearly noted that the diets used in the present study
included 7.2% added lipid to a base of 1% fish oil. Based on the analysed
composition of the diets used in the present study, the determined level of
phospholipid in the lecithin diet was around 5.8% and the krill diet was around 4.0%.
It is unclear whether this level of inclusion could have influenced the growth
performance of the n-6 PL fed fish. In the study of Taylor et al. (2015), the inclusion
of krill oil above 2.6% phospholipid led to a decrease in the growth of Atlantic
salmon; however, no effect of krill oil was seen in the present study. They reasoned
that the difference could be due to the reduced energy availability of phospholipid-
rich oils compared to that of neutral lipid oils that are mostly triacylglycerol (Taylor
et al., 2015).
In larval fish, the digestibility of diets containing phospholipids is
consistently better than control diets, mostly owing to their emulsifying effect
leading to better absorption by the developing gut system (Coutteau et al., 1997;
Tocher et al., 2008). In contrast, the juvenile barramundi fed soybean lecithin in the
present study had the lowest total lipid digestibility potentially owing to its physical
characteristics rather than its chemical composition. However, the most abundant
fatty acid in the phospholipid fraction of the n-6 PL diet was 18:2n-6 and this was
also significantly less digestible leading to the lowest total C18PUFA digestibility.
These reductions in digestibility most likely also led to the poor FCR observed in
that treatment group. However, it is unclear whether these effects are caused by the
phospholipid composition or another feature of the ingredient as the same effects
were not replicated in the krill oil fed fish.
A further interesting result of the present study was that the n-6 NL fed fish
ate less and consequently grew less than the n-3 treatments with no difference in
FCR. The digestibility was also high for the total lipid and all the dominant fatty
acids in fish fed the n-6 NL diet. This reduction in growth might be explained by
several mechanisms. Firstly, soybean oil lacks any measureable n-3 LC-PUFA and to
some extent is also likely to be pro-inflammatory due to the high proportion of
omega-6 fatty acids (Brown and Hart, 2011; Turchini et al., 2009). However, the
reduced growth response should also have been expected in the n-6 PL fed fish as the
fatty acid composition of the raw ingredients is quite similar.
Of all the studies investigating the use of soybean oil, growth reduction is
rarely reported in fish (Brown and Hart, 2011; Glencross, 2009). In a study by Raso
115 | P a g e
and Anderson (2003) they found that although juvenile barramundi fed soybean oil
grew slightly less than the controls it was not confirmed statistically and the FCR
also remained unchanged. However, in a select group of marine species, such as the
black sea bream (Acanthopagrus schlegeli), Japanese flounder (Paralichthys
olivaceous), red sea bream (Pagrus auratus), silver bream (Rhabdosargus sarba) and
cobia (Rachycentron canadum), feeding exclusively with soybean oil has led to
differences in growth performance. Most simply put, this was likely caused by
essential fatty acid (EFA) deficiency rather than another unique feature of the
soybean oil itself (Brown and Hart, 2011; Trushenski et al., 2012).
Another more recent hypothesis is that certain fatty acids including 18:2n-6
may influence feed intake by modulating the expression of ‘satiety’ and ‘hunger’
hormones via various signalling pathways, for example neuropeptide Y or Agouti-
related protein (NPY and AGRP respectively), however, this is yet to be thoroughly
explored in teleost fish (Coccia et al., 2014; Liland et al., 2013; Schwartz et al.,
2000). Therefore, in light of the many possibilities, the fish fed the n-6 NL (soybean
oil) diet in the present study were likely to be EFA deficient. However, the same
effect did not manifest itself in the n-6 PL fish, indicating a marginal improvement
owing to the phospholipid content of the diet.
In the present study, the whole body fatty acid composition mostly resembled
the diet profiles as previously reported in the vast majority of studies (Rosenlund et
al., 2011). Despite the varied NL and PL composition of the diets used in the present
study, it was clear that the proportion of NL and PL in the whole body was tightly
regulated. However, there were some changes to the fatty acid composition of both
the NL and PL fraction of the whole body, consistent with other studies. In larval fish
such as sea bream, increasing marine derived PL led to better assimilation of n-3 FA
whereas soybean lecithin PL increased assimilation of n-6 FA (Saleh et al., 2015).
Alhazzaa et al. (2011b) correlated the up regulation of fatty acid synthesis genes
(FADS2 and ELOVL5) with highly selective fatty acid retention in muscle and liver
PL of juvenile barramundi fed vegetable oils. Other studies have also demonstrated
that the LC-PUFA composition of the PL fraction of certain tissues is tightly
regulated in the absence of adequate dietary supply (Skalli et al., 2006). In the
present study, this effect was seen with the n-6 NL (soybean oil) fish able to retain
numerically more n-3 LC-PUFA than the n-6 PL fish. Moreover, 18:2n-6 was
preferentially retained in the n-6 NL fed fish.
116 | P a g e
In the present study, the apparent in vivo whole body mass-balance of specific
fatty acids was calculated to determine discrete differences in metabolism (Turchini
et al., 2007). The LC-PUFA β-oxidation activity was highest in the n-3 NL followed
by the n-3 PL fish, typical of when barramundi and other species are fed excess LC-
PUFA (Salini et al., 2015c; Stubhaug et al., 2007). The high elongation of SFA and
delta-9 desaturation activity in the n-6 NL fish suggests an attempt to generate 18:1n-
9 as an available energy source. However, there was no corresponding increase in β-
oxidation of MUFA and moreover the same effect was not recorded in the n-6 PL
fish. It is unclear as to why they would invest energy into elongation and delta-9
desaturation processes with no further downstream effect; however, it clearly
indicates a modified metabolic function that could be part of a compensatory
mechanism when EFA are deficient. Moreover, it is difficult to correlate the mass-
balance computations with the quantitative gene expression analysis used in the
present study as different enzymes are involved throughout the fatty acid metabolic
pathway. However, the mass-balance results do correlate with those previously
reported in barramundi and other species fed alternative oils (Francis et al., 2007;
Salini et al., 2015c; Turchini et al., 2013).
The biochemical analysis of the plasma and the hepatic gene expression
potentially indicate a modified metabolic pattern, particularly as a result of the n-6
lipids, soybean lecithin and to a lesser extent soybean oil. However, there were no
dramatic sub-clinical pathologies noted indicating the potential relevance of these
changes. Creatinine kinase (CK) levels were numerically highest in the n-3 NL fed
fish and although there is a general lack of data in this and other teleost species it
could be argued that these fish were in a diseased state (Nanji, 1983; Sandnes et al.,
1988). However, it may also be reasoned that these are the normal enzyme levels as
they are the control group of this study. The elevated glutamate dehydrogenase
(GLDH) activity, creatinine level and total protein in the plasma of the n-6 PL fish
are potentially indicative of organ failure or dehydration that can be characterised by
the depletion of hepatic LC-PUFA stores (Van Waes and Lieber, 1977; Videla et al.,
2004). However, the plasma enzyme markers (eg GLDH and ALT) are typically used
in combination to confirm a clinical diagnosis and in the present study there is not
adequate evidence to support this. In contrast, none of the plasma markers were
elevated in the n-6 NL fed fish. In addition, several authors have reasoned that
together elevated GLDH and plasma urea are implicated in osmoregulatory processes
leading to clinical pathologies such as subcutaneous haemorrhaging (Glencross and
117 | P a g e
Rutherford, 2011; Morton et al., 2014). However, in the present study these
pathologies were not present and moreover the plasma urea content was unaffected
suggesting that the lipid sources used were nutritionally adequate unlike those of the
previously mentioned studies. Further work is clearly warranted in this area to
resolve some of the discrepancies relating to clinical diagnosis in barramundi and
other teleosts.
The hepatic expression of genes related to fatty acid synthesis (Lc ACYL and
Lc FAS) and delta-6 and delta-9 desaturation (Lc FADS2 and Lc SCD), were also
significantly up regulated in n-6 PL fed fish. These transcriptional changes suggest
that there was potentially a metabolic modification in the liver and that the fish might
have a limited ability to respond to the soybean lecithin. Recent studies have shown
similar nutritional regulation of fatty acid metabolism related genes with soybean oil
(Li et al., 2016); however, the same effect of soybean oil (diet n-6 NL) was not
replicated to the same extent in the present study. Interestingly, the expression of Lc
CPT1α, which is considered to be the initial step in the mitochondrial β-oxidation of
fatty acids, was dependent on the omega status being significantly down regulated in
the n-3 PL fish compared to the n-3 NL fish (Frøyland et al., 1998). Moreover, the
apparent in vivo β-oxidation of SFA, MUFA and PUFA was also significantly lower
in the n-3 PL treatment which may indicate an active anti-oxidant effect of
phospholipid rich lipids of marine origin such as krill (Saito and Ishihara, 1997).
118 | P a g e
6.0 Rapid effects of essential fatty acid deficiency on growth and
development parameters and transcription of key fatty acid
metabolism genes in juvenile barramundi Lates calcarifer
Adapted from
Salini, M.J., Turchini, G.M., Wade, N., Glencross, B.D., 2015. Rapid effects of
essential fatty acid deficiency on growth and development parameters and
transcription of key fatty acid metabolism genes in juvenile barramundi Lates
calcarifer. Br. J. Nutr. 114, 1784-1796
119 | P a g e
120 | P a g e
121 | P a g e
6.1 Aims
Essential fatty acids (EFA) play an important physiological role in both larval and
juvenile barramundi while less is known about the effects on larger fish. Despite the
efforts of numerous studies so far, there remains to be a clear analysis of the onset
and progression of EFA deficiency in barramundi. Therefore, the aim of this
experiment was to document the aetiology of essential fatty acid deficiency in
juvenile barramundi. It was hypothesised that once the endogenous reserves of LC-
PUFA were progressively depleted then sub-clinical EFA deficiency symptoms
would develop before further gross clinical signs become evident.
6.2 Materials and methods
The methodology used in the present experiment are detailed in the section 2.0
General methodology. Ethical clearance was approved for the experimental
procedures by the CSIRO Animal Ethics Committee (Application A10/2013). The
chemical composition of the main dietary ingredients is presented in Table 6.1. The
formulation and chemical composition of the three diets are presented in Table 6.2.
Real-Time PCR primers specific to each gene are presented in Table 6.3.
122
| Pa
ge
Tab
le 6
. 1 C
hem
ical
com
posi
tion
of in
gred
ient
s use
d in
exp
erim
enta
l die
ts, a
ll va
lues
are
g/k
g un
less
oth
erw
ise
stat
ed.
Any
val
ues <
0.01
are
repo
rted
as 0
.1; N
A, N
ot a
naly
sed;
ND
, Not
det
ecte
d #
Fish
mea
l was
def
atte
d us
ing
hexa
ne.
Fish
m
eal#
Poul
try
mea
l So
y is
olat
e W
heat
gl
uten
W
heat
flo
ur
Cas
ein
Whe
at
star
ch
Fish
oi
l O
live
oil
Palm
oi
l Pa
lm
flake
Com
posi
tion
Dry
mat
ter
984
958
958
927
839
924
836
992
987
999
994
Pr
otei
n 78
9 64
1 89
5 82
3 11
2 87
0 5
4 4
3 4
A
sh
163
138
46
1 6
11
3 1
ND
N
D
ND
Lipi
d 46
15
1 57
55
22
5
1 95
6 97
3 96
3 98
6
Car
bohy
drat
e 2
70
2 12
1 86
0 11
3 99
2 39
23
34
10
Gro
ss e
nerg
y (M
J/kg
) 18
.9
20.4
21
.8
21.2
15
.3
21.9
14
.5
39.3
39
.5
39.5
39
.3
Fatty
aci
ds (%
) ^
Tota
l FA
(mg/
g lip
id)
625.
0 63
8.8
NA
N
A
NA
N
A
NA
59
6.4
879.
5 86
7.8
749.
8
22:6
n-3
19.9
N
D
NA
N
A
NA
N
A
NA
14
.2
ND
N
D
ND
22:5
n-3
2.2
ND
N
A
NA
N
A
NA
N
A
2.1
ND
N
D
ND
20:5
n-3
9.0
0.5
NA
N
A
NA
N
A
NA
11
.3
ND
N
D
ND
20:4
n-6
2.5
0.6
NA
N
A
NA
N
A
NA
1.
5 N
D
ND
N
D
18
:3n-
3 0.
8 1.
0 N
A
NA
N
A
NA
N
A
1.0
1.0
ND
N
D
18
:2n-
6 1.
7 11
.0
NA
N
A
NA
N
A
NA
2.
0 11
.0
6.7
ND
18:1
15
.5
43.8
N
A
NA
N
A
NA
N
A
18.6
73
.8
34.6
0.
4
18:0
8.
7 8.
6 N
A
NA
N
A
NA
N
A
5.1
3.0
4.6
51.3
16:0
25
.8
25.4
N
A
NA
N
A
NA
N
A
22.9
9.
9 51
.9
46.2
SFA
39
.8
36.1
N
A
NA
N
A
NA
N
A
36.4
13
.4
58.6
99
.6
M
UFA
22
.2
50.8
N
A
NA
N
A
NA
N
A
29.1
74
.6
34.7
0.
4
PUFA
3.
4 12
.0
NA
N
A
NA
N
A
NA
4.
9 12
.0
6.7
ND
LC-P
UFA
34
.5
1.2
NA
N
A
NA
N
A
NA
29
.7
ND
N
D
ND
Tota
l n-3
32
.7
1.6
NA
N
A
NA
N
A
NA
30
.5
1.0
ND
N
D
To
tal n
-6
5.2
11.6
N
A
NA
N
A
NA
N
A
4.1
11.0
6.
7 N
D
123
| Pa
ge
^ A
ll fa
tty a
cids
are
pre
sent
ed a
s a p
erce
ntag
e of
the
tota
l fat
ty a
cids
. Qua
ntita
tive
data
can
be
obta
ined
by
mul
tiply
ing
the
tota
l FA
(mg/
g lip
id) b
y sp
ecifi
c fa
tty a
cids
(%).
18:
1, su
m o
f 18:
1n-7
, 18:
1n-9
cis
, 18:
1n-9
tran
s; sa
tura
ted
fatty
aci
ds (S
FA),
sum
of 1
2:0,
14:
0, 1
6:0,
18:
0, 2
0:, 2
2:0,
24:
0;
mon
ouns
atur
ated
fatty
aci
ds (M
UFA
), su
m o
f14:
1n-5
, 16:
1n-7
, 18:
1n-7
, 18:
1n-9
(cis
and
tran
s), 2
0:1n
-7, 2
0:1n
-9, 2
2:1n
-9, 2
4:1n
-9; p
olyu
nsat
urat
ed
fatty
aci
ds (P
UFA
), su
m 1
8:2n
-6 (c
is a
nd tr
ans)
, 18:
3n-6
, 18:
3n-3
, 18:
4n-3
; lon
g ch
ain
poly
unsa
tura
ted
fatty
aci
ds (L
C-P
UFA
), su
m 2
0:2n
-6, 2
0:3n
-6,
20:4
n-6,
22:
4n-6
, 2-:3
n-3,
20:
5n-3
, 22:
5n-3
, 22:
6n-3
; n-3
, sum
of o
meg
a 3
PUFA
and
LC
-PU
FA; n
-6, s
um o
f om
ega
6 PU
FA a
nd L
C-P
UFA
.
124 | P a g e
Table 6. 2 Formulation and composition of experimental diets, all values are g/kg DM unless stated.
a Ridley aquafeeds, Narangba, QLD, Australia. Fish meal defatted using hexane. b Manildra Group, Rocklea, QLD, Australia c Bulk Powders, www.bulkpowders.com.au d Vitamin and mineral premix (g/kg of premix): vitamin A, 0.75 mg; vitamin D3, 6.3 mg; vitamin E, 16.7 g; vitamin K3, 1.7 g; vitamin B1, 2.5 g; vitamin B2, 4.2 g; vitamin B3, 25 g; vitamin B5, 8.3 g; vitamin B6, 2.0 g; vitamin B9, 0.8 g; vitamin B12, 0.005 g; biotin, 0.17 g; vitamin C, 75 g; choline, 166.7 g; inositol, 58.3 g; ethoxyquin, 20.8 g; copper, 2.5 g; ferrous iron, 10.0 g; magnesium, 16.6 g; manganese, 15.0 g; zinc, 25.0 g e Yttrium oxide; Stanford Materials, Aliso Viejo, California, United States f Sydney Essential Oil Co. (Sydney, NSW, Australia)
FO CTRL FO LOW FO FREE Formulation Defatted fish meal a 150 150 150 Poultry meal a 150 150 150 Soy protein isolate b 150 150 150 Wheat gluten b 150 150 150 Wheat flour b 109 109 109 Casein c 100 100 100 Pregelled wheat starch 80 80 80 DL-Methionine 10 10 10 Di-calcium phosphate 10 10 10 Pre-mix vitamins d 8 8 8 Yttrium oxide e 1 1 1 Fish oil a 82 10 0 Olive oil f 0 36 41 Palm Oil f 0 18 20 Palm Flake f 0 18 20 Composition Dry matter 940 939 945 Protein 598 588 603 Ash 64 64 66 Lipid 126 123 122 Carbohydrate 204 219 202 Gross energy (MJ/kg) 21.5 21.3 21.6 Fatty acids (% total) Total FA (mg/g lipid) 681.8 708.4 717.4 22:6n-3 10.6 2.0 0.9 22:5n-3 1.6 0.1 0.1 20:5n-3 8.1 1.3 0.5 20:4n-6 1.2 0.3 0.1 18:3n-3 1.3 1.1 1.0 18:2n-6 10.0 13.0 13.2 18:1 22.7 38.8 41.1 18:0 5.5 11.9 12.6 16:0 22.9 27.0 27.6 SFA 34.5 41.2 41.8 MUFA 31.3 41.1 42.5 PUFA 12.7 14.1 14.3 LC-PUFA 21.5 3.7 1.4 Total n-3 22.9 4.4 2.4 Total n-6 11.3 13.4 13.2
125
| Pa
ge
Tab
le 6
. 3 R
eal-t
ime
qPC
R p
rim
er p
airs
for
targ
et g
enes
invo
lved
in fa
tty a
cid
met
abol
ism
.
Targ
et g
ene
Abb
revi
atio
n EC
num
ber
Prim
er n
ame
Sequ
ence
Le
ngth
Fa
tty a
cid
met
abol
ism
Lat
es c
alca
rife
r
Fatty
aci
d sy
ntha
se
Lc F
AS
EC 2
.3.1
.85
FAS
qPC
R.F
or 1
TG
AA
TCTC
AC
CA
CG
CTT
CA
G
20
FAS
qPC
R.R
ev 1
A
GG
CA
GC
AA
TAG
AA
CC
CTC
A
20
St
eroy
l CoA
des
atur
ase
Lc S
CD
EC
1.1
4.19
.1
SCD
qPC
R.F
or 1
C
CTG
GTA
CTT
CTG
GG
GTG
AA
20
SC
D q
PCR
.Rev
1
AA
GG
GG
AA
TGTG
TGG
TGG
TA
20
C
arni
tine
palm
itoyl
trans
fera
se
Lc C
PT1a
EC
2.3
.1.2
1
CPT
1A q
PCR
.For
1
TGA
TGG
TTA
TGG
GG
TGTC
CT
20
CPT
1A q
PCR
.Rev
1
CG
GC
TCTC
TTC
AA
CTT
TGC
T 20
ATP
citr
ate
lyas
e Lc
ACY
L EC
2.3
.3.8
Lc
al A
CY
L F1
C
AA
CA
CC
ATT
GTC
TGTG
CTC
20
Lc
al A
CY
L R
1 G
AA
ATG
CTG
CTT
AA
CA
AA
GTC
C
21
Fa
tty a
cid
elon
gatio
n 5
Lc E
LOVL
5 EC
2.3
.1.n
8
Lcal
ELO
VL5
F1
ATC
CA
GTT
CTT
CTT
AA
CC
GT
20
Lcal
ELO
VL5
R1
GG
TTTC
TCA
AA
TGTC
AA
TCC
AC
22
Fatty
aci
d de
satu
rase
6
Lc F
ADS2
EC
1.1
4.19
.- Lc
al F
AD
S2 F
1 TC
ATA
CTA
CC
TTC
GC
TAC
TTC
TC
23
Lcal
FA
DS2
R1
AC
AA
AC
CA
GTG
AC
TCTC
CA
G
20
Refe
renc
e
Lu
cife
rase
Lu
c N
A
Luc
qPC
R F
G
GTG
TTG
GG
CG
CG
TTA
TTTA
20
Lu
c qP
CR
R
CG
GTA
GG
CTG
CG
AA
ATG
C
18
El
onga
tion
fact
or 1
alp
ha
EF1α
N
A
Lcal
EF1α
F A
AA
TTG
GC
GG
TATT
GG
AA
C
19
Lcal
EF1α
R
GG
GA
GC
AA
AG
GTG
AC
GA
C
18
NA
. Not
ava
ilabl
e
126 | P a g e
6.3 Results
6.3.1 Growth and feed utilisation
During the 56 d growth assay, the fish responded readily to the experimental diets
and growth in the control group was consistent with the predicted model growth,
achieving 106% of the modelled potential (Glencross and Bermudes, 2012). When
analysed by one-way ANOVA there was a significant difference in live-weight after
two weeks of feeding on the experimental diets, with the fish fed the FO LOW and
FO FREE diets being smaller from those fed the FO CTRL diet. By six weeks of
feeding there was a significant difference among all three groups of fish and this
remained the same at eight weeks. The same results were found with weight gained
(Table 6.4). When the live-weight, weight gain and FCR data were analysed with a
repeated measures design there was a significant interaction effect of the diets after
controlling for time (repeated) measurements (Table 6.5). There was no difference in
feed intake among the groups of fish, however, there was a significant difference in
feed conversion (FCR) between the FO CTRL and FO FREE fed fish (Table 6.4 and
6.5). There were no differences in terms of survival with only one fish removed from
the system.
The total lipid was significantly more digestible in the FO CTRL fed fish than
the FO FREE fed fish (Table 6.6). There were several significant differences in
specific fatty acid digestibility. The n-3 LC-PUFA including EPA and DHA were all
completely digested by the fish fed FO LOW and FO FREE diets whereas the FO
CTRL fed fish digested less of these fatty acids (Table 6.6). Although significant, the
numerical differences were minor. The digestibility of total saturated fatty acids
(SFA) was significantly different with fish fed the FO FREE diet able to digest more
than the fish fed the FO CTRL diet.
127 | P a g e
Table 6. 4 Growth performance and feed utilisation of barramundi fed experimental diets for eight weeks.
FCR, Feed conversion ratio P SEM, Pooled standard error mean, P<0.001*** P<0.01** P<0.05*. Superscript letters indicate significant differences among means. One-way ANOVA df 2,6, post-hoc Tukey's HSD Percentage data were arcsine transformed prior to analysis Any values <0.01 are reported as 0.1; NA, Not analysed.
FO CTRL FO LOW FO FREE P
SEM ANOVA Live-weight initial (g) 46.9 46.9 46.8 0.07 F=0.1, P=0.89 Live-weight WK2 (g) 92.7b 85.8a 87.4a 1.18 F=10.8 * Live-weight WK4 (g) 139.2b 130.6a 125.9a 2.07 F=23.7 ** Live-weight WK6 (g) 185.0c 173.9b 162.9a 3.40 F=22.3 ** Live-weight WK8 (g) 238.3c 218.9b 202.8a 5.26 F=56.4 *** Gain (g/fish) 191.4c 172.1b 156.0a 5.24 F=58.5 *** Feed intake (g/fish) 209.6 196.3 207.9 3.91 F=1.2, P=0.36 FCR 1.10a 1.14ab 1.33b 0.04 F=12.9 ** Protein retention (%) 34.1a 30.3b 27.8b 1.01 F=13.7 ** Lipid retention (%) 74.8a 61.7a 44.8b 4.61 F=24.2 ** Energy retention (%) 37.8a 34.5a 29.8b 1.30 F=11.4 ** Survival (%) 98 100 100 0.01 NA Abnormalities (%) 4.0a 2.0a 41.0b 0.08 F=6.8 * Behaviour index 1.9 1.7 1.8 2.8 F=4.0, P=0.08
128 | P a g e
Table 6. 5 Split-plot analysis of variance for repeated measures design of growth performance and feed utilisation parameters in juvenile barramundi.
Two-way repeated measures ANOVA, df residuals 6,24 (live-weight), df residuals 6,18 (Gain, FCR, Intake). FCR, Feed conversion ratio
Source of variation Degrees of freedom F value P (>F) Live-weight (g) Diet 2 44.6 < 0.001 Week 4 7341 < 0.001 Diet:Week 8 26.4 < 0.001 Gain (g/fish) Diet 2 48.7 < 0.001 Week 3 4659 < 0.001 Diet:Week 6 20.9 < 0.001 Feed intake (g/fish) Diet 2 2.3 0.18 Week 3 1681 < 0.001 Diet:Week 6 0.9 0.52 FCR Diet 2 11.7 < 0.01 Week 3 345.8 < 0.001 Diet:Week 6 11.5 < 0.001
129 | P a g e
Table 6. 6 Apparent digestibility of macro nutrients and fatty acids present in the experimental diets.
P SEM, Pooled standard error mean P<0.05 *. Superscript letters indicate significant differences among means. One-way ANOVA df 2,6, post-hoc Tukey's HSD. Percentage data were arcsine transformed prior to analysis Any values <0.01 are reported as 0.1; NA, Not analysed.
FO CTRL FO LOW FO FREE PSEM ANOVA Dry matter (%) 64.3 70.8 65.4 1.5 F=4.3, P=0.10 Protein (%) 91.6 93.0 94.1 0.4 F=4.4, P=0.08 Total lipid (%) 90.7a 87.1ab 84.6b 1.5 F = 8.1 * Energy (%) 87.3 85.7 88.7 0.7 F=2.4, P=0.19 22:6n-3 (%) 98.5 100.0 100.0 0.6 NA 22:5n-3 (%) 100.0 100.0 100.0 0.0 NA 20:5n-3 (%) 99.0 100.0 100.0 0.2 NA 20:4n-6 (%) 100.0 100.0 100.0 0.0 NA 18:3n-3 (%) 98.6 100.0 100.0 0.6 NA 18:2n-6 (%) 95.8 96.0 95.6 0.6 F=0.2, P=0.84 18:1 (%) 92.1 93.9 92.9 0.6 F=1.6, P=0.30 18:0 (%) 76.7a 70.9ab 65.6b 2.6 F=9.4* 16:0 (%) 81.5 77.0 73.7 2.1 F=4.7, P=0.9 SFA (%) 82.3a 76.0ab 71.8b 2.4 F = 8.0 * MUFA (%) 92.6 94.0 92.8 0.5 F=1.1, P=0.41 PUFA (%) 96.5 96.3 96.0 0.6 F=0.5, P=0.66 LC-PUFA (%) 98.9 100.0 100.0 0.4 NA Total n-3 (%) 98.9 100.0 100.0 0.4 NA Total n-6 (%) 96.3 96.1 95.6 0.5 F=0.5, P=0.64
130 | P a g e
6.3.2 Biochemical analysis
There were significant differences in macro nutrient retention of the fish after eight
weeks of feeding with the control fed fish retaining more protein, lipid and gross
energy than the FO LOW and FREE fed fish (Table 6.4). Generally, the fatty acid
composition of the diets was reflected in the whole fish and also the liver tissue
(Table 6.7). There were significant differences in most of the dominant fatty acids in
the whole-body with the only exceptions being 18:3n-3 and 16:0 (Table 6.7).
Similarly, in the liver, there were significant differences in most of the dominant
fatty acids with the exception of 18:2n-6, 18:3n-3 and 16:0 (Table 6.7).
After eight weeks, there was a significant decrease in the retention of SFA,
MUFA and PUFA in the FO LOW and FO FREE fed fish compared to the FO CTRL
fed fish while the LC-PUFA retention was highest in the FO LOW fed fish (Figure
6.1). In terms of calculated β-oxidation values there was significantly less oxidation
of total SFA and MUFA in the FO CTRL fed fish. There was no difference in PUFA
oxidation yet significantly more LC-PUFA was oxidised in the FO CTRL fed fish
(Figure 6.1).
131
| Pa
ge
Tab
le 6
. 7 In
itial
and
fina
l fat
ty a
cid
com
posi
tion
of w
hole
-bod
y an
d liv
er ti
ssue
from
juve
nile
bar
ram
undi
. All
fatt
y ac
id d
ata
are
pres
ente
d as
pe
rcen
tage
of t
otal
fatt
y ac
ids (
%) u
nles
s oth
erw
ise
stat
ed.
^ Pl
ease
refe
r to
Tabl
e 1
for d
etai
ls. P
SEM
, Poo
led
stan
dard
err
or m
ean
P<0.
001*
** P
<0.0
1**
P<0
.05*
. Sup
ersc
ript l
ette
rs in
dica
te si
gnifi
cant
diff
eren
ces a
mon
g m
eans
. One
-way
AN
OV
A d
f 2,6
, pos
t-hoc
Tuk
ey's
HSD
Pe
rcen
tage
dat
a w
ere
arcs
ine
trans
form
ed p
rior t
o an
alys
is
Any
val
ues <
0.01
are
repo
rted
as 0
.1; N
A, N
ot a
naly
sed.
W
hole
-bod
y
Li
ver
In
itial
FO
C
TRL
FO
LOW
FO
FR
EE
P SE
M
AN
OV
A
Initi
al
FO
CTR
L FO
LO
W
FO
FREE
P
SEM
A
NO
VA
To
tal l
ipid
(wet
w
eigh
t %)
5.1
9.5
9.4
8.7
0.2
F=2.
9, P
=0.1
4 N
A
NA
N
A
NA
N
A
NA
To
tal F
A (m
g/g
Lipi
d) ^
68
1.0
616.
6a 77
7.2b
729.
6ab
25.8
F=
17.4
**
NA
N
A
NA
N
A
NA
N
A
22:6
n-3
2.1
5.5a
2.0b
1.3c
0.7
F=39
.2**
* 4.
4 4.
8a 2.
2b 0.
9c 0.
6 F=
202.
2***
22
:5n-
3 0.
8 1.
6a 0.
7b 0.
1c 0.
2 F=
88.9
***
1.1
1.4a
0.5b
0.3c
0.2
F=16
7.2*
**
20:5
n-3
1.4
3.7a
1.0b
0.6b
0.5
F=70
.4**
* 1.
7 2.
7a 0.
7b 0.
2c 0.
4 F=
440.
8***
20
:4n-
6 0.
4 0.
7a 0.
1b 0.
1b 0.
1 F=
34.5
***
0.7
0.8a
0.5b
0.2c
0.1
F=94
.9**
*
18:3
n-3
1.0
1.0
0.9
0.9
0.1
F=0.
9, P
=0.4
4 0.
6 0.
5 0.
4 0.
5 0.
1 F=
1.25
, P=
0.35
18
:2n-
6 9.
7 8.
8a 11
.2b
11.4
b 0.
4 F=
136.
9***
6.
4 4.
8 6.
0 6.
8 0.
4 F=
3.0,
P=0
.12
18:1
37
.9
30.1
a 41
.4b
43.9
c 2.
1 F=
475.
1***
34
.8
31.1
a 37
.9b
40.7
c 1.
4 F=
116.
1***
18
:0
7.8
7.4a
9.2b
8.9b
0.3
F=94
.3**
* 11
.5
10.6
a 12
.9b
11.8
ab
0.4
F=14
.1**
16
:0
26.1
26
.9
25.8
25
.7
0.2
F=3.
9, P
=0.0
8 27
.8
30.8
31
.3
31.0
0.
4 F=
0.2,
P=0
.85
SFA
38
.5
39.3
a 37
.3b
36.5
b 0.
5 F=
10.8
* 42
.9
45.5
46
.9
45.1
0.
6 F=
0.7,
P=0
.53
MU
FA
45.7
37
.7a
45.0
b 47
.3c
1.5
F=15
3.3*
**
40.0
37
.9a
41.3
b 44
.1c
0.9
F=47
.6**
* PU
FA
11.0
11
.7a
14.0
b 14
.3b
0.4
F=60
.2**
* 8.
5 6.
2 7.
1 8.
4 0.
5 F=
2.6,
0.1
5 LC
-PU
FA
4.7
11.4
a 3.
8b 2.
0b 1.
5 F=
57.2
***
8.6
10.4
a 4.
7b 2.
4c 1.
2 F=
349.
0***
To
tal n
-3
4.3
11.9
a 4.
4b 2.
7b 1.
5 F=
54.0
***
8.6
9.7a
4.1b
2.6c
1.2
F=36
1.0*
**
Tota
l n-6
11
.4
11.2
a 13
.4b
13.5
b 0.
5 F=
92.0
***
8.5
6.8
7.7
8.2
0.4
F=2
.3, P
=0.1
9
132 | P a g e
Figure 6. 1 (A-D) Mass-balance computations of fatty acid retention and β-oxidation in juvenile barramundi, mean (±SEM) n=3. Saturated fatty acid (a; SFA) retention F=82.5***, β-oxidation F=5.2*; Monounsaturated fatty acid (b; MUFA) retention F=44.5***, β-oxidation F=7.0*; Polyunsaturated fatty acid (c; PUFA) retention F=10.8*, β-oxidation F=1.4, P=0.32; Long-chain polyunsaturated fatty acid (d; LC-PUFA) retention F=6.1*, β-oxidation F=241.5***. Significant differences are indicated by letters (*, P<0.05; **, P<0.01; ***, P<0.001), one - way ANOVA df 2,6., post-hoc Tukey’s HSD.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Retention β-oxidation
LC-P
UFA a
b
a
a
b
c
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Retention β-oxidation
MU
FA
a
b
c
a
b
b
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Retention β-oxidation
PU
FA
a
a
b
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Retention β-oxidation
SFA
bb
a
c
b
a
(A) (B)
(C) (D)
133 | P a g e
6.3.3 Clinical observations
A range of abnormalities were observed during the experiment. These included red
fins (typically caudal and pectoral), red skin, fin erosion, lesions and non-
contributing (feed refusal) fish. The fish fed the FO FREE diet were significantly
more affected by a range of abnormalities than the fish fed the FO LOW and FO
CTRL diets (Table 6.4). There was no significant difference in the behavioural
response of fish assessed by the methods described earlier (Table 6.4).
6.3.4 Sub-clinical parameters
A range of plasma chemistry parameters were assessed and some significant
differences were observed among the dietary treatments (Table 6.8 and Table 6.9). In
all measured parameters, with the exception of glutamate dehydrogenase (GLDH)
and creatinine, there was a significant time effect. In most parameters the numerical
differences were relatively minor. Phosphate, iron, haemoglobin and haptoglobin all
increased significantly after week six. There was a significant diet effect on plasma
cholesterol with the lowest values recorded in the FO LOW and FREE fed fish.
There was also a significant diet effect in plasma iron being highest in the FO LOW
fed fish. Similarly, plasma haptoglobin was highest in the FO FREE fed fish (Table
6.8).
Expression of lipid metabolism related genes in the liver of the FO CTRL and
FO FREE fish were analysed by real-time quantitative PCR and several significant
differences were observed (Figure 6.2). Repeated measures analysis showed that
there were no significant effects on the expression of Lc CPT1α and Lc ELOVL5
genes. There was a significant diet effect on the expression of Lc ACYL with the
highest expression in the FO FREE fed fish at week four. Similarly, the expression of
Lc FAS was approximately 2.5-fold higher in the FO FREE fish. The expression of
Lc SCD was significantly affected by the dietary treatment with levels of expression
in the FO FREE fish approximately 3- to 4-fold higher than the FO CTRL fish. There
was a significant diet effect in the expression of Lc FADS2 with approximately 2-
fold higher expression among the FO FREE fed fish. There were no significant time
or interaction effects for the genes analysed.
134
| Pa
ge
Tab
le 6
. 8 P
lasm
a ch
emis
try
para
met
ers i
n ju
veni
le b
arra
mun
di fe
d ex
peri
men
tal d
iets
, sam
pled
fort
nigh
tly fo
r ei
ght w
eeks
.
CK
, Cre
atin
e ki
nase
; ALT
, Ala
nine
tran
sfer
ase;
GLD
H, G
luta
mat
e de
hydr
ogen
ease
P
SEM
, Poo
led
stan
dard
err
or m
ean
Pre-
supe
rscr
ipt l
ette
rs in
dica
te si
gnifi
cant
diff
eren
ces a
mon
g th
e di
ets a
nd p
ost-s
uper
scrip
t let
ters
indi
cate
diff
eren
ces o
ver t
ime.
FO
CTR
L W
K2
FO
CTR
L W
K4
FO
CTR
L W
K6
FO
CTR
L W
K8
FO
LOW
W
K2
FO
LOW
W
K4
FO
LOW
W
K6
FO
LOW
W
K8
FO
FREE
W
K2
FO
FREE
W
K4
FO
FREE
W
K6
FO
FREE
W
K8
P SE
M
CK
(U/L
) 54
94.0
a
1375
.0 b
27
79.0
b
7486
.3 b
48
57.0
a
2099
.3 b
26
44.0
b
6558
.3 b
74
67.3
a
2862
.3 b
40
42.7
b
4406
.7 b
56
3.98
A
LT (U
/L)
15.0
a 3.
3 b
5.7
b 8.
7 b
9.3
a 6.
7 b
5.0 b
10
.7 b
6.
7 a
4.7 b
8.
0 b
8.7 b
0.
74
GLD
H (U
/L)
14.3
8.
3 7.
7 10
.0
20.7
14
.0
12.0
22
.0
15.0
13
.7
10.3
19
.7
1.12
U
rea
(mm
ol/L
) 2.
1 a
2.1
a 2.
7 ab
2.
9 b
2.0
a 2.
1 a
2.1
ab
3.1
b 2.
3 a
2.1
a 2.
8 ab
2.
8 b
0.09
C
reat
inin
e (u
mol
/L)
23.3
24
.3
22.7
22
.7
23.0
23
.0
22.7
24
.3
22.0
21
.7
22.0
23
.0
0.22
C
alci
um (m
mol
/L)
3.0
a 3.
4 b
3.2
ab
3.2
b 2.
9 a
3.1
b 3.
1 ab
3.
3 b
3.0
a 3.
2 b
3.1
ab
3.2
b 0.
03
Mag
nesi
um (m
mol
/L)
1.2
a 1.
2 a
1.1
a 1.
8 b
1.1
a 1.
0 a
1.0
a 2.
0 b
1.1
a 1.
2 a
1.1
a 2.
0 b
0.08
Ph
osph
ate
(mm
ol/L
) 4.
0 a
3.6
a 3.
3 b
3.6
a 3.
5 a
3.5
a 3.
2 b
3.8
a 3.
5 a
3.6
a 3.
3 b
3.5
a 0.
06
Cho
lest
erol
(mm
ol/L
) x 5.
9 a
x 6.
6 b
x 6.
1 ab
x
5.6
a y 4.
4 a
y 4.
6 b
y 4.
6 ab
y 4.
3 a
y 4.
2 a
y 4.
5 b
y 4.
0 ab
y 3.
8 a
0.16
To
tal P
rote
in (g
/L)
46.7
a 52
.5 b
47.6
ab
48.1
ab
42.8
a 45
.7 b
47.1
ab
47.5
ab
45.4
a 48
.9 b
47.9
ab
47.9
ab
0.47
A
lbum
in (g
/L)
13.4
a 15
.8 b
14.1
b 13
.6 ab
12
.7 a
14.0
b 14
.1 b
13.8
ab
13.6
a 14
.3 b
14.3
b 14
.0 ab
0.
16
Iron
(um
ol/L
) x 20
.5 a
x 23
.3 b
x 18
.9 b
x 21
.5 b
y 26
.4 a
y 27
.2 b
y 26
.2 b
y 22
.9 b
x 11
.7 a
x 17
.3 b
x 16
.6 b
x 23
.6 b
1.05
H
aem
oglo
bin
(mg/
dL)
17.3
a 2.
7 b
4.7
ab
26.0
a 8.
7 a
2.7
b 4.
0 ab
17
.3 a
10.7
a 2.
0 b
6.3
ab
8.7
a 1.
47
Hap
togl
obin
(mg/
mL)
x 0.
3 a
x 0.
3 ab
x
0.3
b x
1.3
c x
0.3
a x 0.
3 ab
x 0.
4 b
x 1.
4 c
y 0.
3 a
y 0.
4 ab
y 0.
4 b
y 1.
3 c
0.07
135
| Pa
ge
Tab
le 6
. 9 S
plit-
plot
ana
lysi
s of v
aria
nce
for
repe
ated
mea
sure
s des
ign
of p
lasm
a ch
emis
try
para
met
ers i
n ju
veni
le b
arra
mun
di.
CK
, Cre
atin
e ki
nase
; ALT
, Ala
nine
tran
sfer
ase;
GLD
H, G
luta
mat
e de
hydr
ogen
ease
Tw
o-w
ay re
peat
ed m
easu
res A
NO
VA
, df o
f res
idua
ls 6
,18.
So
urce
of
varia
tion
Deg
rees
of
fr
eedo
m
F va
lue
P (>
F)
Sour
ce o
f va
riatio
n
Deg
rees
of
fr
eedo
m
F va
lue
P (>
F)
CK
(U/L
) D
iet
2 0.
71
0.85
Phos
phat
e (m
mol
/L)
Die
t 2
0.56
0.
6 W
eek
3 5.
55
<0.0
1
Wee
k 3
3.17
<0
.05
Die
t:Wee
k 6
0.67
0.
67
D
iet:W
eek
6 0.
9 0.
52
ALT
(U/L
) D
iet
2 0.
25
0.79
Cho
lest
erol
(m
mol
/L)
Die
t 2
145.
2 <0
.001
W
eek
3 4.
23
<0.0
5
Wee
k 3
4.85
<0
.05
Die
t:Wee
k 6
1.67
0.
19
D
iet:W
eek
6 0.
77
0.6
GLD
H (U
/L)
Die
t 2
5 0.
05
To
tal P
rote
in
(g/L
) D
iet
2 4.
79
0.06
W
eek
3 3.
06
0.05
Wee
k 3
6.64
<0
.01
Die
t:Wee
k 6
0.43
0.
84
D
iet:W
eek
6 1.
77
0.16
U
rea
(mm
ol/L
) D
iet
2 0.
5 0.
63
A
lbum
in (g
/L)
Die
t 2
1.33
0.
33
Wee
k 3
8.35
<0
.01
W
eek
3 5.
77
<0.0
1 D
iet:W
eek
6 1.
03
0.44
Die
t:Wee
k 6
1.36
0.
28
Cre
atin
ine
(um
ol/L
) D
iet
2 2.
77
0.14
Iron
(um
ol/L
) D
iet
2 14
.6
<0.0
1 W
eek
3 0.
85
0.48
Wee
k 3
5.45
<0
.01
Die
t:Wee
k 6
1.1
0.4
D
iet:W
eek
6 3.
19
<0.0
5 C
alci
um
(mm
ol/L
) D
iet
2 0.
74
0.51
Hae
mog
lobi
n (m
g/dL
) D
iet
2 0.
68
0.54
1 W
eek
3 6.
44
<0.0
1
Wee
k 3
5.48
7 <0
.01
Die
t:Wee
k 6
0.62
0.
72
D
iet:W
eek
6 0.
63
0.7
Mag
nesi
um
(mm
ol/L
) D
iet
2 0.
25
0.78
Hap
togl
obin
(m
g/m
L)
Die
t 2
6.63
<0
.05
Wee
k 3
11.8
1 <0
.001
Wee
k 3
4313
.2
<0.0
01
Die
t:Wee
k 6
0.17
0.
98
D
iet:W
eek
6 2.
41
0.07
136 | P a g e
Figure 6. 2 (A-F) Expression of lipid metabolism genes in the liver of juvenile barramundi. All data are normalised to EF1α and Luc reference genes, log 2 transformed and expressed relative to the initial fish (WK 0), mean (±SEM) n=6. The FO CTRL (control) groups are indicated by light bars and the FO FREE groups are indicated by dark bars. Two-way repeated measures ANOVA, df of residuals 10,30. Lc ACYL, Diet F=15.5**, Week F=2.3, P=0.09, Diet:Week F=1.0, P=0.39; Lc CPT1a, Diet F=0.2, P=0.69, Week F=0.6, P=0.65, Diet:Week F=06, P=0.62; Lc FAS, Diet F=48.5***, Week F=1.4, P=0.28, Diet:Week F=0.8, P=0.50; Lc SCD, Diet F=126.6***, Week F=0.6, P=0.62, Diet:Week F=0.6, P=0.62; Lc ELOVL5, Diet F=0.2, P=0.66, Week F=0.4, P=0.78, Diet:Week F=0.2, P=0.92; Lc FADS2, Diet F=75.4***, Week F=0.7, P=0.59, Diet:Week F=0.2, P=0.91. Please refer to Table 6.3 for individual gene details.
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
Rela
tive
fold
chan
ge (l
og2)
Lc ACYL
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
Rela
tive
fold
chan
ge (l
og2)
Lc CPT1α
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
Rela
tive
fold
chan
ge (l
og2)
Lc ELOVL5
WK 8WK 6WK 4WK 2 -3.75
-3.25
-2.75
-2.25
-1.75
-1.25
-0.75
-0.25
0.25Re
lativ
e fo
ld ch
ange
(log
2)
Lc FADS2
WK 8WK 6WK 4WK 2
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
Rela
tive
fold
chan
ge (l
og2)
Lc FAS
-1
0
1
2
3
4
5
6
7
Rela
tive
fold
chan
ge (l
og2)
Lc SCD
(A) (B)
(C) (D)
(E) (F)
****** ******
******
**
**
***
***
*** ******
***
137 | P a g e
6.4 Discussion
The onset of essential fatty acid (EFA) deficiency on growth and feed utilisation in
juvenile barramundi was evident after only two weeks of feeding the experimental
diets. After eight weeks growth was clearly different among all three groups
suggesting that a primary feature of EFA deficiency is growth potential. In addition,
there was a significant difference in the treatment by time interaction term for growth
performance parameters. This interaction shows a clear progression towards the
development of EFA deficiency in the FO LOW and FO FREE fed fish. This is in
agreement with other studies showing reduced growth when carnivorous marine fish
were fed with high levels of FO substitution (Glencross et al., 2003b; Izquierdo et al.,
2005; Montero et al., 2005). In addition, consistent with the feed intake but with
reduced growth, the feed conversion of FO FREE fed fish was significantly poorer.
The ability to define a response to a nutrient relies on being able to define any
symptoms that may occur in its absence. The defatted fish meal used in the present
study contained trace amounts of lipid and LC-PUFA. However, the inclusion of
15% defatted fish meal was based on previous recommendations to achieve what is
considered normal growth for this species (Glencross et al., 2011b; Glencross and
Bermudes, 2012).
Early studies with barramundi demonstrated that decreased dietary lipid is
associated with reduced growth and in some cases EFA deficiency was inferred
(Buranapanidgit and Boonyaratpalin, 1988; Catacutan and Coloso, 1995; Williams et
al., 2003; Williams et al., 2006). In the present study, the macro nutrient levels were
kept constant as barramundi, like most other species, have an interdependent demand
for energy and specific nutrients (Glencross, 2006). The total lipid level was
formulated to be lower than the known optimal requirement (>14%) for fish of this
size (<100 g) so that in this experimental design the lipid would theoretically be
limiting and therefore cause the supply of this nutrient (and vagaries in its
composition) to be the point of sensitivity in the study (Glencross, 2006). Therefore,
the differences in the present study are directly attributable to the fatty acid profile of
the diets, resulting in EFA deficiency.
In the present study, the digestibility of total lipid was lowest in the FO FREE
fed fish consistent with other studies investigating FO replacement (Caballero et al.,
2002; Ng et al., 2004; Torstensen et al., 2000). The apparent digestibility of specific
fatty acids was not greatly modified by the dietary treatments with the exception of
the saturated fatty acids. In the study of Olsen et al. (1998), 18:0 was reported to be
138 | P a g e
less digestible than other saturates such as 16:0 and 14:0 and in agreement, the
reduction of total lipid digestibility of the present study is likely to be caused by the
18:0 composition of the feeds. Several studies have concluded that changes in lipid
or fatty acid digestibility can result in reduced growth (Hansen et al., 2008;
Karalazos et al., 2011; Menoyo et al., 2007) whereas others have found no such
effect (Caballero et al., 2002; Torstensen et al., 2000). The whole-body and liver
fatty acid profiles in the present study showed some clear differences in terms of
their composition; however, the tissue composition largely resembled that of the fed
diet, also consistent with other studies (Glencross et al., 2014b; Pratoomyot et al.,
2008; Turchini et al., 2011b). A caveat of the present study was that the initial EFA
composition of the fish was reflective of the commercial diet fed prior to
experimentation. There was a slight reduction of total SFA in the whole body and a
lack of any change in the liver, despite the different digestibility values and growth.
Moreover, the LC-PUFA composition of the tissues in the FO FREE fed fish was
almost entirely depleted after eight weeks suggesting that the reduced digestibility of
total SFA had no bearing on the overall composition of the fish.
Examination of the mass-balance relationship of dietary nutrients or indeed
specific fatty acids in the tissues was used to reveal discrete differences in their
utilisation. Improved protein retention and consequently weight gain was evident in
the control group, consistent with previous studies on barramundi (Williams et al.,
2006). The disproportionately low lipid and energy retention of the FO LOW and FO
FREE fed fish is a response to the diets without adequate LC-PUFA. The FO CTRL
fed fish, selectively retained more of the dietary SFA, MUFA and PUFA than the
other fish, however, LC-PUFA were catabolised when in surplus to optimal tissue
concentration, similar to observations from other carnivorous species such as
Atlantic salmon, European sea bass, Murray cod and rainbow trout (Eroldogan et al.,
2013; Stubhaug et al., 2007; Turchini and Francis, 2009; Turchini et al., 2011b).
The aetiology of deficiency was less apparent in the FO LOW fed fish. The
total LC-PUFA concentration in the FO LOW diet with 1% added FO was equivalent
to 4.3 g/kg of the diet compared to 25.6 g/kg in the control diet. Currently available
requirement data for barramundi suggest that an adequate dietary LC-PUFA supply
should be at least 12 g/kg in growing barramundi (Williams et al., 2006). In
agreement, Glencross and Rutherford (2011) reasoned that provided EFA are kept in
balance the total LC-PUFA requirement could be further revised. Despite the very
low inclusion of LC-PUFA (FO LOW and FO FREE diets) there were no apparent
139 | P a g e
effects of the diets on survival or behavioural changes. However, a range of physical
abnormalities were observed in the FO FREE fed fish including; erosion of the fins,
reddening of the fins and extremities, gross lesions and physical deformities,
symptoms which are associated with EFA deficiency in fish (Castell et al., 1972).
This observation suggests that even at very low inclusion levels of FO there was a
marked improvement in the physical health of fish despite the reduced growth.
Moreover, the whole-body composition data suggest that the FO LOW fed fish
maintained a proportion of LC-PUFA similar to that of the initial fish fed a
commercially available diet. Therefore in support of the previous studies on this
species, we too support that LC-PUFA supply should be at least 12 g/kg in growing
barramundi.
A range of plasma biochemical markers were used in the assessment of EFA
deficiency over the eight week time course. The results suggest that only minor
changes occurred and mostly on a temporal basis. There were no significant diet or
interaction effects observed for the enzyme markers creatine kinase (CK), alanine
transferase (ALT) or glutamate dehydrogenase (GLDH). Elevated ALT and GLDH
activity is considered a reliable marker of liver cell necrosis that can be characterised
by the depletion of hepatic LC-PUFA stores (Morton et al., 2014; Van Waes and
Lieber, 1977; Videla et al., 2004). However, in support of previous work with the
same species, CK, ALT and GLDH levels in the present study were not affected by
the lipid composition of the diets (Glencross and Rutherford, 2011). In the
identification of underlying organ failure or disease there can be a concomitant
change in concentration of circulating urea, calcium, magnesium, phosphate and
albumin (Hjelte et al., 1990). However, in the present study these markers only
differed significantly on a temporal basis, generally increasing after week two. It
could be argued that the general increase in the markers is due to an adaptation to the
experimental diets rather than a treatment effect.
In the present study, there was a marked decrease in plasma cholesterol levels
in the FO LOW and FREE fed fish after only two weeks of feeding the experimental
diets. Increasing DHA alone in barramundi did not result in any change in plasma
cholesterol (Morton et al., 2014). While the replacement of FO with vegetable oil in
Atlantic salmon diets led to hypocholesterolemia (Glencross et al., 2014b; Richard et
al., 2006). In light of these findings, it is likely that cholesterol derived from FO is
beneficial in fish, including the barramundi, as it can potentially modulate energy
expenditure and FA metabolic pathways (Norambuena et al., 2013a).
140 | P a g e
Consistent with other studies, nutritionally regulated gene expression in the
FO CTRL fed fish appear to follow a normal progression of desaturation and
elongation of FA to their longer chain and less-saturated derivatives (Mohd-Yusof et
al., 2010; Tu et al., 2013; Wade et al., 2014). Transcription of ATP citrate lyase (Lc
ACYL) produces acetyl-CoA which is a substrate for many biosynthetic pathways
including lipogenesis and in the present study Lc ACYL was up-regulated in the EFA
deficient fish (Dias et al., 1998). There was no significant effect in the expression of
elongation (Lc ELOVL5) or carnitine palmitoyltransferase (Lc CPT1α); however,
there was a tendency towards higher expression in the FO FREE fed fish. CPT1α is
considered a key enzyme in the regulation of mitochondrial oxidation and in the
present study CPT1α was down regulated in both treatments compared to the initial
fish (Torstensen et al., 2000). This effect is likely to be caused by the low lipid levels
of all three diets, a strategy that was intended to highlight the potential effects of
EFA deficiency. This study also found that desaturase enzymes (Lc SCD and Lc
FADS2) in barramundi were significantly up-regulated in fish fed the FO FREE diet.
This is in agreement with other studies, on a range of species, showing an up-
regulation of desaturase enzymes (including both SCD and FADS2) in response to
reduced FO (Alhazzaa et al., 2011b; Betancor et al., 2014; González-Rovira et al.,
2009; Tocher et al., 2006a). Fatty acid synthase (Lc FAS) expression in the present
study was also significantly up-regulated in fish fed the FO FREE diet. Although the
FAS enzyme system has rarely been investigated in fish in response to FO
replacement, it is an important step in the initial synthesis of palmitate from acetyl-
CoA.
141 | P a g e
7.0 Eicosapentaenoic acid, arachidonic acid and eicosanoid
metabolism in juvenile barramundi Lates calcarifer
Adapted from
Salini, M.J., Wade, N.M., Araújo, B.C., Turchini, G.M., Glencross, B.D., 2016.
Eicosapentaenoic acid, arachidonic acid and eicosanoid metabolism in juvenile
barramundi Lates calcarifer. Lipids. 51(8), 973-988
142 | P a g e
143 | P a g e
144 | P a g e
7.1 Aims
The present study hypothesised that increasing the dietary inclusion level of EPA or
ARA independently may lead to improvements in growth performance, feed
conversion and also the utilisation efficiency of supplied EPA and ARA. Therefore, a
two part, dose-response experimental design was used to determine the effect of
increasing the dietary EPA or ARA. To achieve this, commercial preparations of an
EPA rich fish oil and an ARA rich fungal oil were incrementally added to a series of
barramundi diets. It was also hypothesised that high levels of ARA may alter the
transcription of nutritionally inducible genes involved in eicosanoid synthesis. The
genes regulating eicosanoid synthesis have not been identified in barramundi, and no
information is available on their nutritional regulation.
7.2 Materials and methods
The methodology used in the present experiment are detailed in the section 2.0
General methodology. Ethical clearance was approved for the experimental
procedures by the CSIRO Animal Ethics Committee (Application A05/2014). The
chemical composition of the main dietary ingredients is presented in Table 7.1. The
formulation and chemical composition of the ten diets are presented in Table 7.2.
The pairs of degenerate primers that were subsequently synthesised by Sigma-
Aldrich are presented in Table 7.3. The real-time PCR primers specific to each target
gene are presented in Table 7.4.
145
| Pa
ge
Tab
le 7
. 1 C
ompo
sitio
n of
key
ingr
edie
nts u
sed
in d
iet f
orm
ulat
ions
(g/k
g D
M).
Fatt
y ac
ids a
re p
rese
nted
as m
ole
perc
enta
ge (m
ole%
).
Fi
sh m
eala
Poul
try m
eal
Fish
oil
Palm
flak
e O
live
oil
AR
ASC
O®
Incr
omeg
a EP
A T
G50
0 In
crom
ega
DH
A T
G50
0
Dry
mat
ter (
g/kg
) 97
4 96
6 98
5 99
7 99
2 99
1 99
0 99
2 Li
pid
44
157
946
958
911
972
971
975
Ash
19
1 15
1 N
D
ND
N
D
ND
N
D
ND
Pr
otei
n 68
4 66
0 7
7 5
10
6 5
Ener
gy (M
J/kg
) 18
.6
20.9
35
.6
39.5
39
.7
40.1
39
.1
40.1
16
:0
27.3
25
.8
25.1
49
.9
11.3
11
.0
ND
2.
2 18
:0
4.8
8.4
5.2
48.7
3.
9 8.
9 N
D
4.8
18:1
17
.7
40.9
19
.7
ND
71
.6
24.6
1.
3 10
.1
18:2
n-6
1.7
10.5
2.
6 N
D
10.8
7.
8 N
D
1.0
18:3
n-3
0.4
1.4
1.2
ND
1.
0 N
D
ND
N
D
20:4
n-6
0.8
1.1
1.5
ND
N
D
42.3
3.
6 2.
9 20
:5n-
3 14
.7
0.6
10.9
N
D
ND
N
D
74.3
9.
9 22
:5n-
3 2.
8 0.
3 1.
8 N
D
ND
0.
8 2.
2 2.
8 22
:6n-
3 10
.3
0.7
13.5
N
D
ND
N
D
17.0
63
.9
SFA
40
.4
35.9
38
.7
100
15.7
20
.6
ND
6.
9 M
UFA
29
.0
48.8
29
.6
ND
72
.5
24.6
2.
8 12
.6
C18
PUFA
2.
1 12
.0
3.8
ND
11
.8
7.8
ND
1.
0 LC
-PU
FA
28.5
3.
3 27
.8
ND
N
D
47.0
97
.2
79.5
EP
A/A
RA
18
.9
0.5
7.1
ND
N
D
<0.1
20
.4
3.4
* Fi
sh m
eal w
as d
efat
ted
prio
r to
use.
See
met
hods
for d
etai
ls. N
D, n
ot d
etec
ted;
GE,
gro
ss e
nerg
y.
18:1
, sum
of 1
8:1n
-7, 1
8:1n
-9 c
is, 1
8:1n
-9 tr
ans;
satu
rate
d fa
tty a
cids
(SFA
), su
m o
f 12:
0, 1
4:0,
16:
0, 1
8:0,
20:
, 22:
0, 2
4:0;
mon
ouns
atur
ated
fatty
aci
ds (M
UFA
), su
m o
f 14:
1n-5
, 16:
1n-7
, 18:
1n-7
, 18:
1n-9
(cis
and
tran
s), 2
0:1n
-7, 2
0:1n
-9, 2
2:1n
-9, 2
4:1n
-9; p
olyu
nsat
urat
ed fa
tty a
cids
, with
18
carb
on a
tom
s (C
18 P
UFA
), su
m
18:2
n-6
(cis
and
tran
s), 1
8:3n
-6, 1
8:3n
-3, 1
8:4n
-3; l
ong
chai
n po
lyun
satu
rate
d fa
tty a
cids
, with
20
or m
ore
carb
on a
tom
s (LC
-PU
FA),
sum
20:
2n-6
, 20:
3n-6
, 20:
4n-6
, 22
:4n-
6, 2
0:3n
-3, 2
0:5n
-3, 2
2:5n
-3, 2
2:6n
-3; n
-3, s
um o
f om
ega
3 C
18 P
UFA
and
LC
-PU
FA; n
-6, s
um o
f om
ega
6 C
18 P
UFA
and
LC
-PU
FA.
146
| Pa
ge
Tab
le 7
. 2 F
orm
ulat
ion
and
com
posi
tion
of e
xper
imen
tal d
iets
(g/k
g D
M).
Fatt
y ac
ids a
re p
rese
nted
as m
ole
perc
enta
ges (
mol
e%).
EP
A 1
EP
A 5
EP
A 1
0 EP
A 1
5 EP
A 2
0 A
RA
1
AR
A 6
A
RA
12
AR
A 1
8 C
TRL
FO
Fish
mea
l a 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 Po
ultry
mea
l a 97
97
97
97
97
97
97
97
97
97
C
asei
n b
150
150
150
150
150
150
150
150
150
150
Soy
prot
ein
isol
ate
c 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 W
heat
glu
ten
c 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 W
heat
flou
r c 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 15
0 Pr
egel
whe
at st
arch
c 50
50
50
50
50
50
50
50
50
50
M
ethi
onin
e b
5 5
5 5
5 5
5 5
5 5
Prem
ix d
6 6
6 6
6 6
6 6
6 6
Dic
alci
um p
hosp
hate
5
5 5
5 5
5 5
5 5
5 C
holin
e ch
lorid
e 2
2 2
2 2
2 2
2 2
2 St
ay-C
2
2 2
2 2
2 2
2 2
2 Y
ttriu
m e
1 1
1 1
1 1
1 1
1 1
Fish
oil
a 0.
0 0.
0 0.
0 0.
0 0.
0 0.
0 0.
0 0.
0 0.
0 82
.0
Palm
Fla
ke f
31.0
28
.5
25.4
22
.0
19.0
28
.5
23.5
17
.2
11.0
0.
0 O
live
oil f
38.7
36
.0
31.9
28
.0
24.0
36
.0
30.0
21
.9
13.7
0.
0 A
RA
SCO
® g
2.3
1.8
1.2
0.5
0.0
1.8
13.0
27
.6
42.2
0.
0 In
crom
ega™
EPA
TG
500
h 0.
0 7.
7 18
.2
29.0
39
.0
7.7
7.5
7.1
6.9
0.0
Incr
omeg
a™ D
HA
TG
500
h 10
.0
8.0
5.3
2.5
0.0
8.0
8.0
8.2
8.2
0.0
Com
posi
tion
Dry
mat
ter (
g/kg
) 96
1 95
9 95
5 94
6 95
0 95
9 95
2 96
1 96
3 95
9 Li
pid
100
96
106
96
94
96
92
97
96
90
Ash
67
67
66
67
63
67
10
7 68
68
69
Pr
otei
n 58
6 56
6 58
4 56
3 55
9 56
6 56
8 57
7 59
6 57
7
147
| Pa
ge
GE
(MJ/
kg)
21.6
21
.7
21.5
21
.4
21.3
21
.7
21.6
21
.7
21.7
21
.8
Fatty
aci
ds (m
g/g
lipid
) ^ 83
2.7
876.
3 82
8.2
824.
0 85
4.4
876.
3 82
3.8
848.
9 86
6.9
866.
3 16
:0
28.0
28
.0
21.1
20
.8
19.8
28
.0
22.2
19
.7
17.8
25
.0
18:0
17
.4
16.8
16
.4
15.3
13
.0
16.8
14
.7
13.6
11
.5
5.6
18:1
33
.1
31.3
29
.8
29.6
27
.2
31.1
31
.1
29.3
26
.8
21.6
18
:2n-
6 10
.2
10.4
11
.9
7.9
9.7
10.4
11
.7
10.1
10
.9
10.1
18
:3n-
3 0.
8 0.
8 1.
2 0.
8 1.
0 0.
8 1.
0 0.
8 0.
8 1.
5 20
:4n-
6 1.
2 1.
1 1.
2 1.
2 1.
2 1.
0 4.
9 10
.7
15.5
1.
4 20
:5n-
3 1.
1 4.
1 9.
0 16
.1
19.0
4.
1 5.
3 4.
9 5.
3 8.
4 22
:5n-
3 0.
4 0.
4 0.
8 0.
7 0.
8 0.
4 0.
5 0.
7 0.
8 1.
5 22
:6n-
3 4.
0 4.
5 4.
9 5.
5 5.
1 4.
5 4.
6 5.
6 4.
9 10
.0
SFA
46
.7
46.1
38
.9
37.0
34
.0
46.1
38
.3
34.6
30
.6
37.1
M
UFA
34
.6
32.5
31
.8
30.9
29
.0
32.5
32
.8
30.9
28
.5
29.9
C
18PU
FA
11.5
11
.2
13.4
8.
7 10
.8
11.2
13
.1
11.7
12
.9
11.6
LC
-PU
FA
7.2
10.1
15
.9
23.5
26
.3
10.1
15
.8
22.8
28
.0
21.4
EP
A/A
RA
0.
9 3.
8 7.
6 13
.0
15.5
3.
8 1.
1 0.
5 0.
3 6.
1 ^
Ref
er to
Tab
le 7
.1 fo
r def
initi
ons o
f fat
ty a
cids
. Qua
ntita
tive
data
can
be
obta
ined
by
mul
tiply
ing
the
tota
l fat
ty a
cids
(mg/
g lip
id) b
y th
e in
divi
dual
fa
tty a
cid
of in
tere
st.
a R
idle
y A
quaf
eed,
Nar
angb
a, Q
LD, A
ustra
lia.
b B
ulk
Pow
ders
, ww
w.b
ulkp
owde
rs.c
om.a
u c
Man
ildra
Gro
up, R
ockl
ea, Q
LD, A
ustra
lia
d V
itam
in a
nd m
iner
al p
rem
ix (I
U k
g-1
or g
/kg
of p
rem
ix):
vita
min
A, 2
.5M
IU; v
itam
in D
3, 0
.25
MIU
; vita
min
E, 1
6.7
g; v
itam
in K
3, 1
.7 g
; vita
min
B
1, 2
.5 g
; vita
min
B2,
4.2
g; v
itam
in B
3, 2
5 g;
vita
min
B5,
8.3
; vita
min
B6,
2.0
g; v
itam
in B
9, 0
.8; v
itam
in B
12, 0
.005
g; b
iotin
, 0.1
7 g;
vita
min
C, 7
5 g;
cho
line,
166
.7 g
; ino
sito
l, 58
.3 g
; eth
oxyq
uin,
20.
8 g;
cop
per,
2.5
g; fe
rrou
s iro
n, 1
0.0
g; m
agne
sium
, 16.
6 g;
man
gane
se, 1
5.0
g; z
inc,
25.
0 g
e Y
ttriu
m o
xide
, Sta
nfor
d m
ater
ials
, Alis
o V
iejo
, CA
, USA
. f S
ydne
y Es
sent
ial O
il C
o., S
ydne
y, N
SW, A
ustra
lia
g A
RA
SCO
®, M
arte
k B
iosc
ienc
es C
o., C
olum
bia,
MD
, USA
. h
CR
OD
A™
, Sna
ith, E
ast Y
orks
hire
, UK
.
148
| Pa
ge
Tab
le 7
. 3 F
orw
ard
and
reve
rse
prim
er p
airs
(5ʹ –
3ʹ)
used
in th
e cl
onin
g of
eic
osan
oid
met
abol
ism
gen
es in
bar
ram
undi
and
rea
l-tim
e qP
CR
ge
ne e
xpre
ssio
n an
alys
is.
Targ
et
A
cces
sion
Se
quen
ce (F
/R)
Prod
uct s
ize
Deg
ener
ate
prim
ers
Pr
osta
glan
din
G/H
synt
hase
KU
1882
76
TTTG
GG
AA
TGTA
CG
CTA
CG
C
752
bp
TTTG
GG
AA
TGTA
CG
CTA
CG
C
Pros
tagl
andi
n G
/H sy
ntha
se
K
U18
8277
TC
AG
TGTG
CG
TTTC
CA
GTA
CA
G
999
bp
GG
ATT
CTT
TCTC
CA
GA
CA
GC
Pr
osta
glan
din
G/H
synt
hase
KU
1882
78
GTG
ATG
TGC
TGA
AG
GA
GG
TG
997
bp
AG
GA
TTG
CG
GA
CA
TTTC
TTTC
TC
Ara
chid
onat
e 5-
lipox
ygen
ase
K
U18
8279
TT
TAC
CA
TCG
CC
ATC
AA
CA
C
976
bp
GA
GA
TGA
CG
GC
TAC
AG
GG
TG
A
bbre
viat
ion
EC n
umbe
r Se
quen
ce (F
/R)
Am
plic
on si
ze
RT- q
PCR
prim
ers
Pr
osta
glan
din
G/H
synt
hase
Lc
CO
X1a
1.14
.99.
1 A
AC
CG
AG
TCTG
TGA
CA
TCC
T 24
7 bp
C
AA
CG
TGG
GA
TCA
AA
CTT
CA
G
Pros
tagl
andi
n G
/H sy
ntha
se
Lc C
OX1
b 1.
14.9
9.1
CA
GC
CC
TTC
AA
TCA
GTA
CA
G
238
bp
TCTC
AC
CG
AA
TATG
CTA
CC
A
Pros
tagl
andi
n G
/H sy
ntha
se
Lc C
OX2
1.
14.9
9.1
AG
TTTG
TCTT
CA
AC
AC
CTC
TG
264
bp
ATT
TCTC
TGC
TGTT
CTC
AA
TGG
A
rach
idon
ate
5-lip
oxyg
enas
e Lc
ALO
X-5
1.13
.11.
34
TTTA
CC
ATC
GC
CA
TCA
AC
AC
C
228
bp
CTC
TTC
CTT
GC
TGTC
CA
CA
C
Car
nitin
e pa
lmito
ylra
nsfe
rase
Lc
CPT
1a
2.3.
1.21
TG
ATG
GTT
ATG
GG
GTG
TCC
T 19
6 bp
C
GG
CTC
TCTT
CA
AC
TTTG
CT
149
| Pa
ge
Lu
cife
rase
Lu
c N
A
GG
TGTT
GG
GC
GC
GTT
ATT
TA
101
bp
GG
TGTT
GG
GC
GC
GTT
ATT
TA
Elon
gatio
n fa
ctor
1 a
lpha
EF
1α
NA
A
AA
TTG
GC
GG
TATT
GG
AA
C
83 b
p
G
GG
AG
CA
AA
GG
TGA
CG
AC
NA
, Not
ava
ilabl
e
150 | P a g e
7.3 Results
7.3.1 Growth performance and feed utilisation
The results of the 42 d growth assay demonstrated that the barramundi responded
well to the experimental diets in both experiments exceeding over 400% of their
original weight at a rate of more than 1 g/d. The growth parameters for each
experiment were analysed separately using polynomial contrasts (Table 7.4 and 7.5).
When the fish were fed increasing EPA there were no significant contrasts in any of
the growth parameters (Table 7.4). Similarly, the barramundi did not show any
significant contrasts in response to the increasing ARA in the second experiment
(Table 7.5). The only exception being a slight improvement in feed conversion with
the addition of ARA, however, the numerical differences were minor. Survival was
similar among all of the treatments. Several moribund fish were removed from the
system after a weight check at day 28. These fish were considered non-contributing
as they had not consumed any feed. Additionally, the growth performance of the fish
fed different EPA and ARA treatments were compared by one-way ANOVA against
the control. There were no significant differences in any of the growth parameters
measured (data not reported).
151
| Pa
ge
Tab
le 7
. 4 G
row
th p
erfo
rman
ce a
nd fe
ed u
tilis
atio
n of
bar
ram
undi
fed
incr
easi
ng E
PA a
naly
sed
usin
g po
lyno
mia
l con
tras
ts.
^ D
egre
es o
f fre
edom
4, 1
0; li
near
, qua
drat
ic a
nd c
ubic
val
ues a
re P
val
ues a
t an
alph
a le
vel o
f 0.0
5.
BW
, Bod
y w
eigh
t, P
SEM
, Poo
led
stan
dard
err
or m
ean.
D
iets
Poly
nom
ial c
ontra
sts ^
EP
A
1 EP
A
5 EP
A
10
EPA
15
EP
A
20
P SE
M
Line
ar
Qua
drat
ic
Cub
ic
Initi
al w
eigh
t (g)
10
.0
10.4
10
.3
10.3
10
.3
0.00
0.
815
0.55
6 0.
553
Fina
l wei
ght (
g)
55.6
53
.8
54.2
53
.6
53.6
0.
40
0.12
6 0.
437
0.55
0 W
eigh
t gai
ned
(g)
45.3
43
.5
43.9
43
.3
43.3
0.
40
0.12
6 0.
413
0.52
6 B
W g
ain
(%)
439.
5 41
9.7
426.
0 41
9.5
420.
3 3.
80
0.13
2 0.
329
0.44
4 D
aily
gai
n ra
te (g
/d)
1.08
1.
03
1.05
1.
03
1.03
0.
01
0.11
8 0.
417
0.49
6 Fe
ed c
onve
rsio
n 0.
70
0.73
0.
72
0.74
0.
73
0.01
0.
132
0.65
4 0.
820
Surv
ival
(%)
91.1
93
.3
93.3
92
.2
93.3
0.
40
0.64
9 0.
754
0.63
9
152
| Pa
ge
Tab
le 7
. 5 G
row
th p
erfo
rman
ce a
nd fe
ed u
tilis
atio
n of
bar
ram
undi
fed
incr
easi
ng A
RA
ana
lyse
d us
ing
poly
nom
ial c
ontr
asts
.
D
iets
Po
lyno
mia
l con
trast
s ^
A
RA
1
AR
A
6 A
RA
12
A
RA
18
P
SEM
Li
near
Q
uadr
atic
C
ubic
Initi
al w
eigh
t (g)
10
.4
10.2
10
.4
10.4
0.
02
0.46
6 0.
230
0.06
2 Fi
nal w
eigh
t (g)
53
.8
53.7
53
.2
54.8
0.
51
0.43
5 0.
209
0.35
7 W
eigh
t gai
ned
(g)
43.5
43
.5
42.8
44
.4
0.52
0.
466
0.23
9 0.
299
BW
gai
n (%
) 41
9.7
424.
9 41
2.6
428.
7 4.
96
0.62
5 0.
427
0.15
3 D
aily
gai
n ra
te (g
/d)
1.03
1.
04
1.02
1.
06
0.01
0.
466
0.23
9 0.
299
Feed
con
vers
ion
0.73
0.
73
0.72
0.
70
0.01
0.
014
0.05
3 0.
698
Surv
ival
(%)
93.3
93
.3
97.8
98
.9
1.65
0.
017
0.73
0 0.
301
^ M
ultip
le R
2 0.
30, d
f 3,8
; lin
ear,
quad
ratic
and
cub
ic v
alue
s are
P v
alue
s at a
n al
pha
leve
l of 0
.05.
B
W, B
ody
wei
ght;
P SE
M, P
oole
d st
anda
rd e
rror
mea
n.
153 | P a g e
7.3.2 Digestibility analysis of the diets
While no differences were recorded in the digestibility of dry matter, protein or
energy across the treatments, the digestibility of lipid was significantly higher in the
control (FO CTRL) fed fish compared to the EPA 1, EPA 10 and ARA 1 fed fish
(Table 7.6). There was significantly higher digestibility of 18:0 in the EPA 1 fish
compared to the EPA 10, EPA 20 and ARA 18 fish. The digestibility of 18:1 was
significantly higher in the EPA 10, EPA 20 and ARA 18 fish compared to the FO
CTRL fish. Similarly there was a significantly higher digestibility of 18:2n-6 in the
EPA 20 fish compared to the FO CTRL fish. EPA digestibility was significantly
higher in the EPA 20 fish compared to the ARA 1 and FO CTRL fish. The combined
SFA digestibility was significantly highest in the EPA 1 fish compared to the EPA
20 and ARA 18 fish. There was a significantly higher digestibility of MUFA in the
ARA 20 fish compared to the FO CTRL fish. C18PUFA digestibility was
significantly highest in the EPA 1 and EPA 20 compared to the FO CTRL fish. The
digestibility of LC-PUFA was significantly highest in the EPA 1, EPA 10 and EPA
20 fish compared to the ARA 1 fish.
7.3.3 Whole-body composition.
When compared against the control (FO CTRL), there were significant differences in
whole body composition for many parameters (Table 7.7). Whole body dry matter
was significantly higher in the EPA 20 and FO CTRL fish compared to the EPA 1
fish. The lipid composition was significantly higher in the FO CTRL fish compared
to all the other fish groups with the only exception being the ARA 18 fish. While the
energy composition was lowest in the EPA 1 fish compared to all the other fish
groups with the only exception being the ARA 1 fish. There was a significantly
higher 16:0 composition in the FO CTRL fish compared to the EPA 10 and EPA 20
while the ARA 18 fish had the lowest 16:0 composition. The 18:0 composition was
significantly higher in the EPA 1, EPA 10 and ARA 18 fish compared to the EPA 20
fish. The 18:1 composition was significantly highest in the EPA 1 and ARA 1 fish
followed by the EPA 10 fish then followed by the EPA 20 and ARA 18 fish, with the
lowest value recorded in the FO CTRL fish. As expected, there was a significant
elevation of ARA (20:4n-6) in the ARA 18 fed fish. Similarly, the EPA (20:5n-3)
composition was significantly increased with increasing dietary EPA while the FO
CTRL fish had a significantly higher composition of EPA than the ARA 1 and ARA
18 fish. The DHA (22:6n-3) body composition was significantly highest in the FO
154 | P a g e
CTRL fed fish. There was also a significant increase in the EPA/ARA ratio in the
EPA fed fish with the EPA 10 equivalent to the FO CTRL fish, while there was a
significant decrease in the EPA/ARA ratio from the ARA 1 and ARA 18 fish.
155
| Pa
ge
Tab
le 7
. 6 A
ppar
ent d
iges
tibili
ty (%
) of n
utri
ents
and
fatt
y ac
ids i
n di
ets a
naly
sed
by o
ne-w
ay A
NO
VA
.
EP
A
1 EP
A
10
EPA
20
A
RA
1
AR
A
18
FO
CTR
L P
SEM
F
valu
e P
Val
ue ^
Dry
mat
ter
70.2
69
.1
74.0
70
.8
67.3
69
.2
1.1
0.46
0.
800
Prot
ein
92.3
91
.7
93.1
92
.0
91.1
92
.4
0.3
1.05
0.
435
Lipi
d 79
.3 a
85.2
a
89.3
ab
78.3
a
86.4
ab
94.9
b
1.6
11.1
2 0.
001
Ener
gy
82.3
79
.2
84.7
80
.0
81.1
83
.3
0.8
1.27
0.
340
16:0
70
.1
52.7
51
.4
63.6
51
.8
63.8
2.
7 1.
64
0.22
3 18
:0
64.9
a
42.1
cd
28.2
d
53.8
abc
30
.7 d
56
.8 a
bc
3.5
16.5
6 0.
000
18:1
92
.1 a
b 93
.1 a
94.7
a
89.3
ab
93.3
a
84.1
b
1.0
4.87
0.
014
18:2
n-6
95.9
ab
94.9
ab
97.0
a
94.4
ab
95.3
ab
92.5
b
0.4
4.31
0.
017
18:3
n-3
100.
0 10
0.0
100.
0 10
0.0
100.
0 10
0.0
0.0
NA
N
A
20:4
n-6
100.
0 10
0.0
99.0
10
0.0
96.9
10
0.0
0.3
NA
N
A
20:5
n-3
100.
0 97
.8 a
b 99
.2 b
96
.1 a
97.6
ab
96.7
a
0.4
8.70
0.
002
22:5
n-3
100.
0 10
0.0
100.
0 10
0.0
95.3
90
.4
1.1
NA
N
A
22:6
n-3
95.6
96
97
.9
92.9
95
.8
95.7
0.
5 2.
74
0.07
1 SF
A
71.3
a
54.5
ab
49.6
b
63.9
ab
50.7
b
66.9
ab
2.4
4.89
0.
011
MU
FA
92.4
a
93.6
a
96.0
a
89.8
ab
93.3
a
83.7
b
1.1
8.10
0.
002
C18
PUFA
96
.3 a
95.4
ab
97.3
a
94.9
ab
96.0
ab
93.5
b
0.4
5.07
0.
010
LC-P
UFA
98
.2 a
98.2
a
98.9
a
94.5
c
96.9
ab
95.9
bc
0.4
10.1
6 0.
000
^ D
egre
es o
f fre
edom
5, 1
2. S
uper
scrip
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s am
ong
mea
ns.
P SE
M, P
oole
d st
anda
rd e
rror
mea
n.
156
| Pa
ge
Tab
le 7
. 7 W
hole
bod
y an
d fa
tty
acid
com
posi
tion
of b
arra
mun
di a
naly
sed
by o
ne-w
ay A
NO
VA
(g/k
g liv
e-ba
sis)
. Fat
ty a
cids
are
pre
sent
ed a
s m
ole
perc
enta
ges (
mol
e%).
In
itial
fis
h EP
A
1 EP
A
10
EPA
20
A
RA
1
AR
A
18
FO
CTR
L P
SEM
F
stat
istic
P
valu
e ^
Dry
mat
ter
225.
4 26
6.2a
280.
6 ab
284.
6 b
275.
3 ab
280.
4 ab
287.
5 b
2.0
4.95
0.
011
Prot
ein
144.
4 17
3.0
182.
3 18
1.1
182.
2 17
8.0
177.
4 1.
5 0.
95
0.48
2 Li
pid
32.9
55
.0 a
63.9
b
66.0
b
61.2
ab
67.5
bc
73.5
c
1.5
12.7
0.
000
Ener
gy (M
J/kg
) 47
.2
59.9
a
67.0
b
66.4
b
65.2
ab
66.6
b
67.3
b
0.8
4.50
0.
015
16:0
26
.0
26.5
ab
24.7
bc
23.6
bc
25.8
ab
22.8
c
28.7
a
0.9
30.6
0.
000
18:0
7.
5 7.
4 a
7.5 a
6.
5 b
7.1 a
b 7.
5 a
6.7 a
b 0.
2 13
.6
0.00
1 18
:1
32.9
38
.4 a
36.0
b
32.5
c
37.7
a
30.8
c
27.4
d
1.8
3.32
0.
041
18:2
n-6
10.3
12
.2 a
11.2
a
11.1
a
11.9
a
11.3
a
9.4 b
0.
4 22
.8
0.00
0 18
:3n-
3 1.
2 1.
0 1.
0 1.
0 1.
0 0.
8 1.
1 0.
0 1.
0 0.
458
20:4
n-6
0.8
1.0 a
1.
1 a
0.9 a
1.
0 a
10.4
b
0.9 a
1.
6 >9
99.9
0.
000
20:5
n-3
2.6
1.2 a
5.
5 d
11.4
e
2.9 b
3.
0 b
4.2 c
1.
5 25
0.0
0.00
0 22
:5n-
3 1.
2 0.
8 a
1.8 c
2.
4 c
1.4 b
1.
3 b
2.0 c
0.
2 32
.2
0.00
0 22
:6n-
3 5.
1 4.
1 a
4.3 a
4.
4 a
4.2 a
4.
3 a
6.8 b
0.
4 12
1.0
0.00
0 SF
A
37.8
36
.0 b
34.1
cd
32.1
d
34.9
bc
32.0
d
40.1
a
1.3
64.5
0.
000
MU
FA
40.5
42
.5 a
40.0
b
36.1
c
41.8
a
34.3
c
34.7
c
1.5
119.
2 0.
000
C18
PUFA
12
.0
14.1
a
12.9
bc
12.7
bc
13.7
ab
13.4
ab
11.1
c
0.4
22.9
0.
000
LC-P
UFA
9.
6 7.
4 a
13.0
cd
19.1
e
9.7 b
20
.2 e
13.9
d
2.1
146.
9 0.
000
EPA
/AR
A
3.4
1.2 b
5.
2 d
12.2
e
2.9 c
0.
3 a
4.5 d
1.
7 62
4.0
0.00
0 ^
Deg
rees
of f
reed
om 5
, 12.
Sup
ersc
ript l
ette
rs in
dica
te si
gnifi
cant
diff
eren
ces a
mon
g m
eans
. P
SEM
, Poo
led
stan
dard
err
or m
ean.
157 | P a g e
7.3.4 Fatty acid retention efficiency
The retention efficiency of each fatty acid in the control fed fish (FO CTRL) did not
differ from the fish fed either increasing EPA or ARA (Fig. 7.1a-j). There was a
concave quadratic effect of increasing EPA, rising to a plateau at 94.9% retention of
20:4n-6 (Fig. 7.1c; Table 7.8). There was a convex quadratic effect of increasing
ARA, falling to a nadir at 68.8% on the retention efficiency of 20:4n-6 (Fig. 7.1h;
Table 7.8). There was a significant quadratic effect on 20:5n-3 retention in response
to increasing dietary EPA diets with values ranging between 55.6 to 71.2%; whereas
increasing ARA had no effect on 20:5n-3 retention (Fig.7.1d, i; Table 7.8). Retention
efficiency of 22:6n-3 was not affected by increasing dietary EPA with values ranging
between 78.3 to 88.4%; however, there was a significant quadratic effect in response
to increasing dietary ARA with values ranging between 83.8 to 109.3% (Fig. 7.1e, j;
Table 7.8).
7.3.5 Marginal utilisation efficiencies
The marginal utilisation efficiencies are presented for both EPA and ARA fed fish
(Fig. 7.2a, 7.2b respectively). Based on the linear assessment of marginal EPA intake
against marginal EPA gained, a significant difference was observed (P<0.01). The
positive y intercept value indicates that no maintenance demand could be established
for 20:5n-3. Using the live-weight exponent value of 0.687 the EPA fed fish had a
20:5n-3 utilisation efficiency of 62.1% described by the linear equation of y = 0.621x
+ 0.0003, R2 = 0.975 (Fig. 7.2a). Similarly, there was a significant linear relationship
of marginal ARA intake against marginal ARA gain (P < 0.05). The negative y
intercept value suggests that a maintenance value of around 0.012 g/kg0.796/d could
be determined for 20:4n-6. Using the live-weight exponent of 0.796 the ARA fed fish
had a 20:4n-6 utilisation efficiency of 91.9% described by the linear equation of y =
0.919x – 0.011, R2 = 0.965 (Fig. 7.2b). A summary of the maintenance demand and
utilisation efficiencies is presented in Table 7.9.
158 | P a g e
Figure 7. 1 Specific fatty acid retention efficiency by barramundi fed increasing EPA (a-e) or ARA (f-j). The control (FO CTRL) fed fish are represented in each Figure with a triangle (∆). Bars indicate standard error means (n=3).
y = 0.45x2 - 7.8x + 99.9R² = 0.93
0
20
40
60
80
100
120
0 5 10 15 20
y = 0.09x2 - 1.1x + 66.9R² = 0.93
0102030405060708090
100
0 5 10 15 20
y = 0.27x2 - 3.6x + 91.8R² = 0.93
0
20
40
60
80
100
120
0 5 10 15 2020:4n-6 inclusion (g/kg)
y = 0.10x2 - 0.01x + 104.2R² = 0.57
0
20
40
60
80
100
120
140
0 5 10 15 20
y = -0.018x2 + 2.4x + 101.8R² = 0.67
020406080
100120140160
0 5 10 15 20
y = 0.11x2 - 2.1x + 87.8R² = 0.32
0
20
40
60
80
100
120
0 5 10 15 20
22:6
n-3
rete
tnio
n (%
)
20:5n-3 inclusion (g/kg)
y = -0.37x2 + 6.1x + 67.4R² = 0.86
0
20
40
60
80
100
120
0 5 10 15 20
20:4
n-6
rete
ntio
n (%
)
y = 0.15x2 - 3.3x + 76.0R² = 0.77
0
20
40
60
80
100
120
0 5 10 15 20
20:5
n-3
rete
ntio
n (%
)
y = 0.05x2 - 0.1x + 98.2R² = 0.21
0
20
40
60
80
100
120
140
0 5 10 15 20
18:3
n-3
rete
ntio
n (%
)y = 0.11x2 - 0.5x + 101.4
R² = 0.450
20406080
100120140
0 2 4 6 8 10 12 14 16 18 20
18:2
n-6
rete
ntio
n (%
)
(e) (j)
(i)(d)
(c)
(a)
(b)
(f)
(g)
(h)
159 | P a g e
Table 7. 8 Retention efficiency of selected fatty acids in barramundi fed either increasing EPA or ARA analysed by polynomial contrasts.
EPA Polynomial contrasts ^ ARA Polynomial contrasts ^ Linear Quadratic Cubic Linear Quadratic Cubic 18:2n-6 retention 0.000 0.242 0.006 0.000 0.660 0.001 18:3n-3 retention 0.020 0.989 0.104 0.001 0.094 0.001 20:4n-6 retention 0.057 0.006 0.209 0.911 0.000 0.178 20:5n-3 retention 0.167 0.047 0.165 0.303 0.339 0.682 22:6n-3 retention 0.965 0.201 0.061 0.004 0.006 0.180
^ Degrees of freedom EPA 4, 10; ARA 3, 8; linear, quadratic and cubic values are P values at an alpha level of 0.05
160 | P a g e
Figure 7. 2 Marginal utilisation efficiency assessments of 20:5n-3 (a) and 20:4n-6 (b) gain with varying intake by juvenile barramundi. Efficiency functions are described by the linear regression for 20:5n-3 gain y = 0.621x + 0.0003, R2 = 0.975 and 20:4n-6 gain y = 0.919x - 0.011, R2 = 0.965).
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.00 0.05 0.10 0.15 0.20
Mar
gina
l 20:
5n-3
gai
n (g
/kg0
.687
/d)
Marginal 20:5n-3 intake (g/kg0.687/d)
(a)
0.00
0.05
0.10
0.15
0.20
0.25
0.00 0.05 0.10 0.15 0.20 0.25
Mar
gina
l 20:
4n-6
gai
n (g
/kg0
.796
/d)
Marginal 20:4n-6 intake (g/kg0.796/d)
(b)
161 | P a g e
Table 7. 9 Summary of maintenance demand and utilisation efficiencies by barramundi fed either increasing EPA or ARA.
Maintenance demand (g/kg
LWx/t)
Efficiency constant k
Intake:Gain ratio R2 P
value
20:5n-3; EPA 0.000 0.621 1.610 0.975 <0.01
20:4n-6; ARA 0.011 0.919 1.088 0.966 <0.05
162 | P a g e
7.3.6 Gene identification and quantitative expression
Partial cDNA sequences of Lc COX1a, Lc COX1b, Lc COX2 and Lc ALOX-5 were
identified through degenerate PCR. BLAST similarity searches showed that
barramundi gene orthologs shared between 88 and 91% similarity with other teleost
fish at the amino acid level, and protein alignments showed similarity with other
teleost fish (Supplementary Figure 1a-d). The expression of eicosanoid pathway
genes (Lc COX1a, Lc COX1b, Lc COX2 and Lc ALOX-5), as well as the
mitochondrial ß-oxidation gene (Lc CPT1a) were analysed by quantitative real-time
RT-PCR in the liver and kidney of fish fed the highest and lowest EPA and ARA
diets and compared with the control fed fish.
The expression of Lc COX1b was significantly upregulated in the liver of the
EPA 1 fed fish compared the EPA 20 fed fish. (Fig. 7.3a). No other genes were
nutritionally regulated in the liver or kidney tissue of fish fed increasing EPA. In the
case of the ARA fed fish, the expression of Lc COX2 was significantly upregulated in
the liver of the ARA 20 fish compared to the ARA 1 fish (Fig. 7.4a). Similarly, the
expression of Lc ALOX-5 in the liver was significantly upregulated in the ARA 20 fish
compared to the ARA 1 fish (Fig. 7.4a). There was a significant down regulation of
the Lc COX1b in the kidney of the ARA 1 fish compared to the ARA 20 fish. The
expression of Lc COX2 was significantly upregulated in the kidney of the ARA 18 fish
compared to the ARA 1 fish. The Lc CPT1a expression in the kidney tissue was
significantly downregulated in the ARA 1 compared to the ARA 18 fish (Fig. 7.4b).
163 | P a g e
Figure 7. 3 Eicosanoid pathway and mitochondrial fatty acid oxidation gene expression in the liver (a) and kidney (b) of juvenile barramundi fed increasing EPA. Gene expression is normalised to the EF1α and Luc reference genes and expressed relative to the control fish. Data were analysed by students t-test with significant differences defined as P<0.05.
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Rel
ativ
e ex
pres
sion
(log
2)
Liver EPA 1EPA 20
*
(a)
-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0
Lc COX1a Lc COX1b Lc COX2 Lc ALOX-5 Lc CPT1a
Rel
ativ
e ex
pres
sion
(log
2)
Kidney EPA 1
EPA 20(b)
164 | P a g e
Figure 7. 4 Eicosanoid pathway and mitochondrial fatty acid oxidation gene expression in the liver (a) and kidney (b) of juvenile barramundi fed increasing ARA. Gene expression is normalised to the EF1α and Luc reference genes and expressed relative to the control fish. Data were analysed by students t-test with significant differences defined as P<0.05.
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
Rel
ativ
e ex
pres
sion
(log
2)
Liver ARA 1ARA 18*
***
(a)
-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5
Lc COX1a Lc COX1b Lc COX2 Lc ALOX-5 Lc CPT1α
Rel
ativ
e ex
pres
sion
(log
2)
Kidney ARA 1ARA 18
****
***
(b)
165 | P a g e
7.4 Discussion
The growth performance of juvenile barramundi in the present study, using a pair-fed
feeding regime, was not affected by increasing dietary EPA or ARA content.
Moreover, there were no significant differences in growth performance compared to
a control diet containing only fish oil. Although it is an unlikely real-world scenario
to include high levels of any one LC-PUFA, these observations and those of a
previous study (Glencross and Rutherford, 2011), confirm that there are no growth
stimulatory effects owing to any individual LC-PUFA in juvenile barramundi . In
many species, EPA can exert cardio-protective benefits such as lowering
triglycerides and low-density lipoprotein levels (Aarsland et al., 1990; Cottin et al.,
2011). Whereas ARA on the other hand, is an essential and necessary precursor to
the 2-series and 4-series eicosanoids that mediate homeostasis during times of
environmental or physiological stress (Bell and Sargent, 2003).
Studies examining the dose-response of EPA in juvenile or growing fish are
relatively scarce and mostly concentrate on larval fish requirements (Izquierdo,
1996). Consistent with the present study, several studies in larval and juvenile fish
such as the Atlantic salmon Salmo salar (Thomassen et al., 2012), cobia
Rachycentron canadum (Trushenski et al., 2012), gilthead sea bream Sparus aurata
(Atalah et al., 2011), Senegalese sole Solea senegalensis (Villalta et al., 2008),
striped jack Pseudocaranx dentex (Watanabe et al., 1989) and turbot Scophthalmus
maximus (Bell et al., 1995) all concluded that EPA does not stimulate an improved
growth response. However, larval and juvenile red seabream Pagrus major were
found to have an EPA requirement with linear improvements in growth and survival
observed (Furuita et al., 1996; Takeuchi et al., 1990). The results of the present study
are consistent with the general consensus that size and species differences exist and
that an appropriate balance between these essential fatty acids is probably more
critical than their absolute inclusion (Tocher, 2003).
Also in agreement with the present study, a range of larval and juvenile
marine species such as Atlantic cod Gadus morhua (Bransden et al., 2005), gilthead
sea bream (Alves Martins et al., 2012; Atalah et al., 2011; Fountoulaki et al., 2003),
Senegalese sole (Villalta et al., 2005) and turbot (Estévez et al., 1999), were able
tolerate a wide range of dietary ARA levels with growth and survival found to be
independent of ARA inclusion. Recent studies have further demonstrated that
balanced ARA and EPA levels in the diet are more critical than either of these
166 | P a g e
individually in terms of growth and other metabolic processes (Norambuena et al.,
2015).
Consistent with recent studies on juvenile barramundi held under similar
conditions, the diets in the present study were readily digested (Salini et al., 2015c).
There was a significant reduction in total lipid digestibility in the fish fed the diets
with the lowest levels of EPA and ARA compared to the control fish however this is
likely to be caused by the unavoidable higher SFA content of those diets.
Interestingly, the digestibility of the total SFA appears higher in those same
treatments as the fish attempt to cope with the changed fatty acid composition.
Despite this, the energy digestibility remained the same and confirms that there was
no dramatic effects caused by the different dietary strategies. Many studies, including
the present study, have demonstrated that the fatty acid composition of the tissues is
representative of the profile of the fed diet (Rosenlund et al., 2011). However, the
efficiency by which fatty acids are retained may represent a more metabolic or
biological importance to the fish. The DHA retention of the fish in the present study
fed increasing EPA did not change. However, the EPA and ARA retention were
inversely related and responded in a curvilinear fashion. Although the effect was not
dramatic, it indicated a point of sensitivity as EPA in the diet increased and the
barramundi retained more of the endogenous ARA. Glencross and Rutherford (2011)
reported disproportionate EPA and ARA retention in barramundi; however, this was
likely an effect of the increasing DHA level in combination with either EPA or ARA.
Atlantic salmon were also shown to conserve ARA (Norambuena et al., 2015),
whereas Senegalese sole attempted to synthesise ARA when dietary supply was
limited (Norambuena et al., 2013b).
Past work in our laboratory has suggested that the marginal efficiency of LC-
PUFA utilisation is low in growing barramundi, indicative of a higher turnover of
these fatty acids relating to their biological importance (Salini et al., 2015a). The
present study describes an assessment of the marginal efficiency of EPA and ARA
on restrictively pair-fed barramundi, while simultaneously controlling for other LC-
PUFA (rather than FO substitution). Under these experimental conditions, we report
the maintenance requirement of ARA to be 0.012 g/kg LW0.796/d, with a marginal
efficiency value of 91.9%. Although the requirement was low, this potentially
confirms that ARA is unusual in its characteristic metabolic requirement for this
species compared to other LC-PUFA. EPA on the other hand is apparently not
167 | P a g e
required for maintenance in juvenile barramundi, suggesting that their maintenance
demands are easily met by endogenous reserves. The differences between the
marginal efficiency values of these studies warrant further investigation. In addition,
the EPA retention figures were modest with a calculated intake to gain ratio of 1.6:1,
further supporting this concept. This is consistent with other studies on barramundi
and red sea bream in that EPA is only modestly retained unless there is a gross
imbalance of other LC-PUFA (Glencross et al., 2003c; Glencross and Rutherford,
2011).
There is a very large body of research into the mechanisms of action of n-3
LC-PUFA on inflammatory pathways (Rangel-Huerta et al., 2012). The data from
the present study take the first steps in our understanding in barramundi by
identifying the key eicosanoid pathway enzymes and quantifying their nutritional
regulation. The current work verifies that barramundi possess at least two variants of
the cyclooxygenase 1 enzyme (Lc COX1a, Lc COX1b), a single cyclooxygenase 2
(Lc COX2) and an arachidonate 5-lipoxygenase (Lc ALOX-5). The fish in the present
study were not exposed to any form of physical or environmental stressors (apart
from being in research aquaria) that could cause an inflammatory response and
therefore the only trigger for a response is of a nutritional nature.
The present study demonstrated that either a high or low inclusion of EPA did
not modify the expression of any eicosanoid pathway gene in either the liver or the
kidney tissue. However, the only exception to this was the increased expression of Lc
COX1b in the liver of the EPA 1 fed fish. Conversely, there was a significant
downregulation of this gene in the kidney tissue of the ARA 1 fed fish. COX1
isoforms are widely distributed and generally constitutively expressed, however this
homeostatic function is now considered to be an oversimplified view (Rouzer and
Marnett, 2009). In agreement with the present study, there were non-significant (P =
0.06) yet quite measurable differences in COX1b expression in larval tongue sole
(Cynoglossus semilaevis) fed increasing ARA (Yuan et al., 2015). Further supporting
this notion, there was a nutritionally induced upregulation of COX1 and COX2
expression in rat mammory gland tissue by n-6 rich safflower oil (Badawi et al.,
1998). The results of the present study confirm that the Lc COX1b gene in
barramundi is inducible and the differences warrant further investigation into the
potential for pathophysiological effects. Some explanation for these inconsistencies
may be found in whole-body composition of wild barramundi collected from
168 | P a g e
different environments (Nichols et al., 2014; Sinclair et al., 1983). These authors
found that Northern Australian barramundi, maintain a characteristically high ARA
content regardless of their environment. Therefore, it is suggested that barramundi
can modify the transcription of these genes depending on the potentially transient
nature of dietary ARA supply.
The EPA derived or anti-inflammatory eicosanoids are known to be
correlated with changes in transcription of fatty acid synthesis and β-oxidation genes
(Aarsland et al., 1990; Sijben and Calder, 2007) . However, the Lc CPT1α expression
in the present study was not affected by the inclusion level of either EPA or ARA,
with the exception of a slight decrease in the kidney tissue of the ARA 1 fish. This
may suggest that some other characteristic, perhaps of the fish oil base used in the
control diet caused this response. Additionally, Lc ALOX-5 expression was
upregulated in the liver tissue of the ARA 18 fish consistent with a modified
metabolic function. In a recent study, changes in ALOX-5 were also observed in
marine fish larvae in response to increasing dietary ARA (Yuan et al., 2015). In
contrast to the present study, these authors reported improved growth of larvae,
despite the potential for excessive generation of pro-inflammatory leukotrienes and
lipoxins (Rowley et al., 1995).
Past studies have demonstrated that nutritionally inducible COX2 genes exist
in several teleost species (Montero et al., 2015a; Yuan et al., 2015; Zuo et al., 2015).
The role of Lc COX2 as an active and rapidly inducible gene has undoubtedly played
a role in the modified metabolic function of the ARA fed fish during the present
study. The Lc COX2 expression was significantly increased in both liver and kidney
tissues suggesting that the highest inclusion of ARA was excessive or that the EPA
to ARA ratio was unbalanced.
To conclude, the present study supports that there is no phenotypic response
by barramundi to the addition of either EPA or ARA to the diets. However, there are
some metabolic changes to the retention and marginal utilisation efficiencies as a
result of the diets. Increasing the dietary EPA or ARA level also modulated the
expression of several eicosanoid metabolism and fatty acid oxidation genes. Further
studies should focus on the most appropriate balance of dietary LC-PUFA in light of
the current levels of fish oil substitution in aquafeeds. Further refinements could also
be made to factorial growth and feed utilisation models for barramundi with respect
to specific fatty acids and their marginal utilisation efficiencies.
169 | P a g e
8.0 Defining the allometric relationship between size and nutrient
turnover in barramundi Lates calcarifer
Adapted from
Salini, M.J., Poppi, D.A., Turchini, G.M., Glencross, B.D., 2016. Defining the
allometric relationship between size and nutrient turnover in barramundi Lates
calcarifer. Comp. Biochem. Physiol. Part A. 201, 79-86
170 | P a g e
171 | P a g e
172 | P a g e
8.1 Aims
Bioenergetic modelling of the nutrient flows in barramundi has been extensively
researched and ‘user friendly’ simulation programs are used routinely (Glencross,
2008; Glencross and Bermudes, 2010; 2011; 2012). One of the key assumptions and
constraints in the application of these models is that the live-weight (LW) exponent
values are constant. Studies have shown with barramundi, over a range of normal
temperatures, that this is generally the case for the energy, protein and lipid
exponents (Glencross and Bermudes, 2011). However, it is assumed that when
broken down into constituent fatty acids the body weight exponents are also
equivalent to that of the lipid as a complete nutrient.
A further refinement of those factorial bioenergetic models could include
consideration of the individual fatty acids and potentially amino acids in order to
better understand their utilisation in terms of productivity on a size independent
basis. Therefore, the aim of the present study was to determine the allometric scaling
effect of individual fatty acids in barramundi for use in future bioenergetic studies. In
addition, a re-evaluation of previously published data is used to refine the fatty acid
demands for maintenance and growth of barramundi using in silico predictive
modelling.
8.2 Materials and methods
The methodology used in the present experiment are detailed in the section 2.0
General methodology. Ethical clearance was approved for the experimental
procedures by the CSIRO Animal Ethics Committee (Application A3/2015).
8.3 Results
8.3.1 Fish compositional changes
The initial and final weights of the fish are presented in Table 8.1. In all size groups
of fish weight loss was between 12.9% for the smallest fish to 5.3% for the largest
fish. The condition factor was also lower in the fish after fasting. No fish died during
the experiment. The initial and final chemical composition of the fish were analysed
and reported in Table 8.1. The energy density of the barramundi of varying size
before and after fasting was best fitted to a power function with high R2 values of
0.845 and 0.844 respectively (Fig. 8.1). There was a decrease in the energy density of
the fasted fish (Fig. 8.1). The lipid density of the barramundi was best fitted to a
logarithmic function with initial and final R2 values of 0.711 and 0.744 respectively
173 | P a g e
(Fig. 8.2). The logarithmic response after fasting appears to be driven by Treatment
F.
The individual fatty acid density of barramundi for each of the treatment size classes
is presented in Table 8.2 and the LC-PUFA density is plotted in Fig. 8.3. There was a
general increase in the fatty acid density with increasing size, concomitant with the
lipid composition of the fish (Table 8.1). The response after fasting was best fitted to
a logarithmic function that appears to be driven by the lipid content of Treatment F.
174
| Pa
ge
Tab
le 8
. 1. P
erfo
rman
ce p
aram
eter
s and
che
mic
al c
ompo
sitio
n fo
r in
itial
and
fina
l bar
ram
undi
of v
aryi
ng si
zes.
Dat
a ar
e pr
esen
ted
as m
ean
± SE
M a
nd c
ompo
sitio
n da
ta a
re p
rese
nted
on
a w
et-w
eigh
t bas
is.
Tr
eatm
ent A
Tr
eatm
ent B
Tr
eatm
ent C
Tr
eatm
ent D
Tr
eatm
ent E
Tr
eatm
ent F
Bi
omet
ric
para
met
ers
Initi
al w
eigh
t (g/
fish)
10
.5 ±
0.1
19
.2 ±
0.1
28
.3 ±
0.1
12
2.4
± 0.
1 21
7.6
± 0.
4 44
3.7
± 1.
5 Fi
nal w
eigh
t (g/
fish)
9.
2 ±
0.1
17.3
± 0
.1
25.6
± 0
.1
114.
3 ±
0.1
206.
5 ±
0.3
42
0.0
± 1.
0 W
eigh
t los
s (g/
fish)
1.
4 ±
0.1
1.9
± 0.
0 2.
7 ±
0.2
8.1
± 0.
2 11
.1 ±
0.2
23
.7 ±
1.6
W
eigh
t los
s (%
) 12
.9 ±
0.7
9.
9 ±
0.1
9.6
± 0.
5 6.
6 ±
0.2
5.1
± 0.
1 5.
3 ±
0.3
Con
ditio
n in
itial
*
1.2
± 0.
0 1.
2 ±
0.0
1.3
± 0.
1 1.
2 ±
0.0
1.2
± 0.
1 1.
2 ±
0.0
Con
ditio
n fin
al*
1.1
± 0.
0 1.
0 ±
0.0
1.1
± 0.
1 1.
1 ±
0.0
1.1
± 0.
1 1.
1 ±
0.0
Initi
al c
ompo
sitio
n
D
ry m
atte
r (%
) 24
.4
24.4
27
.2
27.2
29
.7
30.9
Pr
otei
n (%
) 16
.0
15.2
17
.1
17.2
17
.9
19.2
A
sh (%
) 3.
8 3.
8 3.
7 3.
1 4.
1 4.
8 Li
pid
(%)
3.8
4.0
6.0
6.3
7.2
6.4
Gro
ss e
nerg
y (M
J/kg
) 5.
1 5.
2 6.
2 6.
5 7.
0 6.
9 Fi
nal c
ompo
sitio
n
D
ry m
atte
r (%
) 21
.7 ±
0.7
22
.0 ±
0.1
25
.6 ±
0.3
27
.4 ±
0.3
28
.1 ±
0.3
29
.6 ±
0.2
Pr
otei
n (%
) 15
.1 ±
0.6
15
.4 ±
0.1
16
.7 ±
0.4
17
.6 ±
0.5
18
.2 ±
0.3
18
.5 ±
0.1
A
sh (%
) 4.
5 ±
0.1
4.4
± 0.
3 4.
2 ±
0.1
3.7
± 0.
1 4.
2 ±
0.1
5.4
± 0.
1 Li
pid
(%)
1.6
± 0.
2 1.
9 ±
0.1
4.3
± 0.
1 5.
3 ±
0.2
5.2
± 0.
2 5.
1 ±
0.1
Gro
ss e
nerg
y (M
J/kg
) 4.
2 ±
0.1
4.4
± 0.
1 5.
4 ±
0.1
6.3
± 0.
1 6.
4 ±
0.1
6.4
± 0.
1 *C
ondi
tion
fact
or=w
eigh
t/len
gth^
3
175
| Pa
ge
Tab
le 8
. 2 F
atty
aci
d de
nsity
in th
e fis
h (m
g/g/
fish)
aft
er 2
1 da
ys o
f fas
ting.
Dat
a w
ere
fitte
d to
loga
rith
mic
func
tions
and
pre
sent
ed a
s mea
n ±
SEM
. Tr
eatm
ent A
Tr
eatm
ent B
Tr
eatm
ent C
Tr
eatm
ent D
Tr
eatm
ent E
Tr
eatm
ent F
Eq
uatio
n R2
16:0
30
.0 ±
1.0
35
.1 ±
0.7
66
.0 ±
2.9
97
.9 ±
0.5
94
.8 ±
0.9
99
.1 ±
0.3
y=
19.7
28(±
0.10
1)ln
(x)
– 9.
833(
±0.3
98)
0.88
3
18:0
10
.8 ±
0.4
11
.6 ±
0.2
18
.6 ±
0.8
28
.1 ±
0.3
27
.0 ±
0.4
27
.2 ±
0.1
y=
4.93
5(±0
.028
)ln(x
) +
0.46
6(±0
.104
) 0.
873
18:1
n-9
39.3
± 0
.5
47.8
± 0
.2
90.0
± 4
.0
164.
3 ±
0.3
161.
0 ±
0.4
147.
8 ±
0.1
y=34
.580
(±0.
245)
ln(x
) –
32.3
92(±
0.82
4)
0.84
9
18:2
n-6
14.0
± 0
.0
17.3
± 0
.1
28.2
± 1
.1
57.9
± 0
.1
58.3
± 0
.5
51.9
± 0
.1
y=12
.511
(±0.
083)
ln(x
) –
13.0
06(±
0.28
1)
0.85
8
18:3
n-3
1.3
± 0.
3 1.
8 ±
0.1
3.5
± 0.
0 5.
8 ±
0.2
5.9
± 0.
2 5.
0 ±
0.2
y=1.
195(
±0.0
08)ln
(x)
– 0.
985(
±0.0
27)
0.79
9
20:4
n-6
2.6
± 1.
8 2.
5 ±
0.9
2.6
± 0.
1 3.
6 ±
1.3
4.2
± 2.
9 3.
1 ±
2.4
y=0.
325(
±0.0
04)ln
(x)
+ 1.
771(
±0.0
18)
0.53
1
20:5
n-3
4.1
± 6.
2 5.
0 ±
1.6
8.6
± 0.
3 9.
0 ±
5.5
8.8
± 7.
2 10
.1 ±
5.0
y=
1.40
8(±0
.009
)ln(x
) +
1.88
4(±0
.045
) 0.
763
22:5
n-3
3.3
± 1.
1 3.
7 ±
0.4
5.5
± 0.
1 6.
5 ±
0.9
6.3
± 1.
4 7.
1 ±
0.6
y=0.
954(
±0.0
05)ln
(x)
+ 1.
510(
±0.0
23)
0.86
7
22:6
n-3
13.9
± 4
.4
15.2
± 1
.0
20.2
± 0
.5
22.2
± 2
.7
21.7
± 4
.7
21.8
± 1
.8
y=2.
224(
± 0.
017)
ln(x
) +
10.6
49(±
0.0
73)
0.75
6
176 | P a g e
Figure 8. 1 Energy density of barramundi of varying live-weight before (●=4.221(±0.010)x0.088(±0.001), R2 = 0.845) and after (○=3.359(±0.011)x0.118(±0.001), R2 = 0.844) fasting for 21 days.
012345678
0 100 200 300 400
Ener
gy (k
J/g)
Live-weight (g)
177 | P a g e
Figure 8. 2 Lipid density of the barramundi of varying live-weight before (●=0.807(±0.019)ln(x) + 2.312(±0.002), R2 = 0.711) and after (○=0.981(±0.014)ln(x) – 0.083(±0.003), R2 = 0.744) fasting for 21 days.
012345678
0 100 200 300 400
Lipi
d (%
)
Live-weight (g)
178 | P a g e
Figure 8. 3 Fatty acid density (mg/g lipid) in barramundi of varying live-weight after fasting for 21 days. Values were fitted to a logarithmic curve and equations are presented in Table 8.2.
179 | P a g e
8.3.2 Determination of metabolic live-weight exponents
Somatic losses of energy, lipid and individual fatty acids were well described by the
function a*Xb (Table 8.3). A bootstrapping approach was used to generate
replications of coefficient (slope) and exponent values of energy, lipid and fatty acid
loss. An energy live-weight (LW) exponent of 0.817 was derived based on energy
losses after fasting and the equation is presented in Eqn. 8.1. Similarly, a lipid loss
LW exponent of 0.895 was derived based on lipid loss after fasting and the equation
is presented in Eqn. 8.2. The relationships between fatty acid losses and the
geometric mean weight over the range of sizes in the present study are presented in
Fig. 8.4 (A and B).
Energy loss (kJ/fish/day) = 0.104(±0.003)*(Live-weight) 0.817(±0.010), R2 = 0.949 (8.1)
Lipid loss (g/fish/day) = 0.002(±0.000)*(Live-weight) 0.895(±0.007), R2 = 0.985 (8.2)
There was a significant difference in the derived LW exponent values for specific
fatty acids confirming that they are for the most part, different from that of the total
lipid (LW0.895). However, there was no difference in the exponent values of 16:0 and
18:3n-3 and 18:0 and 20:4n-6 (ARA) (Table 8.3). The LW exponent values for
22:6n-3 (DHA), 22:5n-3 (DPA) and 20:5n-3 (EPA), 0.792, 0.748 and 0.687
respectively, were all significantly lower than that of lipid while 18:1n-9 was higher
at 0.954 (Table 8.3). The weighted exponent values were calculated and the sum of
all fatty acids presented was equal to 0.854 ± 0.033 (Table 8.3; sum not presented)
180 | P a g e
Table 8. 3 Coefficient and exponent values derived from the power function (y=aXb) of fatty acid loss over a wide range of fish sizes from ~10 g to ~440 g. Replication was derived by manually bootstrapping each individual value and presented as mean ± SEM (n=18).
Coefficient (a)
Exponent (b) R2 Weighted
Exponent* Energy 0.104 ± 0.003 0.817 ± 0.010 0.949 NA Lipid 0.002 ± 0.000 0.895 ± 0.007a 0.985 NA
16:0 0.346 ± 0.010 0.890 ± 0.024a 0.992 0.208 ± 0.012 18:0 0.107 ± 0.003 0.876 ± 0.010b 0.991 0.061 ± 0.004 18:1n-9 0.399 ± 0.011 0.954 ± 0.008c 0.992 0.350 ± 0.007 18:2n-6 0.182 ± 0.005 0.915 ± 0.009d 0.992 0.120 ± 0.003 18:3n-3 0.026 ± 0.001 0.899 ± 0.007a 0.991 0.013 ± 0.000 ARA 0.012 ± 0.000 0.796 ± 0.007b 0.942 0.009 ± 0.001 EPA 0.114 ± 0.002 0.687 ± 0.005e 0.990 0.021 ± 0.001 DPA 0.038 ± 0.001 0.748 ± 0.008f 0.985 0.015 ± 0.001 DHA 0.138 ± 0.003 0.792 ± 0.006g 0.990 0.058 ± 0.004
P value NA <2.2-16 NA NA NA, not analysed. Superscript letters indicate significant differences. * Calculated as geometric mean weight of each fatty acid x exponent (b).
181 | P a g e
8.3.3 Metabolic demands for fatty acids
A re-evaluation of the marginal utilisation efficiencies of individual fatty acids using
the fatty acid LW exponents derived from the present experiment is presented in
Table 8.4. This re-evaluation was performed on data from three separate experiments
(Glencross and Rutherford, 2011; Salini et al., 2015a; Salini et al., 2016). The linear
equations of the marginal intake to marginal gain ratio were extrapolated to zero (0 =
b(x) + a) in order to obtain estimated maintenance requirement values. In the first
experiment, the LC-PUFA all produced negative requirement values whereas all
other shorter-chain length and more saturated fatty acids have a determined
requirement. In the subsequent studies, the marginal utilisation efficiencies for ARA,
EPA and DHA were higher, contrasting those of the first study. There was no
requirement value established for EPA and DHA, however, there was a maintenance
requirement of 0.012 g/kg0.880/d determined for ARA.
Iteratively determined fatty acid maintenance, fatty acid gain, fatty acid for growth
and total requirements are presented in Table 8.5. For each of the size classes the
values are presented for barramundi fed either 100% fish oil or 100% poultry oil
diets adapted from Salini et al. (2015a).
182 | P a g e
Table 8. 4 Re-evaluation of the marginal efficiency of fatty acid utilisation in barramundi. Data were transformed to LW exponent values determined from the present study. Maintenance requirements and intake to gain ratio for each fatty acid are presented
Fatty acid Slope Intercept R2 Req# 1/k* Salini et al., (2015a)
16:0 2.258 -0.536 0.712 0.237 0.443 18:0 1.539 -0.068 0.837 0.044 0.650 18:1 1.111 -0.178 0.951 0.161 0.900 18:2n-6 0.821 -0.025 0.751 0.031 1.218 18:3n-3 1.040 -0.010 0.881 0.010 0.962 ARA 0.192 0.005 0.427 -0.025 5.222 EPA 0.305 0.005 0.953 -0.017 3.279 DPA 0.340 0.008 0.783 -0.023 2.943 DHA 0.271 0.014 0.952 -0.050 3.693
Salini et al., (2016) ARA 0.919 -0.011 0.965 0.012 1.088 EPA 0.621 0.000 0.975 -0.000 1.610
Glencross and Rutherford, (2011) DHA 1.065 0.046 0.961 -0.043 0.939
# Maintenance requirement (g/kgx/d) determined by extrapolation to 0 = b (x) + a. * Intake to gain ratio.
183 | P a g e
Figure 8. 4 Fatty acid (A and B) loss in fasted barramundi of varying live-weight. Data are (n=3) mean ± SEM. Values were fitted to a power function and equations are presented in Table 8.3.
184
| Pa
ge
Tab
le 8
. 5 F
atty
aci
d de
man
ds in
gro
win
g ba
rram
undi
fed
eith
er 1
00%
fish
oil
(FO
) or
100%
pou
ltry
oil (
PO) d
iets
mai
ntai
ned
at 3
0 °C
. C
alcu
latio
ns a
re b
ased
on
the
pred
ictiv
e gr
owth
mod
els a
nd u
tilis
atio
n ef
ficie
ncie
s fro
m p
ublis
hed
stud
ies f
or th
is sp
ecie
s
Fish
live
wei
ght (
g/fis
h)
50
100
500
1000
20
00
50
100
500
1000
20
00
Expe
cted
gro
wth
(g/d
ay)1
2.13
2.
88
5.81
7.
85
10.6
1 2.
13
2.88
5.
81
7.85
10
.61
Die
t2 FO
FO
FO
FO
FO
PO
PO
PO
PO
PO
16
:0 d
eman
ds
16
:0-m
aint
(mg/
fish/
d)3
0.00
4 0.
007
0.03
0 0.
055
0.10
3 0.
004
0.00
7 0.
027
0.05
1 0.
094
16:0
gai
n (m
g/fis
h/d)
4 0.
028
0.04
2 0.
108
0.16
3 0.
245
0.02
5 0.
038
0.09
9 0.
150
0.22
5 16
:0-g
row
th (m
g/fis
h/d)
5 0.
012
0.01
8 0.
048
0.07
2 0.
109
0.01
1 0.
017
0.04
4 0.
066
0.10
0 16
:0-to
tal (
mg/
fish/
d)6
0.01
6 0.
026
0.07
8 0.
127
0.21
1 0.
015
0.02
3 0.
071
0.11
7 0.
194
18:0
dem
ands
7
18:0
-mai
nt (m
g/fis
h/d)
0.
000
0.00
0 0.
002
0.00
3 0.
006
0.00
0 0.
000
0.00
2 0.
003
0.00
5 18
:0 g
ain
(mg/
fish/
d)
0.00
8 0.
012
0.03
2 0.
048
0.07
2 0.
008
0.01
2 0.
031
0.04
7 0.
070
18:0
-gro
wth
(mg/
fish/
d)
0.00
5 0.
008
0.02
1 0.
031
0.04
7 0.
005
0.00
8 0.
020
0.03
0 0.
046
18:0
-tota
l (m
g/fis
h/d)
0.
005
0.00
8 0.
022
0.03
4 0.
052
0.00
5 0.
008
0.02
2 0.
033
0.05
1 18
:1 d
eman
ds7
18
:1-m
aint
(mg/
fish/
d)
0.00
3 0.
006
0.02
7 0.
051
0.09
9 0.
004
0.00
7 0.
032
0.06
2 0.
120
18:1
gai
n (m
g/fis
h/d)
0.
038
0.05
7 0.
148
0.22
2 0.
335
0.04
6 0.
069
0.17
9 0.
269
0.40
6 18
:1-g
row
th (m
g/fis
h/d)
0.
034
0.05
1 0.
133
0.20
0 0.
302
0.04
1 0.
062
0.16
1 0.
242
0.36
5 18
:1- t
otal
(mg/
fish/
d)
0.03
7 0.
057
0.15
9 0.
251
0.40
1 0.
045
0.06
9 0.
193
0.30
4 0.
485
18:2
dem
ands
7
18:2
-mai
nt (m
g/fis
h/d)
0.
000
0.00
0 0.
001
0.00
2 0.
005
0.00
0 0.
000
0.00
2 0.
004
0.00
7 18
:2 g
ain
(mg/
fish/
d)
0.01
0 0.
014
0.03
7 0.
056
0.08
5 0.
014
0.02
1 0.
055
0.08
4 0.
126
18:2
-gro
wth
(mg/
fish/
d)
0.01
2 0.
017
0.04
5 0.
068
0.10
3 0.
017
0.02
6 0.
067
0.10
2 0.
153
18:2
-tota
l (m
g/fis
h/d)
0.
012
0.01
8 0.
047
0.07
1 0.
108
0.01
8 0.
026
0.06
9 0.
105
0.16
0 18
:3 d
eman
ds7
18
:3-m
aint
(mg/
fish/
d)
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
18:3
gai
n (m
g/fis
h/d)
0.
001
0.00
1 0.
004
0.00
5 0.
008
0.00
2 0.
002
0.00
6 0.
009
0.01
3
185
| Pa
ge
18:3
-gro
wth
(mg/
fish/
d)
0.00
1 0.
001
0.00
3 0.
005
0.00
8 0.
001
0.00
2 0.
006
0.00
9 0.
013
18:3
-tota
l (m
g/fis
h/d)
0.
001
0.00
1 0.
004
0.00
5 0.
008
0.00
1 0.
002
0.00
6 0.
009
0.01
3 AR
A de
man
ds7
A
RA
-mai
nt (m
g/fis
h/d)
0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 A
RA
gai
n (m
g/fis
h/d)
0.
001
0.00
1 0.
003
0.00
5 0.
007
0.00
1 0.
001
0.00
2 0.
003
0.00
5 A
RA
-gro
wth
(mg/
fish/
day)
0.
004
0.00
6 0.
016
0.02
3 0.
035
0.00
3 0.
004
0.01
1 0.
016
0.02
4 A
RA
-tota
l (m
g/fis
h/da
y)
0.00
4 0.
006
0.01
6 0.
024
0.03
6 0.
003
0.00
4 0.
011
0.01
6 0.
024
EPA
dem
ands
7
EPA
-mai
nt (m
g/fis
h/d)
0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 EP
A g
ain
(mg/
fish/
d)
0.00
5 0.
007
0.01
8 0.
027
0.04
0 0.
001
0.00
1 0.
003
0.00
5 0.
008
EPA
-gro
wth
(mg/
fish/
day)
0.
015
0.02
2 0.
058
0.08
7 0.
131
0.00
3 0.
004
0.01
1 0.
017
0.02
5 EP
A-to
tal (
mg/
fish/
day)
0.
015
0.02
2 0.
058
0.08
7 0.
131
0.00
3 0.
004
0.01
1 0.
017
0.02
5 D
PA d
eman
ds7
D
PA-m
aint
(mg/
fish/
d)
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
DPA
gai
n (m
g/fis
h/d)
0.
001
0.00
2 0.
006
0.00
9 0.
013
0.00
1 0.
001
0.00
3 0.
004
0.00
6 D
PA-g
row
th (m
g/fis
h/da
y)
0.00
4 0.
007
0.01
7 0.
026
0.03
9 0.
002
0.00
3 0.
008
0.01
2 0.
017
DPA
-tota
l (m
g/fis
h/da
y)
0.00
4 0.
007
0.01
7 0.
026
0.03
9 0.
002
0.00
3 0.
008
0.01
2 0.
017
DH
A de
man
ds7
D
HA
-mai
nt (m
g/fis
h/d)
0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 0.
000
0.00
0 D
HA
gai
n (m
g/fis
h/d)
0.
005
0.00
7 0.
019
0.02
9 0.
043
0.00
2 0.
003
0.00
8 0.
012
0.01
8 D
HA
-gro
wth
(mg/
fish/
day)
0.
018
0.02
7 0.
070
0.10
6 0.
159
0.00
8 0.
011
0.02
9 0.
044
0.06
7 D
HA
-tota
l (m
g/fis
h/da
y)
0.01
8 0.
027
0.07
0 0.
106
0.15
9 0.
008
0.01
1 0.
029
0.04
4 0.
067
1 M
odel
led
daily
gro
wth
bas
ed o
n 30
°C w
ater
tem
pera
ture
(Gle
ncro
ss, 2
008;
Gle
ncro
ss a
nd B
erm
udes
, 201
2).
2 D
ata
for t
he c
alcu
latio
n of
fatty
aci
d de
man
ds w
ere
take
n fro
m p
revi
ousl
y pu
blis
hed
stud
ies (
Salin
i et a
l., 2
015)
. 3
Mai
nten
ance
dig
estib
le fa
tty a
cid
requ
irem
ents
bas
ed o
n ex
trapo
late
d va
lues
(Tab
le 4
), pe
r exp
onen
t tra
nsfo
rmed
fatty
aci
d bo
dy w
eigh
t (Ta
ble
3) a
nd
mul
tiplie
d by
the
who
le b
ody
fatty
aci
ds (g
/kg/
fish)
. 4
Fatty
aci
d co
nten
t of t
he m
odel
led
live-
wei
ght g
ain.
5
Dig
estib
le fa
tty a
cid
dem
and
base
d on
the
gain
thro
ugh
mod
elle
d gr
owth
div
ided
by
the
utili
satio
n ef
ficie
ncy
of th
at fa
tty a
cid.
6
Com
bine
d di
gest
ible
dem
and
for b
oth
mai
nten
ance
and
gro
wth
. 7
Ref
er to
16:
0 de
man
ds.
186 | P a g e
8.4 Discussion
One of the key assumptions of nutritional modelling is that the allometric scaling
exponent values for biological variables ascribed to transform live-weight (LW) are
constant (Glencross and Bermudes, 2011; Lupatsch et al., 2003). In reality, exponent
values for the metabolic LW for energy in aquatic species usually fit around an
average value of 0.80, which has been adopted and used routinely (Bureau et al.,
2002; Cho and Kaushik, 1990; Cui and Liu, 1990; Lupatsch et al., 2003; NRC,
2011). Similarly for protein, a range of LW exponents have been used to describe the
allometric relationship and the value of 0.70 is routinely used under normal
physiological conditions (Lupatsch et al., 1998; Pirozzi et al., 2010). Arguably the
average of the weighted LW exponents for lipid and protein energy should be equal
to that of gross energy, therefore 0.90 can be ascribed to LW exponent of lipid
(Glencross and Bermudes, 2011). The development of predictive models of energy
transactions that also consider the individual compounds of nutrients rather than
aggregates of energy would help in understanding the discrete biochemical
relationships that exist and some attempts at compartmentalising these have been
made in monogastric animals (Birkett and Lange, 2007). However, these are not
common in the literature or in practice for aquatic species. The present study
therefore investigated the allometric scaling effect of individual fatty acids in
barramundi held at a constant temperature.
In the present study, the assessment of somatic energy before and after fasting was
highly consistent with the study of Glencross and Bermudes (2011). This suggests
that over the variable size range of fish used in the present study and held at an
optimal temperature, fasting losses are quite predictable, further validating the
methodology to be extended to individual fatty acids. One caveat of the present study
was that the size class selection of the fish was limited to two initial shipments of
fish that were held in stock aquaria and subsequently graded. Therefore we cannot
conclude on what may happen outside this range or within the range if additional
treatments were available. The increasing live-weight as a function of energy and
lipid density of the fish was best fitted to power and natural logarithmic equations
respectively. The lower than expected analytical values obtained for lipid in
Treatment F are likely related to the nutritional status of the fish prior to the
commencement of the study however there is no consistent explanation for this.
Consistent with other studies, the loss of lipid was concomitant to the loss of energy,
187 | P a g e
confirming that lipid is preferentially metabolised under fasting conditions in order to
retain protein (Glencross and Bermudes, 2011; Lupatsch et al., 1998).
The somatic losses determined in the present study were best described by power
functions, following the equation y=a*Xb, where a represents a temperature
dependent coefficient, X is the live-weight (LW) and b the scaling exponent. An
important finding of the present study was that the energy LW exponent value (0.817
± 0.010) is consistent with the commonly reported value of 0.80. This is an important
finding as previous studies have found that even slight variations in the reported
exponent values can lead to substantially different outcomes when applied to the
determination of maintenance energy demands (Pirozzi, 2009). The lipid exponent
value of 0.895 ± 0.007 was also highly consistent with the values previously
described for barramundi (Glencross and Bermudes, 2011). One caveat of the present
study was that only a single temperature range was examined; however, it is reported
that provided barramundi are held within their normal temperature range then the
values should be mostly consistent (Glencross and Bermudes, 2011).
The fatty acid allometric scaling exponent values derived in the present study were
significantly different and ranged from 0.687 ± 0.005 for EPA to 0.954 ± 0.008 for
18:1n-9. With the exception of ARA, all the LC-PUFA exponent values were
significantly lower than the more dominant shorter-chain length and more saturated
fatty acids. The individual fatty acids presented as weighted exponent values are also
consistent with that of lipid as a complete nutrient (0.854 ± 0.033 vs. 0.895 ± 0.007
respectively). The lower exponent values recorded for the LC-PUFA suggest that
there is likely to be a greater turnover of these fatty acids in the juvenile fish
indicating more specific biological demands. While the higher exponents (eg. 18:1n-
9) suggest there is less effect of size and lower biological demands for those fatty
acids. Additionally, the LC-PUFA with lower exponent values also have marginal
utilisation efficiencies that are considerably lower than other more dominant fatty
acids (Salini et al., 2015a). This lends further support to the theory that they are more
biologically important and that they are selectively retained in the tissues,
corroborating evidence from past studies in barramundi (Glencross and Rutherford,
2011; Salini et al., 2015c). Moreover, the significance of LC-PUFA is also be
supported by their anti-inflammatory role in the production of eicosanoids and
specialised pro-resolving mediators (Bannenberg and Serhan, 2010; Rowley et al.,
1995; Serhan, 2010).
188 | P a g e
The energy from metabolizable food in juvenile animals can only really be
partitioned into maintenance and growth as reproductive effort is essentially zero
(Lucas, 1996; NRC, 2011). Moreover, the concept of maintenance and growth
demands are additive in terms of productivity (Bureau et al., 2002; Clarke and
Fraser, 2004). A range of data pools were used in the analysis of the present study in
order to iteratively determine the metabolic demands for specific fatty acids in
growing barramundi. The partial (marginal) efficiency values from Salini et al.
(2015a) were re-calculated with the newer exponent values derived in the present
study. The LW exponent of lipid (0.90) was applied to individual fatty acids and
acknowledged as an assumption in that earlier study. This re-calculation allowed a
more accurate determination of maintenance fatty acid demands given the
acknowledged impact that this transformation can have on the determination of that
parameter (Pirozzi, 2009). We also assessed the suitability of marginal efficiency
values for ARA, EPA and DHA determined from subsequent studies (Glencross and
Rutherford, 2011; Salini et al., 2016). However, these values were inconsistent with
those of Salini et al. (2015a) and not included in the final analysis (Table 5). Reasons
for these inconsistencies are likely to be due to the large differences in the initial size
of the fish and the feeding regime utilised.
The results of the present study demonstrate that the different fatty acids are utilised
with different efficiencies. However, contrary to what might be expected, the levels
of LC-PUFA required in barramundi fed a fish oil based diet are numerically higher
than those fed a poultry oil based diet. This apparent difference is driven largely by
the deposition demands of individual fatty acids rather than catabolism or other
processes. Based on the demands (requirements) for maintenance presented in Table
8.5, we could conclude that the LC-PUFA requirements are negligible (Birkett and
Lange, 2007). This conclusion may be more generally applied to larger growing
barramundi however, evidence suggests that essential fatty acid requirements are
more pronounced during the rapid growth phase of juveniles, and virtually negligible
at larger fish sizes (Salini et al., 2015c).
The relative contribution of the more dominant shorter-chain length and more
saturated fatty acids for the provision of energy is clear. This corroborates with data
recently obtained in barramundi where the monounsaturated and to a lesser extent
saturated fatty acids ‘spared’ LC-PUFA for deposition and were preferentially
utilised as energy sources (Salini et al., 2015b). This supports that the available lipids
189 | P a g e
are partitioned into either those fatty acids directed towards oxidative fates for
generating energy or those directed towards other downstream biological purposes
such as eicosanoid production.
There are many potential assumptions in the application of energetic models
(Glencross, 2008). The current allometric assessment only considers a single
phenotypic parameter (live-weight). Not surprisingly, past reports have concluded
that temperature plays a key role in the metabolism of ectotherms, including fish
(Clarke and Johnston, 1999; Clarke and Fraser, 2004; Glencross and Bermudes,
2011; Pirozzi et al., 2010). With barramundi, Glencross and Bermudes (2011)
demonstrated that the allometric scaling over a range of temperatures did change;
however, the response was not dramatic under normal thermal conditions for the
species. Therefore, we assume that the effect of temperature would be minimised by
using a constant ‘optimal’ temperature of 30 °C.
Additionally, there are many studies investigating the metabolic rate in animals and
these relationships with size can usually be described similarly using non-linear
power equations or variants of these (Clarke and Johnston, 1999; White, 2011). The
assumption in the present study is that the standard metabolic rate does not change
under fasting conditions as this could further impact the somatic losses incurred.
There is evidence to suggest that in fish and crustaceans, the standard metabolic rate
is reduced by up to 50% during fasting and this is due partly to decreased protein
synthesis (O'Connor et al., 2000; Simon et al., 2015). Without an estimation of
oxygen consumption or another measure of standard metabolic rate, we cannot
conclude on what might happen on a temporal basis under fasting conditions.
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Conclusions The series of studies presented within this thesis explored many aspects of lipid
metabolism in barramundi (Lates calcarifer). The experimental chapters each
provide an appropriately referenced, comprehensive discussion section that was
adapted from the corresponding published manuscripts. The conclusions from each
chapter are presented below. The first experiment followed an applied approach in
understanding the effect of diluting FO with PF in barramundi diets. This study also
developed methodology in order to predict the fillet composition of LC-PUFA in an
attempt to meet the minimum intake requirements proposed by the FAO for humans.
The study demonstrated that PF can completely substitute fish oil in growing
barramundi. There were no aberrations to fish growth performance or feed utilisation
parameters, however, there were clear differences in the retention and marginal
efficiencies of LC-PUFA utilisation. The fish responded to increasing PF by
improving the retention of LC-PUFA; however, the marginal efficiency of LC-PUFA
was relatively low, and reasons for this need to be further explored. Moreover the
increasing use of PF had a clear impact on the fillet LC-PUFA content, which needs
to be carefully considered in order to manage the defined impacts on meat LC-
PUFA.
After establishing that the effect of poultry fat (rich in monounsaturated fatty acids)
was minimal in growing barramundi we conducted a more comprehensive
investigation into the effect of fatty acid class on the performance of juvenile
barramundi. The fatty acids can be assembled into classes based on their biochemical
structure, including the saturated fatty acids (SFA) and monounsaturated fatty acids
(MUFA). The results demonstrated that the inclusion of a 2:1 or 1:2 ratio of SFA to
MUFA did not lead to a reduction in growth performance of juvenile barramundi.
However, a range of other metabolic modifications were observed. Notable was the
LC-PUFA sparing effect of both MUFA and SFA. Additionally, SFA and MUFA
were preferentially metabolised and deposited in the whole body and liver tissue
proportional to their respective intake. The low digestibility of specific fatty acids
(18:0 and 16:0) is consistent with other studies and may have an impact in the long
term utilisation of the SFA rich diet. These results clearly indicate that consideration
must be given to the proportion of either SFA or MUFA during diet formulation, as
these two classes of fatty acids can influence the in vivo metabolism of fatty acids
and the final fatty acid composition of the whole fish.
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After consideration of the different classes of fatty acids, a subsequent study was
designed to investigate the effect of different lipid classes, both neutral lipid and
polar lipid (NL and PL respectively), from marine or non-marine sources. Studies
have consistently demonstrated a range of advantages when using phospholipid-rich
lipid sources in diets for larval and juvenile fish. In this study, we reported for the
first time the use of phospholipid-rich krill oil (KO) and also soybean lecithin (SL)
compared to neutral lipid sources including fish oil (FO) and soybean oil (SO). In
support of the vast majority of studies, we demonstrated that the inclusion of either
marine or non-marine phospholipid maintains performance of juvenile barramundi
equivalent to a FO based control diet. An interesting result of the study was the
differences observed between both the non-marine (omega-6 rich) diets. The fish fed
SL avoided gross signs of EFA deficiency which was in contrast to the SO fed fish.
However, the dietary SO affected the FCR and some sub-clinical markers indicating
a modified metabolic function.
After these preliminary studies, we concluded that barramundi of a larger size
(~200g fish) generally tolerated diets mostly devoid of FO. In order to test whether
this would hold true on smaller baramundi, we conducted an experiment that
comprehensively investigated the aetiology of the onset and progression of EFA
deficiency in juvenile barramundi. Juvenile barramundi showed signs of modified
metabolic function and potentially signs of essential fatty acid (EFA) deficiency in
the absence of n-3 LC-PUFA. The EFA deficient diets clearly impacted growth
performance and feed utilisation in as little as two weeks. Discrete differences in the
utilisation of dietary lipid and also specific fatty acids suggest that in the absence of
EFA, signs of deficiency were evident substantiating their essentiality in juvenile
barramundi. In addition, a range of clinical abnormalities manifested in the fish fed
the EFA deficient diet. Transcription of genes involved in fatty acid metabolism
consistently demonstrated that those fish receiving the EFA deficient diet were
affected after as little as two weeks. This lends support to the hypothesis that juvenile
barramundi have a determined requirement for EFA. The importance of developing
and then testing an appropriate hypothesis in nutritional studies is also highlighted.
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Following this study, a dose-response experiment was conducted in order to clarify
the biological role of EPA and ARA in juvenile barramundi. This study builds on an
already published assessment of the quantitative DHA requirements in barramundi
and provides a more complete understanding of LC-PUFA metabolism and
requirements in the species. Past studies had indicated that the effect of DHA on
growth was negligible and our study supports this conclusion in that there was no
phenotypic response by barramundi to the addition of either EPA or ARA to the
diets. However, there were some modification to the retention and marginal
utilisation efficiencies as a result of the diets. Increasing the dietary EPA or ARA
level also modulated the expression of several eicosanoid metabolism and fatty acid
oxidation genes. The role of Lc COX2 as an active and rapidly inducible gene has
undoubtedly played a role in the modified metabolic function of the ARA fed fish
during the present study. It is therefore recommended that further studies should
focus on the most appropriate balance of dietary LC-PUFA in light of the current
levels of fish oil substitution in aquafeeds.
A bioenergetic modelling approach was used in several experiments throughout this
thesis in order to generate an understanding of and eventually predict certain
parameters such as LC-PUFA deposition in the tissues and fatty acid utilisation
efficiencies. Different nutrients will almost certainly have different energetic
efficiencies depending on many factors and therefore, we designed a final
experiment that assessed the allometric scaling effect of specific fatty acids after
fasting for 21 days in barramundi. The underlying assumption so far has been that the
scaling exponent of lipid (0.90) could be applied at a nutrient level to any situation
involving fatty acids, including the calculation of maintenance demands. The results
of the study indicate that there are differences in the utilisation efficiencies of fatty
acids, corroborating evidence from past studies. After re-evaluating data from three
separate experiments, we have concluded that the biggest driver in our understanding
of LC-PUFA metabolism in barramundi is that of deposition demand. We have also
suggested that the metabolizable lipid can be broadly separated into that destined for
catabolism and that which is conserved in the tissues for downstream biological
purposes. Empirically based models should now attempt to consider the energetic
costs associated with the lipid metabolic pathway, as this would be the logical
progression of the current work.
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General conclusions The research presented within this thesis collectively provides the reader and the
scientific community with a coherent, up-to-date body of work that makes a
significant contribution to the understanding of lipid metabolism and physiology in
the barramundi (Lates calcarifer). After several decades of research and commercial
development of the barramundi industry, there is now a greater understanding of
their overall nutritional management. Significant advances were made in
understanding the utilisation of alternative oils, the effect of lipid and fatty acid
classes, essential fatty acid deficiency and long-chain polyunsaturated fatty acid
requirements. In addition, advances were made in the bioenergetic modelling of LC-
PUFA. It is anticipated that the knowledge gained throughout this work will become
more commercially relevant in light of the volatile global fish oil market. While there
are potentially other avenues that could be investigated, the present work addressed
many aspects of lipid nutrition in barramundi from an applied approach towards a
more detailed fundamental understanding of lipid metabolism.
Overall, the results presented within this thesis build on published data and several
recommendations could be made that may become practical solutions for the
growing industry:
A minimum requirement of at least 20 g/kg LC-PUFA in growing barramundi
up to ~200g should be respected.
Larger barramundi are more tolerant to reduced FO, however, a more optimal
LC-PUFA in the flesh could be achieved with appropriate blending strategies.
Barramundi adapt well to oils rich in SFA or MUFA, however, the reduced
digestibility of SFA is a concern.
n-6 fatty acids from soybean oil and ARA rich oil can impact negatively on
performance, suggesting that n-3/n-6 balance is critical in maintaining
metabolic homeostasis.
Factorial modelling of LC-PUFA deposition and LC-PUFA maintenance
requirements and utilisation efficiencies is now made possible.
Future investigations may be warranted particularly from a nutritional health aspect.
There is a growing area of research focusing on the welfare of cultured aquatic
animals. There is also a growing range of products available for the prevention of
disease and nutritional management of fish and examination of many of these is still
194 | P a g e
ongoing. Further to this, as more complex instrumentation is becoming routinely
available in laboratories there is significant potential to expand areas of research
particularly in the nutritional health area, at a cellular or molecular level. For
example the specialised pro-resolving mediators have scarcely been investigated in
fish despite their relative importance in inflammatory response and tissue
homeostasis. Lastly, we still have a limited understanding of the complex vitamin
and mineral interactions in the barramundi and many other species. Therefore, future
research should invest in more understanding of these issues with obvious animal
welfare and commercial benefits.
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Appendix - Supplementary figures
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