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)

Barramundi (Lates calcarifer), a catadromous teleost of significant and growing commercial importance, are reported to have limited fatty acid bioconversion capability and therefore require preformed long-chain PUFA (LC-PUFA) as dietary essential fatty acid (EFA). In this study, 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 either devoid of fish oil (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 8-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 2 weeks, the fish fed the FO FREE and FO LOW diets had significantly lower live-weights, and after 8 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 2 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 2 weeks in juvenile fish.

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

An experiment was conducted with barramundi (Lates calcarifer) juveniles to examine the marginal efficiency of utilisation of long chain-polyunsaturated fatty acids (LC-PUFA). 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 fish over a 12-week period. The replacement of fish oil 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 LOA, 18:2n-6 and LNA, 18:3n-3). By examining the body mass independent retention of different fatty acids with differential levels of intake of each, the marginal efficiencies of the use of these nutrients by this species 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 LC-PUFA levels.

Effect of dietary saturated and monounsaturated fatty acids in juvenile barramundi Lates calcarifer

Barramundi (Lates calcarifer), a catadromous teleost of commercial interest, perform well when fed a wide range of dietary oils. However, the range of alternative oils now being explored is typically rich in saturated and monounsaturated fatty acids (SFA and MUFA). In this study, the response of juvenile barramundi (47.0 g per fish initial weight) fed isolipidic and isoenergetic diets with 82 g kg−1 added oil was tested. The experimental test diets had a 2 : 1 or 1 : 2 ratio of SFA to MUFA (SFA-D and MUFA-D, respectively) compared to a control diet (CTRL-D) fed for 8 weeks. The diets containing mostly olive oil (dietary MUFA-D) and mostly refined palm oil (dietary SFA-D) did not impact the growth performance or feed utilization parameters of the barramundi. The in vivo beta-oxidation activity was consistent with the dietary fatty acid composition, with the most dominant FA being heavily beta-oxidized. 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 utilized in the SFA-D- and MUFA-D-fed fish. This study provides evidence that additional dietary MUFA and SFA are suitable lipid classes for juvenile barramundi and they are both equally efficient at sparing LC-PUFA from an oxidative fate.

The effect of marine and non-marine phospholipid rich oils when fed to juvenile barramundi (Lates calcarifer)

An experiment was conducted to assess the response of juvenile barramundi (Lates calcarifer) to four diets containing either marine- or non-marine derived neutral lipid (NL) or polar lipid (PL) sources for eight weeks in a 2 × 2 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 (FCR) 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 neutral lipid fatty acid composition showed that these mirrored those of the diets and significant interaction terms were noted. However, the whole body polar lipid 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. Statement of relevance: Juvenile barramundi may benefit from dietary phospholipid.

Eicosapentaenoic acid, arachidonic acid and eicosanoid metabolism in juvenile barramundi Lates calcarifer

A two part experiment was conducted to assess the response of barramundi (Lates calcarifer; initial weight = 10.3 ± 0.03 g; mean ± S.D.) fed one of five diets with varying eicosapentaenoic acid (diets 1, 5, 10, 15 and 20 g/kg) or one of four diets with varying arachidonic acid (1, 6, 12, 18 g/kg) against a fish oil control diet. After 6 weeks of feeding, the addition of EPA or ARA did not impact on 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 calculated marginal utilisation efficiencies of EPA and ARA were high (62.1 and 91.9 % respectively) and a dietary ARA requirement was defined (0.012 g/kg(0.796)/day). 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 carnitine palmitoyltransferase 1 (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 nutritionally inducible nature of the cyclooxygenase and lipoxygenase enzymes.

Defining the allometric relationship between size and individual fatty acid turnover in barramundi Lates calcarifer

An experiment was conducted with barramundi (Asian seabass; Lates calcarifer) to examine the allometric scaling effect of individual fatty acids. Six treatment size classes of fish were deprived of food for 21days (Treatment A, 10.5±0.13g; Treatment B, 19.2±0.11g; Treatment C, 28.3±0.05g; Treatment D, 122.4±0.10g; Treatment E, 217.6±0.36g; Treatment F, 443.7±1.48g; mean±SD) with each treatment comprising of fifteen fish, in triplicate. The assessment of somatic losses of whole-body 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 (eicosapentaenoic acid) and 0.954±0.008 for 18:1n-9 (oleic acid). 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. The metabolic demands for maintenance and growth were then iteratively determined for fish over a range of 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.

Fatty acid metabolism in barramundi (Lates Calcarifer)

This PhD contributes towards an complete understanding of lipid metabolism in the barramundi (Lates calcarifer), a culturally important and profitable aquaculture species. The work extended from a more general applied approach through to a complex understanding of molecular mechanisms and lipid bioenergetics.

The influence of dietary fatty acid and fasting on the hepatic lipid metabolism of barramundi (Lates calcarifer)

For many fish species, dietary fish oil (FO) has been substituted with other oils such as poultry oil (PO) without affecting growth performance. However, in barramundi, the mechanisms by which fatty acid metabolism is regulated are poorly understood, and the effects of FO substitution are unknown. This study defined changes in the expression of genes controlling the metabolism of fatty acids in barramundi over a 24-h time period after a single meal. From one to 12 h after a single feeding event, the expression of fatty acid synthesis genes in the liver was upregulated, while genes involved in the β-oxidation showed minimal alteration. However, the expression of β-oxidation genes was significantly correlated with the expression of genes regulating fatty acid synthesis. In a second experiment, the changes in liver fatty acid composition and gene expression were defined after FO was substituted with PO. Liver fatty acid profile reflected the diet composition, with some subtle exceptions supporting the enrichment of certain long-chain polyunsaturated fatty acids in the liver. The fish from all experimental groups preferentially retained more docosahexaenoic acid than eicosapentaenoic acid in the liver, suggesting a bioconversion of this fatty acid to intermediate fatty acids. Replacement of FO with PO significantly regulated genes controlling both fatty acid synthesis and catabolism pathways, potentially related to a higher percentage of monounsaturated fatty acids, in the livers of fish fed these diets. The results demonstrated that diet composition significantly altered the lipid metabolism in barramundi and that there was a balance between direct dietary effects and endogenous synthetic capacity.

Performance of intensively farmed Murray cod Maccullochella peelii peelii (Mitchell) fed newly formulated vs. currently used commercial diets, and a comparison of fillet composition of farmed and wild fish

Murray cod is a top-order carnivore with high culture potential. Currently, there are no commercial diets formulated specifically for Murray cod. In this study, results of two growth trials on Murray cod (80–83.5-g mean initial weight), conducted in commercial settings, using two laboratory-formulated diets (DU1 and DU2; 48.9% and 49.1% protein, and 16.9% and 16.1% lipid, respectively, on a dry matter basis), and two commercial diets, formulated for other species (salmon – CD/S and barramundi – CD/B) but used in Murray cod farming are presented. The two commercial diets had less protein (46.6% and 44.4%) but higher lipid (21.7% and 19.5%). The energy content of the feeds tested was similar (about 20–22 kJ g−1). The growth performance and feed utilization of Murray cod did not differ significantly amongst the diets, but the food conversion ratio and % protein efficiency ratio in fish fed the DU1 and DU2 diets were consistently better. There was significantly less carcass and muscle lipid deposition in fish fed with the latter diets. Of the fatty acids in muscle, the lowest amounts (in μg mg lipid−1) of n-3 (262.5±2.9), n-6 (39.8±0.9) and polyunsaturated fatty acid (PUFA) (302.3±3.8) were observed in fish fed CD/S, and the highest in fish fed DU2 and CD/B. Fatty acids 16:0 and 18:0, 18:1n-9 and 16:1n-7, and 22:6n-3, 20:5n-3, 22:5n-3 and 18:2n-6 were the dominant fatty acids amongst the saturates, monoenes and PUFA, respectively, and accounted for 80.8–88.7% of all identified fatty acids (23) in muscle of Murray cod. The study showed that Murray cod could be cultured successfully on a diet (DU2) containing 20% soybean meal without compromising growth and/or carcass quality. Differences in the proximate composition and fatty acid composition of muscle of wild and farmed Murray cod were observed, the most obvious being in the latter. Wild Murray cod had significantly less (P<0.05) saturates (192.6±1.84 vs. 266.3±3.51), monoenes (156.5±8.7 vs. 207.6±6.19), n-3 (145.2±5.24 vs. 261.8±3.2) but higher n-6 (144.3±2.73 vs. 48.3±1.38) in muscle (all values are in μg mg lipid−1) than in farmed fish. Wild fish also had a much lower n-3 to n-6 ratio (1.0±0.03 vs. 5.4±0.09).

Reductions in nutrient discharge from aquaculture through recycling of water utilising floating media and activated carbon filtration

Nutrient discharge into coastal areas, such as the Great Barrier Reef can result in the degradation of coastal ecosystems. For example, excess nitrogen and phosphorus can damage corals through inducing algal bloom and subsequent shading. Excessive phosphorus can further weaken coral skeletons making them susceptible to damage. Land based industries such as aquaculture can contribute to such problems. This study set out to develop a system whereby water from aquaculture can be constantly reused resulting in minimized waste discharge. A three-stage filtration system utilizing floating media and activated carbon was designed to harness bacterial processes that could reduce both particulate and dissolved compounds to the extent whereby approximately 100% reuse of the wastewater became possible. This involved efficient and effective particulate and biological removal mechanisms in both aerobic and anaerobic zones of the filtration system. This design reduced dissolved nitrogen levels by up to 70% and maintained low phosphorus levels, which allowed the reuse of water for the successful culture of barramundi with a survival rate of 97% over 25 days. This pilot scale study demonstrated the potential of reusing aquaculture wastewater from the viewpoint of reducing nutrient input into coastal environments. Future research will refine these processes and assess the performance of the system at several commercial scale applications.

Powdered activated carbon for fouling reduction of membrane in a pilot-scale recirculation aquaculture system

Recirculating aquaculture systems (RAS) are essential for the reduction in fresh water usage as well as the discharge of nutrients along with aquaculture effluents. A RAS consisting of an anoxic reactor, a membrane bioreactor (MBR) and a UV-disinfection unit was used to process 10,000 L/d of aquaculture effluent providing high-quality treated water for recirculation to a Barramundi fish culture. The system maintained low levels of nitrate (<20 mg/L), nitrite (<3 mg/L) and ammonia (<0.6 mg/L) in the fish tank. Permeate from the membrane that was recirculated to the fish tank contained <21 mg/L of nitrate, <2 mg/L of nitrite and 0 mg/L of ammonia. However, the rate of fouling of the membrane in the MBR was around 1.47 kPa/d, and the membrane in the MBR required cleaning due to fouling after 16 days. Cleaning of the membrane was initiated when the TMP reached around 25 to 30 kPa. In order to reduce the rate of fouling, 500 mg of powdered activated carbon (PAC) per litre of MBR volume was introduced, which decreased the rate of fouling to 0.90 kPa/d. Cleaning of membrane was needed only after 31 days of operation while maintaining the treated effluent quality. Thus the frequency of cleaning could be halved due to the introduction of PAC into the MBR.

Sesamin modulation of lipid class and fatty acid profile in early juvenile teleost, Lates calcarifer, fed different dietary oils

Sesamin, a major sesame seed lignan, has diverse biological functions including the modulation of molecular actions in lipid metabolic pathways and reducing cholesterol levels. Vertebrates have different capacities to biosynthesize long-chain PUFA from dietary precursors and sesamin can enhance the biosynthesis of ALA to EPA and DHA in marine teleost. Early juvenile barramundi, Lates calcarifer, were fed for two weeks on diets rich in ALA or SDA derived from linseed or Echium plantagineum, respectively. Both diets contained phytosterols and less cholesterol compared with a standard fish oil-based diet. The growth rates were reduced in the animals receiving sesamin regardless of the dietary oil. However, the relative levels of n-3 LC-PUFA in total lipid, but not the phospholipid, increased in the whole body by up to 25% in animals fed on sesamin with ALA or SDA. Sesamin reduced the relative levels of triacylglycerols and increased polar lipid, and did not affect the relative composition of phospholipid subclasses or sterols. Sesamin is a potent modulator for LC-PUFA biosynthesis in animals, but probably will have more effective impact at advanced ages. By modulating certain lipid metabolic pathways, sesamin has probably disrupted the body growth and development of organs and tissues in early juvenile barramundi.

Up-regulated Desaturase and Elongase Gene Expression Promoted Accumulation of Polyunsaturated Fatty Acid (PUFA) but Not Long-Chain PUFA in Lates calcarifer, a Tropical Euryhaline Fish, Fed a Stearidonic Acid- and γ-Linoleic Acid-Enriched Diet

The limited activity of Δ6 fatty acid desaturase (FAD6) on α-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids in marine fish alters the long-chain (≥C₂₀) polyunsaturated fatty acid (LC-PUFA) concentration in fish muscle and liver when vegetable oils replace fish oil (FO) in aquafeeds. Echium oil (EO), rich in stearidonic acid (SDA, 18:4n-3) and γ-linoleic acid (GLA, 18:3n-6), may enhance the biosynthesis of n-3 and n-6 LC-PUFA by bypassing the rate-limiting FAD6 step. Nutritional and environmental modulation of the mechanisms in LC-PUFA biosynthesis was examined in barramundi, Lates calcarifer, a tropical euryhaline fish. Juveniles were maintained in either freshwater or seawater and fed different dietary LC-PUFA precursors present in EO or rapeseed oil (RO) and compared with FO. After 8 weeks, growth of fish fed EO was slower compared to the FO and RO treatments. Irrespective of salinity, expression of the FAD6 and elongase was up-regulated in fish fed EO and RO diets, but did not lead to significant accumulation of LC-PUFA in the neutral lipid of fish tissues as occurred in the FO treatment. However, significant concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), but not docosahexaenoic acid (DHA, 22:6n-3), appeared in liver and, to a lesser extent, in muscle of fish fed EO with marked increases in the phospholipid fraction. Fish in the EO treatment had higher EPA and ARA in their liver phospholipids than fish fed FO. Endogenous conversion of dietary precursors into neutral lipid LC-PUFA appears to be limited by factors other than the initial rate-limiting step. In contrast, phospholipid LC-PUFA had higher biosynthesis, or selective retention, in barramundi fed EO rather than RO.