David S. Francis

Fatty acid metabolism (desaturation, elongation and β-oxidation) in rainbow trout fed fish oil- or linseed oil-based diets

In consideration of economical and environmental concerns, fish oil (FO) substitution in aquaculture is the focus of many fish nutritionists. The most stringent drawback of FO replacement in aquafeeds is the consequential modification to the final fatty acid (FA) make-up of the fish fillet. However, it is envisaged that a solution may be achieved through a better understanding of fish FA metabolism. Therefore, the present study investigated the fate of individual dietary FA in rainbow trout (Oncorhynchus mykiss) fed a FO-based diet (rich in 20 : 5n-3) or a linseed oil-based diet (LO; rich in 18 : 3n-3). The study demonstrated that much of the 18 : 3n-3 content from the LO diet was oxidised and, despite the significantly increased accretion of Delta-6 and Delta-5 desaturated FA, a 2- and 3-fold reduction in the fish body content of 20 : 5n-3 and 22 : 6n-3, respectively, compared with the FO-fed fish, was recorded. The accretion of longer-chain FA was unaffected by the dietary treatments, while there was a greater net disappearance of FA provided in dietary surplus. SFA and MUFA recorded a net accretion of FA produced ex novo. In the fish fed the FO diet, the majority of dietary 20 : 5n-3 was accumulated (53.8 %), some was oxidised (14.7 %) and a large proportion (31.6 %) was elongated and desaturated up to 22 : 6n-3. In the fish fed the LO diet, the majority of dietary 18 : 3n-3 was accumulated (58.1 %), a large proportion was oxidised (29.5 %) and a limited amount (12.4 %) was bio-converted to longer and more unsaturated homologues.

A Whole Body, In Vivo, Fatty Acid Balance Method to Quantify PUFA Metabolism (Desaturation, Elongation and Beta-oxidation)

Currently there are several contrasting methods utilized for estimating elongation and desaturation of fatty acids and their general metabolism. The majority of these methods involve an ex vivo approach, requiring expensive and sophisticated equipment, likely to result in considerable variation in enzyme activity between and within species. In the present paper we introduce a further development of the whole-body fatty acid balance method for the estimation of the elongation and desaturation of fatty acids. This method though receiving considerable attention because of its simplicity and reliability has yet to be presented in detail. Theoretically, the whole-body fatty acid balance method can potentially be applied to any organism and requires little more than a gas chromatography unit for fatty acid analysis and elementary calculations. As such in this paper we attempt to spell out in detail the theoretical basis and the methods of application drawing specific examples. Using the present method it is possible to measure the fate of individual fatty acids towards desaturation, elongation and oxidation and calculate the elongase, Δ-6 desaturase and Δ-5 desaturase activities.

LC-PUFA biosynthesis in rainbow trout is substrate limited: use of the whole body fatty acid balance method and different 18:3n-3/18:2n-6 ratios.

Five experimental diets with constant total C18 PUFA and varying 18:3n-3/18:2n-6 ratios were fed to rainbow trout over an entire production cycle. The whole-body fatty acid balance method demonstrated a clear trend of progressively reduced fatty acid bioconversion activity along the n-3 and n-6 pathways, up to the production of 20:5n-3 and 20:4n-6, respectively. This suggests that the pathway exhibits a “funnel like” progression of activity rather than the existence of a single rate limiting step. The production of 22:5n-3 and 22:6n-3 was more active than that of 20:5n-3. However, despite this trend in reduced apparent in vivo net enzyme activity, the efficiency of the various bioconversion steps (measured as % of bioconverted substrate) confirmed an opposing trend. A 3.2-fold higher Δ-6 desaturase affinity towards 18:3n-3 over 18:2n-6 and an 8-fold greater Δ-5 desaturase affinity towards 20:4n-3 over 20:3n-6 were recorded. The main results of the study were that (1) rainbow trout are quite efficient at bioconverting 18:3n-3 to 22:6n-3, and (2) the LC-PUFA biosynthetic pathway is substrate limited. Fillet n-3 LC-PUFA concentrations increased with the increasing dietary supply of 18:3n-3. Despite an almost identical dietary supply of n-3 LC-PUFA, originating from the fish meal fraction of the diets, the fillets of trout fed the diet richest in 18:3n-3 were 2-fold higher in n-3 LC-PUFA than fish fed low 18:3n-3 diets. Nevertheless, fillets of trout fed a fish oil control diet contained more than double the amount of n-3 LC-PUFA compared to fish fed the diets richest in 18:3n-3.

Apparent in vivo Δ-6 desaturase activity, efficiency, and affinity are affected by total dietary C18 PUFA in the freshwater fish murray cod.

Dietary fatty acids are known to modulate fatty acid metabolism in fish. However, the innate capability of fish to bioconvert short chain fatty acids to health promoting long chain fatty acids (LCPUFA) is insufficient to compensate for a reduced dietary intake. While many studies have focused on the dietary regulation of the fatty acid bioconversion pathways, there is little known regarding the effects of the dietary levels of C(18) polyunsaturated fatty acids (PUFA) on fatty acid metabolism. Here, we show a greater degree of apparent enzyme activity (Δ-6 desaturase) in fish fed a diet with higher amounts of dietary C(18) PUFA. In particular, fish receiving high amounts of dietary C(18) PUFA had a greater amount of Δ-6 desaturase activity acting on 18:3n-3 than 18:2n-6. However, with the gradual reduction of dietary C(18) PUFA there was a shift in substrate preference of Δ-6 desaturase from 18:3n-3 to 18:2n-6. This information will provide valuable insight for the implementation of low fish oil diets, which permit the maintenance of n-3 LCPUFA levels in farmed Murray cod.

Echium Oil Provides No Benefit over Linseed Oil for (n-3) Long-Chain PUFA Biosynthesis in Rainbow Trout

The implementation of alternative lipid sources for use in aquaculture is of considerable interest globally. However, the possible benefit of using stearidonic acid (SDA)-rich fish oil (FO) alternatives has led to scientific confusion. Two hundred and forty rainbow trout (Oncorhynchus mykiss) were fed 1 of 4 diets (3 replicate tanks/treatment) containing either FO, linseed oil (LO), echium oil, or mixed vegetable oil (72% LO, 23% sunflower oil, and 6% canola oil) as the dietary lipid source (16.5%) for 73 d to investigate the competition and long-chain PUFA (LC-PUFA) biosynthesis between the fatty acid substrates α-linolenic acid (ALA) and SDA. SDA was more efficiently bioconverted to LC-PUFA compared with ALA. However, when the dietary lipid sources were directly compared, the increased provision of C18 PUFA within the LO diet resulted in no significant differences in (n-3) LC-PUFA content compared with fish fed the other diets. This study therefore shows that, rather than the previously speculated substrate competition, the limiting process in the apparent in vivo (n-3) LC-PUFA biosynthesis appears to be substrate availability. Rainbow trout fed the SDA- and ALA-rich dietary lipid sources subsequently had similar significant reductions in (n-3) LC-PUFA compared with fish fed the FO diet, therefore providing no additional dietary benefit on (n-3) LC-PUFA concentrations.

Effects of Dietary α-Linolenic Acid (18:3n-3)/Linoleic Acid (18:2n-6) Ratio on Fatty Acid Metabolism in Murray Cod (Maccullochella peelii peelii)

Global shortages in fish oil are forcing the aquaculture feed industry to use alternative oil sources, the use of which negatively affects the final fatty acid makeup of cultured fish. Thus, the modulation of fatty acid metabolism in cultured fish is the core of an intensive global research effort. The present study aimed to evaluate the effects of various dietary α-linolenic acid (ALA, 18:3n-3)/linoleic acid (LA, 18:2n-6) ratios in cultured fish. A feeding trial was implemented on the freshwater finfish Murray cod, in which fish were fed either a fish oil-based control diet or one of five fish oil-deprived experimental diets formulated to contain an ALA/LA ratio ranging from 0.3 to 2.9, but with a constant total C₁₈ PUFA (ALA+LA) content. The whole-body fatty acid balance method was used to evaluate fish in vivo fatty acid metabolism. The results indicate that dietary ALA was more actively β-oxidized and bioconverted, whereas LA appears to be more efficiently deposited. LA was β-oxidized at a constant level (~36% of net intake) independent of dietary availability, whereas ALA was oxidized proportionally to dietary supply. The in vivo apparent Δ-6 desaturase activity on n-3 and n-6 PUFA exhibited an increasing and decreasing trend, respectively, in conjunction with the increasing dietary ALA/LA ratio, clearly indicating that this enzymatic activity is substrate dependent. However, the maximum Δ-6 desaturase activity acting on ALA peaked at the substrate level of 3.2186 (μmol g fish⁻¹ day⁻¹), suggesting that additional inclusion of ALA is not only wasteful but counterproductive in terms of n-3 LC-PUFA production. Despite a constant total supply of ALA+LA, the recorded total in vivo apparent Δ-6 desaturase activity on both substrates (ALA and LA) increased in synchrony with the ALA/LA ratio, peaking at 1.54, and a 3.2-fold greater Δ-6 desaturase affinity toward ALA over LA was recorded.

A whole body, in vivo, fatty acid balance method to quantify PUFA metabolism (desaturation, elongation and beta-oxidation) : erratum to : lipids (2007) 42 :1065-1071

Currently there are several contrasting methods utilized for estimating elongation and desaturation of fatty acids and their general metabolism. The majority of these methods involve an ex vivo approach, requiring expensive and sophisticated equipment, likely to result in considerable variation in enzyme activity between and within species. In the present paper we introduce a further development of the whole-body fatty acid balance method for the estimation of the elongation and desaturation of fatty acids. This method though receiving considerable attention because of its simplicity and reliability has yet to be presented in detail. Theoretically, the whole-body fatty acid balance method can potentially be applied to any organism and requires little more than a gas chromatography unit for fatty acid analysis and elementary calculations. As such in this paper we attempt to spell out in detail the theoretical basis and the methods of application drawing specific examples. Using the present method it is possible to measure the fate of individual fatty acids towards desaturation, elongation and oxidation and calculate the elongase, Delta-6 desaturase and Delta-5 desaturase activities.

n-3 LC-PUFA deposition efficiency and appetite-regulating hormones are modulated by the dietary lipid source during rainbow trout grow-out and finishing periods

Largely attributable to concerns surrounding sustainability, the utilisation of omega-3 long-chain polyunsaturated fatty acid-rich (n-3 LC-PUFA) fish oils in aquafeeds for farmed fish species is an increasingly concerning issue. Therefore, strategies to maximise the deposition efficiency of these key health beneficial fatty acids are being investigated. The present study examined the effects of four vegetable-based dietary lipid sources (linseed, olive, palm and sunflower oil) on the deposition efficiency of n-3 LC-PUFA and the circulating blood plasma concentrations of the appetite-regulating hormones, leptin and ghrelin, during the grow-out and finishing phases in rainbow trout culture. Minimal detrimental effects were noted in fish performance; however, major modifications were apparent in tissue fatty acid compositions, which generally reflected that of the diet. These modifications diminished somewhat following the fish oil finishing phase, but longer-lasting effects remained evident. The fatty acid composition of the alternative oils was demonstrated to have a modulatory effect on the deposition efficiency of n-3 LC-PUFA and on the key endocrine hormones involved in appetite regulation, growth and feed intake during both the grow-out and finishing phases. In particular, n-6 PUFA (sunflower oil diet) appeared to ‘spare’ the catabolism of n-3 LC-PUFA and, as such, resulted in the highest retention of these fatty acids, ultimately highlighting new nutritional approaches to maximise the maintenance of the qualitative benefits of fish oils when they are used in feeds for aquaculture species.

Monola oil versus canola oil as a fish oil replacer in rainbow trout feeds: effects on growth, fatty acid metabolism and final eating quality.

Monola oil, a high oleic acid canola cultivar, and canola oil were evaluated as replacers of fish oil at three levels of inclusion (60%, 75% and 90%) in rainbow trout diets. After a 27-week grow-out cycle, the diet-induced effects on growth, fatty acid metabolism and final eating quality were assessed. Overall, no effects were noted for growth, feed utilisation or fish biometry, and the fatty acid composition of fish fillets mirrored that of the diets. Dietary treatments affected fillet lipid oxidation (free malondialdehyde), pigmentation and flavour volatile compounds, but only minor effects on sensorial attributes were detected. Ultimately, both oils were demonstrated to possess, to differing extents, suitable qualities to adequately replace fish oil from the perspective of fish performance and final product quality. However, further research is required to alleviate on-going issues associated with the loss of health promoting attributes (n-3 long chain polyunsaturated fatty acids) of final farmed products.

Fatty Acid-Specific Alterations in Leptin, PPARα, and CPT-1 Gene Expression in the Rainbow Trout

It is known that fatty acids (FA) regulate lipid metabolism by modulating the expression of numerous genes. In order to gain a better understanding of the effect of individual FA on lipid metabolism related genes in rainbow trout (Oncorhynchus mykiss), an in vitro time-course study was implemented where twelve individual FA (butyric 4:0; caprylic 8:0; palmitic (PAM) 16:0; stearic (STA) 18:0; palmitoleic16:1n-7; oleic 18:1n-9; 11-cis-eicosenoic 20:1n-9; linoleic (LNA) 18:2n-6; α-linolenic (ALA) 18:3n-3; eicosapentenoic (EPA) 20:5n-3; docosahexaenoic (DHA) 22:6n-3; arachidonic (ARA) 20:4n-6) were incubated in rainbow trout liver slices. The effect of FA administration over time was evaluated on the expression of leptin, PPARα and CPT-1 (lipid oxidative related genes). Leptin mRNA expression was down regulated by saturated fatty acids (SFA) and LNA, and was up regulated by monounsaturated fatty acids (MUFA) and long chain PUFA, whilst STA and ALA had no effect. PPARα and CPT-1mRNA expression were up regulated by SFA, MUFA, ALA, ARA and DHA; and down regulated by LNA and EPA. These results suggest that there are individual and specific FA induced modifications of leptin, PPARα and CPT-1 gene expression in rainbow trout, and it is envisaged that such results may provide highly valuable information for future practical applications in fish nutrition.