David A.J. Stone

Experimental Evaluation of Fatty Acid Profiles as a Technique to Determine Dietary Composition in Benthic Elasmobranchs

Fatty acid (FA) analysis is a tool for dietary investigation that complements traditional stomach content analyses. Controlled feeding experiments were used to determine the extent to which the FA composition of diet is reflected in the liver and muscle tissue of the Port Jackson shark Heterodontus portusjacksoni. Over 10 wk, two groups of sharks were fed prawns or squid, which have distinct FA profiles. The percentage of total FA was significantly different for shark liver and muscle tissue when comparing controls with prawn- and squid-fed sharks. Compared with experimentally fed sharks, control shark muscle and liver had higher levels of 18:1n-9 and 20:2n-9. When comparing prawn- and squid-fed sharks, only liver tissue showed a significant difference in FA profiles. The livers of prawn-fed sharks were comparatively higher in 18:1n-7, 22:5n-3, 20:0, and 18:1n-9, while the squid-fed sharks had higher levels of 16:0 and 22:6n-3. These FAs in shark liver tissue were all reflective of higher amounts in their respective dietary items, demonstrating the conservative transfer of FA from diet to liver tissue. This study shows that liver and muscle FA profiles can be used as indicators of dietary change through the comparison of controls and fed sharks. The timescale of this study may not have been sufficient for capturing the integration of FA into muscle tissue because only liver FA profiles were useful to distinguish between sharks fed different diets. These findings have important implications for sampling design where FA profiles are used to infer dietary preferences.

Barramundi (Lates calcarifer) desaturase with Δ6/Δ8 dual activities

Barramundi is a commercially farmed fish in Australia. To examine the potential for barramundi to metabolise dietary α-linolenic acid (ALA, 18:3 n-3), the existence of barramundi desaturase enzymes was examined. A putative fatty acid Δ6 desaturase was cloned from barramundi liver and expressed in yeast. Functional expression revealed Δ6 desaturase activity with both the 18 carbon (C18) and C24 n-3 fatty acids, ALA and 24:5 n-3 as well as the C18 n-6 fatty, linoleic acid (LA, 18:2 n-6). Metabolism of ALA was favoured over LA. The enzyme also had Δ8 desaturase activity which raises the potential for synthesis in barramundi of omega-3 (n-3) long chain polyunsaturated fatty acids from ALA via a pathway that bypasses the initial Δ6 desaturase step. Our findings not only provide molecular evidence for the fatty acid desaturation pathway in the barramundi but also highlight the importance of taking extracellular fatty acid levels into account when assessing enzyme activity expressed in Saccharomyces cerevisiae.

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer).

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.

Dietary alpha-linolenic acid does not enhance accumulation of omega-3 long-chain polyunsaturated fatty acids in barramundi (Lates calcarifer)

This study examined the effects of substituting fish oil and fish meal with a blend of alpha-linolenic acid (ALA, 18:3 n-3) rich vegetable oils (14%, w/w) and defatted poultry meal (34%, w/w) in a formulated diet, on growth and tissue fatty acid profiles in barramundi fingerlings. Results indicated that on average, while the ALA levels of the barramundi liver and fillet increased with increasing dietary ALA, there was no corresponding increase in the levels of the omega-3 (n-3) long chain polyunsaturated fatty acid (LCPUFA). Compared to fish consuming a commercial feed, which contained fish meal and fish oil, fish on the ALA diets grew slower, had a lower feed intake and lower n-3 LCPUFA levels in the tissues. Hepatic mRNA expression of Δ6 desaturase (FADS2) and elongase (ELOVL5/2) was ~10 fold and ~3 fold higher, respectively, in all the ALA dietary groups, relative to those fed the commercial feed. However, the level of expression of the two genes was not different between fish fed differing ALA levels. These data demonstrate that increasing the ALA level of the diet is not an appropriate strategy for replacing marine sources of n-3 LCPUFA in barramundi. It was also noted, however, that within the different ALA dietary groups there was a large amount of variation between individual fish in their tissue DHA levels, suggesting a significant heterogeneity in their capacity for conversion of ALA and/or retention of n-3 LCPUFA. When dietary ALA intakes were greater than 0.8% en, tissue DHA levels were inversely related to ALA intake, suggesting that high intake of dietary ALA may inhibit DHA synthesis.

Restricting feed ration has more effect than diet type on the feeding behaviour of greenlip abalone Haliotis laevigata

Abalone farmed in Australia are predominantly fed formulated feeds, while in the wild, their diet consists of a mix of macroalgae. Here we investigated the feeding behaviour of juvenile greenlip abalone, Haliotis laevigata; fed live macroalgae and formulated diets at different rations; and observed their feeding behaviour using a novel ventral video monitoring technique. Four different diets (commercial chip, experimental flake, Ulva sp. and Gracilaria cliftonii) at two feeding rations (excess vs. restricted) were tested. Diet type had no effect on abalone movement, but macroalgal diets resulted in higher feed intakes. Restricting feed rations induced greater movement. Abalone moved little during the light period and moved mostly during darkness, except for animals on the restricted feed ration where feeding commenced during the light period. On farms, this phenomenon may be a useful behavioural indicator for identifying underfed abalone. Despite their lower intake, formulated diets promoted higher energy and nutritional intake, indicating that quantity of feed consumed is not solely indicative of nutritional gain. From a research perspective, the novel ventral monitoring method has created opportunities for further behavioural studies in molluscs.

Dietary fish oil replacement with canola oil up-regulates glutathione peroxidase 1 gene expression in yellowtail kingfish (Seriola lalandi).

The marine carnivore yellowtail kingfish (YTK, Seriola lalandi) was fed diets containing 5% residual fish oil (from the dietary fish meal) plus either 20% fish oil (FO), 20% canola oil (CO), 20% poultry oil (PO), 10% fish oil plus 10% canola oil (FO/CO) or 10% fish oil plus 10% poultry oil (FO/PO) and the effects on fish growth and hepatic expression of two glutathione peroxidase (GPx 1 and GPx 4) and two peroxiredoxin (Prx 1 and Prx 4) antioxidant genes were investigated. Partial (50%) replacement of the added dietary fish oil with poultry oil significantly improved fish growth whereas 100% replacement with canola oil significantly depressed fish growth. The fatty acid profiles of the fish fillets generally reflected those of the dietary oils except that there was apparent selective utilization of palmitic acid (16:0) and oleic acid (18:1n-9) and apparent selective retention of eicospentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The Prx 1 and 4 genes were expressed at 10- and 100-fold the level of the GPx 4 and 1 genes, respectively, and at one-tenth the level of the highly expressed β-actin reference gene. Dietary fish oil replacement with canola oil significantly up-regulated GPx 1 gene expression and there was a non-significant tendency towards down-regulation of Prx 1 and Prx 4. The results are discussed in terms of the effects of fish oil replacement on the peroxidation index of the diets and the resulting effects on the target antioxidant enzymes.

Comparative Histological Changes in the Greenlip Abalone Haliotis laevigata Gastrointestinal Tract in Response to Water Temperature, Different Dietary Protein Levels, and Animal Age

ABSTRACT n The land-based culture of greenlip abalone Haliotis laevigata in southeastern Australia is carried out using seawater that is prone to seasonal temperature fluctuations and is done almost exclusively using artificial feeds. Although some nutrition research has been done to identify the optimum dietary protein level, typically farms use only 1 diet for grow-out after the juveniles are weaned. Little consideration has been given to the effects of fluctuating water temperatures and dietary protein levels on the morphology of the gastrointestinal tract (GIT) of abalone. Because these factors are known to impact growth in other aquatic species, it is important that they are investigated further to improve growth in abalone species. In this study, the histological changes of the GIT of greenlip abalone in response to 2 water temperatures and 4 dietary levels of crude protein (juvenile, 27% and 36%; subadult, 24% and 33%) for juveniles (1.75 g) and subadults (22.93 g) were investigated. The epithelial thickness of the stomach and crop; intestinal villus height, width, and area; lamina propria height; and stomach, crop, and intestinal neutral and acidic goblet cell numbers were measured. The stomach epithelium was significantly thicker at 14°C than 22°C in both juveniles and subadults, whereas the crop epithelium was significantly thicker at 22°C than 14°C in juveniles. The crop epithelial thickness of subadults was reduced by increasing dietary protein; however, juveniles did not show the same response. Juvenile abalone were more sensitive to temperature fluctuations than subadults, whereas significant effects of dietary protein levels were only observed in subadults. The alterations in the morphology of the GIT did not appear to be detrimental to the health and growth of the abalone. Further research is required to investigate the interactive effects of water temperature and dietary ingredients, particularly in regard to antinutritional factors, on the morphology and function of the GIT to improve our understanding of abalone physiology.

Inter-tissue differences in fatty acid incorporation as a result of dietary oil manipulation in Port Jackson sharks (Heterodontus portusjacksoni).

Fatty acid profile analysis is a tool for dietary investigation that may complement traditional stomach contents analysis. While recent studies have shown that the liver of sharks fed different diets have differing fatty acid profiles, the degree to which diet is reflected in shark blood serum and muscle tissue is still poorly understood. An 18-week controlled feeding experiment was undertaken using captive Port Jackson sharks (Heterodontus portusjacksoni). Sharks were fed exclusive diets of artificial pellets treated with fish or poultry oil and sampled every 6xa0weeks. The fatty acid profiles from liver, blood serum, and muscle were affected differently, with the period from which significant differences were observed varying by tissue and diet type. The total fatty acid profiles of fish oil and poultry oil fed sharks were significantly different from week 12 onwards in the liver and blood serum, but significant differences were only observed by week 18 in the muscle tissue of sharks fed different diets. The drivers of dissimilarity which aligned with dietary input were 14:0, 18:2n-6, 20:5n-3, 18:1n-9 and 22:6n-3 in the liver and blood serum. Dietary fatty acids accumulated more consistently in the liver than in the blood plasma or muscle, likely due to its role as the central organ for fat processing and storage. Blood serum and muscle fatty acid profiles were influenced by diet, but fluctuated over-time. The low level of correlation between diet and muscle FA profiles is likely a result of low levels of fat (<1xa0%) in the muscle and the domination of structural, cell-membrane phospholipids in shark muscle tissues. Our findings describe inter-tissue differences in the incorporation of fatty acids from the diet to consumer, which should be taken into account when interpreting dietary patterns from fatty acid profiles.

Dietary fish oil replacement with palm or poultry oil increases fillet oxidative stability and decreases liver glutathione peroxidase activity in barramundi (Lates calcarifer)

Complete dietary fish oil replacement with palm or poultry oil in barramundi (Lates calcarifer) had no detrimental effects on growth or hepatosomatic index of juvenile fish up to an average size of ~50xa0g. However, it significantly decreased the omega-3 (n-3) long-chain polyunsaturated fatty acid content of the fish muscle (fillet) lipids. This was particularly true for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which are recognised for their health beneficial effects in the human diet. As a result of their decreased EPA and DHA content, the peroxidation index of the muscle lipids was also decreased. This was associated with increased simulated retail storage shelf life as indicated by decreased thiobarbituric acid reactive substances in muscle samples from fish fed the palm or poultry oil-based diets. Concomitantly, glutathione peroxidase (GPx) activity, but not glutathione S-transferase (GST) activity or reduced glutathione concentration, was significantly reduced in the liver of barramundi fed the palm or poultry oil-based diets as compared with the fish fed the fish oil-based diet. Furthermore, GPx and GST activity were very low in muscle, much lower than in gastrointestinal tract, liver or swim bladder. Therefore, we propose that liver GPx activity may be a good predictor of fillet shelf life in barramundi and other fish species.

From egg to hatchling: preferential retention of fatty acid biomarkers in young-of-the-year Port Jackson sharks Heterodontus portusjacksoni.

The muscle and liver fatty acid composition of young-of-the-year (YOY) Port Jackson sharks Heterodontus portusjacksoni were investigated to determine the effects of a known dietary lipid source v. maternal input as demonstrated by egg yolk fatty acid profiles. Ten Heterodontus portusjacksoni egg yolks were collected in situ and compared with four hatched H. portusjacksoni fed a known diet in a controlled feeding experiment of 185 days. This study demonstrated that fatty acids are probably conservatively transferred from egg yolks to YOY H. portusjacksoni, while diet did not have a large effect on the fatty acid composition of the liver or muscle.