Fish oil replacement in current aquaculture feed : is cholesterol a hidden treasure for fish nutrition?

Teleost fish, as with all vertebrates, are capable of synthesizing cholesterol and as such have no dietary requirement for it. Thus, limited research has addressed the potential effects of dietary cholesterol in fish, even if fish meal and fish oil are increasingly replaced by vegetable alternatives in modern aquafeeds, resulting in progressively reduced dietary cholesterol content. The objective of this study was to determine if dietary cholesterol fortification in a vegetable oil-based diet can manifest any effects on growth and feed utilization performance in the salmonid fish, the rainbow trout. In addition, given a series of studies in mammals have shown that dietary cholesterol can directly affect the fatty acid metabolism, the apparent in vivo fatty acid metabolism of fish fed the experimental diets was assessed. Triplicate groups of juvenile fish were fed one of two identical vegetable oil-based diets, with additional cholesterol fortification (high cholesterol, H-Chol) or without (low cholesterol, L-Chol), for 12 weeks. No effects were observed on growth and feed efficiency, however, in fish fed H-Col no biosynthesis of cholesterol, and a remarkably decreased apparent in vivo fatty acid b-oxidation were recorded, whilst in LChol fed fish, cholesterol was abundantly biosynthesised and an increased apparent in vivo fatty acid b-oxidation was observed. Only minor effects were observed on the activity of stearyl-CoA desaturase, but a significant increase was observed for both the transcription rate in liver and the apparent in vivo activity of the fatty acid D-6 desaturase and elongase, with increasing dietary cholesterol. This study showed that the possible effects of reduced dietary cholesterol in current aquafeeds can be significant and warrant future investigations.

Bioconversion of α-Linolenic Acid into n-3 Long-Chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation

This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-3 (EPA) production from 50 mM ALA initial concentration was achieved after 3 days of incubation. Next, it was established that a gradual increase in the ALA concentration from 0 up to 125mM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA) and 22:6n-3 (DHA) in 3 day incubations. Of interest, ALA bioconversion products were observed in the culture medium. Therefore, in vitro experiments disregarding the medium fatty acid content are underestimating the metabolism efficiency. The novel application of the fatty acid mass balance (FAMB) method on cell culture system (cells with medium) enabled quantifying the apparent enzymatic activities for the biosynthesis of n-3 LC-PUFA. The activity of the key enzymes was estimated and showed that, under these conditions, 50% (Km) of the theoretical maximal (Vmax = 3654 mmol.g21 of cell protein.hour21) Fads2 activity on ALA can be achieved with 81 mM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) was the slowest amongst other biosynthesis steps. Therefore, the possible improvement of Elovl2 activity is suggested toward a more efficient DHA production from ALA. The present study proposed and described an ad hoc optimised cell culture conditions and methodology towards achieving a reliable experimental platform, using FAMB, to assist in studying the efficiency of ALA bioconversion into n-3 LC-PUFA in vitro. The FAMB proved to be a powerful and inexpensive method to generate a detailed description of the kinetics of n-3 LC-PUFA biosynthesis enzymes activities in vitro.

The A622 gene in Nicotiana glauca (tree tobacco): evidence for a functional role in pyridine alkaloid synthesis

Nicotiana glauca (Argentinean tree tobacco) is atypical within the genus Nicotiana, accumulating predominantly anabasine rather than nicotine and/or nornicotine as the main component of its leaf pyridine alkaloid fraction. The current study examines the role of the A622 gene from N. glauca (NgA622) in alkaloid production and utilises an RNAi approach to down-regulate gene expression and diminish levels of A622 protein in transgenic tissues. Results indicate that RNAi-mediated reduction in A622 transcript levels markedly reduces the capacity of N. glauca to produce anabasine resulting in plants with scarcely any pyridine alkaloids in leaf tissues, even after damage to apical tissues. In addition, analysis of hairy roots containing the NgA622-RNAi construct shows a substantial reduction in both anabasine and nicotine levels within these tissues, even if stimulated with methyl jasmonate, indicating a role for the A622 enzyme in the synthesis of both alkaloids in roots of N. glauca. Feeding of Nicotinic Acid (NA) to hairy roots of N. glauca containing the NgA622-RNAi construct did not restore capacity for synthesis of anabasine or nicotine. Moreover, treatment of these hairy root lines with NA did not lead to an increase in anatabine levels, unlike controls. Together, these results strongly suggest that A622 is an integral component of the final enzyme complex responsible for biosynthesis of all three pyridine alkaloids in Nicotiana.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

In situ synthesis of natural rubber latex-supported gold nanoparticles for flexible SERS substrates

Natural rubber latex (NRL) from Hevea brasiliensis was used as a matrix to synthesize gold nanoparticles (AuNPs), leading to an organic-inorganic hybrid latex of NRL-supported AuNPs (AuNPs@NRL). The in situ and environmentally friendly preparation of AuNPs in an NRL matrix was developed by thermal treatment without using any other reducing agents or stabilizers because natural rubber particles and non-rubber components present in serum can serve as supporters for the synthesized AuNPs. As a result, the nanosized and well-dispersed AuNPs not only are decorated on the surface of natural rubber particles, but also can be found in the serum of NRL. The size of the AuNPs presented in NRL matrix can be controlled by adjusting the concentration of NRL. Furthermore, the flexible surface-enhanced Raman scattering (SERS) substrates made from the AuNPs@NRL through vacuum filtration presented good enhancement of the Raman probe molecule of 4-mercaptopyridine and outstanding SERS reproducibility. The capability of synthesizing the bio-supported nanohybrid latex provides a novel green and simple approach for the fabrication of flexible and effective SERS substrates.

Predictive growth budgets in terns and gulls

Energy budgets for nestling growth are presented for sandwich tern Sterna sandvicensis, common tern S. hirundo, Arctic tern S. paradisaea, and herring gull Larus argentatus. Energy used in the production of body tissue averaged 27% (of which 7% for biosynthesis) while BMR accounted for 45%, the remainder being cost of activity and thermoregulation (28%). Where quantified, cost of temperature regulation accounted for only 10% of the total expenditure under field conditions. A regression made of metabolic energy (ME) intake over the entire nestling period against body mass of the fledgling based on eight studies of gulls and terns resulted in ME=35.14×M1.0105. -from Authors

Synthetic biology based metabolic engineering of saccharomyces cerevisiae for omega 6 and 3 PUFA production

Metabolic engineering of PUFA biosynthesis pathway using codon optimized DGA1 (Diacylglycerol acyltransferase), FAA3 (Acyl-CoA synthetase), desaturase genes named D9D, D12D, D5D, D6D, D17D and D6E elongase gene was studied in S. cerevisiae. Engineered yeast strains successfully demonstrated increase in lipid accumulation, and heterologous biosynthesis of linoleic, γ-linolenic, dihomo γ-linolenic, arachidonic and eicosapentaenoic acid.

Dietary creatine supplementation during pregnancy: a study on the effects of creatine supplementation on creatine homeostasis and renal excretory function in spiny mice

Recent evidence obtained from a rodent model of birth asphyxia shows that supplementation of the maternal diet with creatine during pregnancy protects the neonate from multi-organ damage. However, the effect of increasing creatine intake on creatine homeostasis and biosynthesis in females, particularly during pregnancy, is unknown. This study assessed the impact of creatine supplementation on creatine homeostasis, body composition, capacity for de novo creatine synthesis and renal excretory function in non-pregnant and pregnant spiny mice. Mid-gestation pregnant and virgin spiny mice were fed normal chow or chow supplemented with 5 % w/w creatine for 18 days. Weight gain, urinary creatine and electrolyte excretion were assessed during supplementation. At post mortem, body composition was assessed by Dual-energy X-ray absorptiometry, or tissues were collected to assess creatine content and mRNA expression of the creatine synthesising enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) and the creatine transporter (CrT1). Protein expression of AGAT and GAMT was also assessed by Western blot. Key findings of this study include no changes in body weight or composition with creatine supplementation; increased urinary creatine excretion in supplemented spiny mice, with increased sodium (P < 0.001) and chloride (P < 0.05) excretion in pregnant dams after 3 days of supplementation; lowered renal AGAT mRNA (P < 0.001) and protein (P < 0.001) expressions, and lowered CrT1 mRNA expression in the kidney (P < 0.01) and brain (P < 0.001). Creatine supplementation had minimal impact on creatine homeostasis in either non-pregnant or pregnant spiny mice. Increasing maternal dietary creatine consumption could be a useful treatment for birth asphyxia.

Changes in lipid class content and composition of Isochrysis sp. (T-Iso) grown in batch culture

© 2015, Springer International Publishing Switzerland. For decades, the microalgae Isochrysis spp. have been widely utilised as a live feed in aquaculture practices. This species possesses a number of favourable characteristics, notably its long-chain omega-3 polyunsaturated fatty acid (LC n-3 PUFA) content; primarily docosahexaenoic acid (DHA, 22:6n-3). This article describes the lipid class content and composition of this microalga grown in batch culture, covering the entirety of lag, log and stationary growth phases. The total lipid was highest in the lag phase (27 pg/cell). Total lipid significantly decreased in the exponential growth (7 pg/cell), then steadily increasing for the remainder of growth. The increase in total lipid was due to the accumulation of neutral lipid in the form of triacylglycerides. The DHA content (pg/cell) of the neutral lipid remained relatively unchanged for the duration of growth, with the influx of fatty acids being primarily myristic and palmitic acids. DHA (pg/cell) was found at relatively uniform amounts across all lipid classes. However, the DHA content as a percentage differed greatly between classes. The polar lipid class had a significantly higher DHA content, which peaked at 38 % of all polar lipid in log growth. The primary PUFA species present in the glycolipid class was stearidonic acid (18:4n-3). This work gives an overview of the lipid content and composition of Isochrysis sp. (T-Iso) over the entirety of its growth under batch culture. The lipid profile for this species at different stages of culture provides a basal data set that is useful for comparative studies using this organism.

Arachidonic acid and eicosapentaenoic acid metabolism in juvenile Atlantic Salmon as affected by water temperature

Salmons raised in aquaculture farms around the world are increasingly subjected to sub-optimal environmental conditions, such as high water temperatures during summer seasons. Aerobic scope increases and lipid metabolism changes are known plasticity responses of fish for a better acclimation to high water temperature. The present study aimed at investigating the effect of high water temperature on the regulation of fatty acid metabolism in juvenile Atlantic salmon fed different dietary ARA/EPA ratios (arachidonic acid, 20:4n-6/ eicosapentaenoic acid, 20:5n-3), with particular focus on apparent in vivo enzyme activities and gene expression of lipid metabolism pathways. Three experimental diets were formulated to be identical, except for the ratio EPA/ARA, and fed to triplicate groups of Atlantic salmon (Salmo salar) kept either at 10°C or 20°C. Results showed that fatty acid metabolic utilisation, and likely also their dietary requirements for optimal performance, can be affected by changes in their relative levels and by environmental temperature in Atlantic salmon. Thus, the increase in temperature, independently from dietary treatment, had a significant effect on the β-oxidation of a fatty acid including EPA, as observed by the apparent in vivo enzyme activity and mRNA expression of pparα -transcription factor in lipid metabolism, including β-oxidation genes- and cpt1 -key enzyme responsible for the movement of LC-PUFA from the cytosol into the mitochondria for β-oxidation-, were both increased at the higher water temperature. An interesting interaction was observed in the transcription and in vivo enzyme activity of Δ5fad-time-limiting enzyme in the biosynthesis pathway of EPA and ARA. Such, at lower temperature, the highest mRNA expression and enzyme activity was recorded in fish with limited supply of dietary EPA, whereas at higher temperature these were recorded in fish with limited ARA supply. In consideration that fish at higher water temperature recorded a significantly increased feed intake, these results clearly suggested that at high, sub-optimal water temperature, fish metabolism attempted to increment its overall ARA status -the most bioactive LC-PUFA participating in the inflammatory response- by modulating the metabolic fate of dietary ARA (expressed as % of net intake), reducing its β-oxidation and favouring synthesis and deposition. This correlates also with results from other recent studies showing that both immune- and stress- responses in fish are up regulated in fish held at high temperatures. This is a novel and fundamental information that warrants industry and scientific attention, in consideration of the imminent increase in water temperatures, continuous expansion of aquaculture operations, resources utilisation in aquafeed and much needed seasonal/adaptive nutritional strategies.

Isolation and functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 desaturase activity

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C18 precursors.