The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinisation of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-14C]18:2n-6 and [1-14C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolised by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while Δ6 desaturase activity was increased significantly with cell age, Δ5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures

Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-14C]Stearic (18:0), [1-14C]linoleic (18:2n-6) and [1-14C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of phosphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial Δ9, Δ6 and Δ5 fatty acyl desaturase activities. Although, as expected, the cells were more active towards [1-14C]18:3n-3, the cells were unusually active towards [1-14C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit Δ9 and Δ6 desaturase. Changes in the distribution of radioactivity between individual phospholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor in fish and considering the importance of eicosanoids in the biochemistry of skin ,it is suggested that these agents may have direct effects on fish skin that may have important consequences for fish health in general

Replacement of dietary fish oil with increasing levels of linseed oil: Modification of flesh fatty acid compositions in Atlantic salmon (Salmo salar) using a fish oil finishing diet

Five groups of Atlantic salmon smolts, of initial mean weight 127 ± 3g, were fed increasing levels of dietary linseed oil (LO) in a regression design. The control diet contained capelin oil (FO) only and the same oil was blended with LO to provide the experimental diets. After an initial growth period of 40 weeks all treatment groups were switched to a finishing diet containing only FO for a further 24 weeks. Growth, flesh total lipid content and astaxanthin content were not affected by dietary oil composition. The fatty acid compositions of flesh total lipids were linearly correlated with dietary fatty acid compositions (r2 = 0.88-1.00, P < 0.0001). Inclusion of the LO at 50% of added dietary lipid reduced flesh docosahexaenoic (22:6n-3; DHA) and eicosapentaenoic acids (20:5n-3; EPA) to 65 and 58%, respectively, of the concentrations in fish fed FO. When inclusion of dietary LO reached 100% the flesh DHA and EPA concentrations were reduced to 38 and 30%, respectively, of values in fish fed FO. Differences between diet fatty acid concentration and flesh fatty acid concentration showed that 16:0, 18:1n-9 and especially DHA were preferentially retained by salmon whereas 18:2n-6, 18:3n-3 and 22:1n-11 were selected against and presumably utilised for energy production. Feeding a finishing diet containing FO alone for 16 weeks restored flesh DHA and EPA concentrations in fish previously fed 50 and 100% LO to around 80% of their values in fish fed FO throughout. Flesh DHA and EPA concentrations in fish fed up to 50% LO were in excess of recommended intake values for these fatty acids. By utilising FO finishing diets for at least 16 weeks similar flesh DHA and EPA concentrations could be achieved in fish previously fed up to 100% LO for 40 weeks. This study suggests that LO can be used as a substitute for FO in salmon feeds during seawater growth and that any reductions in DHA and EPA can be overcome by feeding FO for a period before harvest

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n-3), to eicosapentaenoic acid, 20:5(n-3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C18 to C20 polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n-3), and its elongation product, 20:4(n-3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 uM), U-14C (1 uM) or deuterated (d5; 25 uM) fatty acids. Stearidonic acid, 18:4(n-3), was metabolised to 20:5(n-3) in both cells lines, but more so in AS than in TF cells. Delta-5 desaturation was more active in TF cells than in AS cells, whereas C18 to C20 elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n-3)) were produced by both cell lines, although there was significant production of 22:5(n-3) in both cultures, especially when 20:4(n-3) was supplemented. We conclude that limited elongation of C18 to C20 fatty acids rather than limited fatty acyl Delta-5 desaturation accounts for the limited rate of conversion of 18:3(n-3) to 20:5(n-3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C18 to C20 PUFA by the turbot and the Atlantic salmon in vivo

Effects of diets containing linseed oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes in Atlantic salmon (Salmo salar)

We hypothesized that replacing fish oil with 18:3n-3-rich linseed oil may enable salmon to maintain the levels of tissue n-3HUFA levels through a combination of increased desaturation activity and increased substrate fatty acid provision. To this end we investigated desaturation/elongation of [1-14C18:3n-3 in hepatocytes and intestinal enterocytes, and determined the extent to which 18:3n-3 was oxidized and desaturated by measuring both simultaneously in a combined assay. Salmon smolts were stocked randomly into five seawater pens and fed for 40 weeks on diets in which the fish oil was replaced in a graded manner by linseed oil. At the end of the trial, fatty acyl desaturation/elongation and oxidation activities were determined in isolated hepatocytes and intestinal enterocytes using [1-14C]18:3n-3 as substrate, and samples of liver and intestinal tissue were collected for analysis of lipid and fatty acid composition. The results showed that, despite increased desaturation of [1-14C]18:3n-3 in hepatocytes, provision of dietary 18:3n-3 did not prevent the decrease in tissue n-3HUFA in fish fed linseed oil. Intestinal enterocytes were a site of significant fatty acid desaturation but, in contrast to hepatocytes, the activity was not increased by feeding linseed oil and was generally lower in fish fed linseed oil compared to fish fed only fish oil. In contrast, oxidation of [1-14C]18:3n-3 in enterocytes was generally increased in fish fed linseed oil compared to fish fed the diet containing only fish oil. However, oxidation of [1-14C]18:3n-3 in hepatocytes was 4- to 8-fold lower than in enterocytes and was not affected by diet. Furthermore, oxidation of [1-14C]18:3n-3 in enterocytes exceeded desaturation irrespective of dietary treatment, whereas similar amounts of [1-14C]18:3n-3 were desaturated and oxidized in hepatocytes from fish fed only fish oil and desaturation exceeded oxidation by 3-fold in fish fed the diet containing 100% linseed oil. The molecular mechanisms underpinning these results were discussed

Genomic selection strategies for clonally propagated crops

For genomic selection (GS) in clonal breeding programs to be effective, parents should be selected based on genomic predicted cross-performance unless dominance is negligible. Genomic prediction of cross-performance enables efficient exploitation of the additive and dominance value simultaneously. Here, we compared different GS strategies for clonally propagated crops with diploid (-like) meiotic behavior, using strawberry as an example. We used stochastic simulation to evaluate six combinations of three breeding programs and two parent selection methods. The three breeding programs included (1) a breeding program that introduced GS in the first clonal stage, and (2) two variations of a two-part breeding program with one and three crossing cycles per year, respectively. The two parent selection methods were (1) parent selection based on genomic estimated breeding values (GEBVs) and (2) parent selection based on genomic predicted cross-performance (GPCP). Selection of parents based on GPCP produced faster genetic gain than selection of parents based on GEBVs because it reduced inbreeding when the dominance degree increased. The two-part breeding programs with one and three crossing cycles per year using GPCP always produced the most genetic gain unless dominance was negligible. We conclude that (1) in clonal breeding programs with GS, parents should be selected based on GPCP, and (2) a two-part breeding program with parent selection based on GPCP to rapidly drive population improvement has great potential to improve breeding clonally propagated crops

Dynamical Masses for Low-Mass Pre-Main Sequence Stars: A Preliminary Physical Orbit for HD 98800 B

We report on Keck Interferometer observations of the double-lined binary (B) component of the quadruple pre-main sequence (PMS) system HD 98800. With these interferometric observations combined with astrometric measurements made by the Hubble Space Telescope Fine Guidance Sensors (FGS), and published radial velocity observations we have estimated preliminary visual and physical orbits of the HD 98800 B subsystem. Our orbit model calls for an inclination of 66.8 $\pm$ 3.2 deg, and allows us to infer the masses and luminosities of the individual components. In particular we find component masses of 0.699 $\pm$ 0.064 and 0.582 $\pm$ 0.051 M_{\sun} for the Ba (primary) and Bb (secondary) components respectively. Modeling of the component SEDs finds temperatures and luminosities in agreement with previous studies, and coupled with the component mass estimates allows for comparison with PMS models in the low-mass regime with few empirical constraints. Solar abundance models seem to under-predict the inferred component temperatures and luminosities, while assuming slightly sub-solar abundances bring the models and observations into better agreement. The present preliminary orbit does not yet place significant constraints on existing pre-main sequence stellar models, but prospects for additional observations improving the orbit model and component parameters are very good.Comment: 20 pages, 6 figures, ApJ in press; tables 2 and 3 to be included in ApJ versio

Symmetry between absorption and amplification in disordered media

We address the issue of whether amplification, like absorption, suppresses wave transmission at large gain, as has been claimed in previous studies of wave propagation in active random media. A closer examination reveals that the paradoxical symmetry between absorption and amplification is an artifact of unphysical solutions from the time-independent wave equation. Solutions from the time-dependent equation demonstrate clearly that when gain is above the threshold, the amplitude of both the transmitted and the reflected wave actually increases with time, apparently without bound. The implications of the current finding is discusse