R.J. Henderson

Polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr-smolt transformation and the effects of dietary linseed and rapeseed oils

Duplicate groups of Atlantic salmon parr were fed diets containing either fish oil (FO), rapeseed oil (RO), linseed oil (LO) or linseed oil supplemented with arachidonic acid (20:4n-6; AA) (LOA) from October (week 0) to seawater transfer in March (week 19). From March to July (weeks 20–34) all fish were fed a fish oil-containing diet. Fatty acyl desaturation and elongation activity in isolated hepatocytes incubated with [1-14C]18:3n-3 increased in all dietary groups, peaking in early March about one month prior to seawater transfer. Desaturation activities at their peak were significantly greater in fish fed the vegetable oils, particularly RO, compared to fish fed FO. Docosahexaenoic acid (22:6n-3:DHA) and AA in liver and gill polar lipids (PL) increased in all dietary groups during the freshwater phase whereas eicosapentaenoic acid (20:5n-3; EPA) increased greatly in all groups after seawater transfer. The AA/EPA ratio in tissue PL increased up to seawater transfer and then decreased after transfer. AA levels and the AA/EPA ratio in gill PL were generally higher in the LOA group. The levels of 18:3n-3 in muscle total lipid were increased significantly in the LO, LOA and, to a lesser extent, RO groups prior to transfer but were reduced to initial levels by the termination of the experiment (week 34). In contrast, 18:2n-6 in muscle total lipid was significantly increased after 18 weeks in fish fed the diets supplemented with RO and LO, and was significantly greater in the FO and RO groups at the termination of the experiment. Gill PGF production showed a large peak about two months after transfer to seawater. The production of total PGF post-transfer was significantly lower in fish previously fed the LOA diet. However, plasma chloride concentrations in fish subjected to a seawater challenge at 18 weeks were all lower in fish fed the diets with vegetable oils. This effect was significant in the case of fish receiving the diet with LOA, compared to those fed the diet containing FO. The present study showed that during parr-smolt transformation in Atlantic salmon there is a pre-adaptive increase in hepatocyte fatty acyl desaturation/elongation activities that is controlled primarily by environmental factors such as photoperiod and temperature but that can also be significantly modulated by diet. Feeding salmon parr diets supplemented with rapeseed or linseed oils prevented inhibition of the desaturase activities that is induced by feeding parr diets with fish oils and thus influenced the smoltification process by altering tissue PL fatty acid compositions and eicosanoid production. These effects, in turn, had a beneficial effect on the ability of the fish to osmoregulate and thus adapt to salinity changes.

Partial decontamination of rotifers with ultraviolet radiation: the effect of changes in the bacterial load and flora of rotifers on mortalities in start-feeding larval turbot

Abstract Exposure of the rotifer Brachionus plicatilis to ultraviolet radiation in 1 mm deep liquid layers at an energy intensity of 38 mW cm −2 reduced the rotifer bacterial load by >90% within 2 min. Such radiation doses had no significant effect upon rotifer viability, fatty acid composition, swimming or feeding activity. The surviving bacterial flora of irradiated rotifers was similar to that of un-irradiated rotifers and there was no evidence of major differences in sensitivity to ultraviolet radiation between different bacteria. For irradiation of large numbers of rotifers, a flow-through cell was used, operated with rotifer densities of 200 ml −1 and a flow rate of 1.5 l min −1 . In two separate field trials involving groups of 34,000 turbot larvae per group, higher survival was found in groups receiving ultraviolet-irradiated rotifers in which the bacterial load was reduced by 88%. This was attributed to the slower rate of colonisation of the larval gut by bacteria, as a consequence of the lower bacterial load on the rotifers. Attempts to introduce specified bacteria into the larval turbot gut in significant numbers by colonisation of either normal or irradiated rotifers with particular bacteria were unsuccessful.

The determination of lipase and phospholipase activities in gut contents of turbot (Scophthalmus maximus) by fluorescence-based assays

To study the potential of fluorescence based assays in the study of lipid digestion in fish, acyl esters of 4-methylumbelliferone and 1-acyl-2-[6 (7 nitro-1,3 benzoxadiazol-4-yl)amino]caproyl labeled phosphatidylcholine compounds (NBD-PC) were used as substrates for the assay of neutral lipase and phospholipase, respectively, in the gut contents of turbot. 4-Methylumbelliferyl hepatanoate (4-MUH) was hydrolysed at a higher rate than the butyrate or oleate esters whilst the hexanoic (C6) ester of NBD-PC was a more convenient substrate for the phospholipase assay than the dodecanoic (C12) ester. Neutral lipase activity was almost 10% higher when 50 mm potassium phosphate buffer pH 7.8 was used instead of 0.01 m citrate/sodium phosphate buffer pH 7.2. Both assays were very sensitive: neutral lipase and phospholipase activities were detectable at a minimum protein concentration in the digesta of 0.04 and 1.25 mg/ml, respectively. When the variations in lipolytic activities with gut segment and with size of fish were examined neutral lipase activity was always found to be higher in the hindgut and rectum segments than in the foregut. Although phospholipase activity was also found to be highest in the hindgut of the largest fish examined (av. wt. 182.3g), in fish of average weight 8g fish the activity was similar in all three segments. In the digesta from the whole gut of smaller fish (av. wt. 0.2, 0.6 and 1.43g) neutral lipase and phospholipase activities increased with increasing body mass when expressed as per ml of digesta. It is concluded that fluorescence-based assays are applicable to the study of lipid digestion in fish of different size.

Lipid digestion in turbot (Scophthalmus maximus). I: Lipid class and fatty acid composition of digesta from different segments of the digestive tract

The lipid content and lipid composition of digesta from the stomach, foregut, hindgut and rectum of juvenile turbot fed a commercial diet were determined in order to examine the process of lipid digestion in this species. The moisture content of the digesta increased along the digestive tract from 71.5% in the stomach to 89.6% in the rectum. The lipid content of the digesta increased initially from 15.7% of the dry weight in the stomach to 36.1% in the foregut but thereafter decreased through 23.2% in the hindgut to 9.1% in the rectum. The proportion of triacylglycerols (TAG) in the total lipid of the digesta decreased from 63% in the stomach to 17.4% in the rectum whereas that of free fatty acids (FFA) increased from 10016 to 48.9%. The highest proportions of monoacylglycerols (MAG), diacylglycerols (DAG) and most phospholipids were observed in the lipid of the hindgut digesta. In addition, a fall in levels of neutral and phospholipid classes as digesta moved from hindgut to rectum signified absorption.Analysis of the fatty acid composition of the lipid classes TAG, DAG and MAG suggest a polyunsaturated fatty acid specificity for hydrolysis may exist. Saturated, monounsaturated and polyunsaturated fatty acids (PUFA) accounted for 17.9%, 45.4% and 37.0%, respectively of the FFA present in the foregut whereas the corresponding values for the rectum were 32.6%, 51.9% and 16.3%. Overall, the results suggest a PUFA specifity for hydrolysis may exist alongside the positional non-specific lipolytic activity associated with the hindgut regions of the digestive tract of turbot and that PUFA, released by lipolysis are more effectively absorbed from the digesta than monounsaturated and saturated fatty acids.

Lipids of arctic charr,Salvelinus alpinus (L.) I. Dietary induced changes in lipid class and fatty acid composition

Arctic charr (Salvelinus alpinus L.) were fed either a commercial diet or six experimental test diets containing coconut oil and different polyunsaturated fatty acids (PUFA) at a level of 1% by dry weight. Best growth rates were observed with the commercial diet, worst with diet containing coconut oil with no PUFA. An increase in hepatic lipid, hepatic sterol esters and muscular moisture content, and a decrease in muscular lipid was generally found in fish fed the test diets compared to those maintained on the commercial diet.Phosphatidylcholine was the dominant polar lipid (PL) class in all tissues examined. Extensive modification of dietary saturated fatty acids into 18:1 (n-9) was observed in tissue triacylglycerols (TAG) of fish fed test diets. No changes occurred with the commercial diet.Dietary PUFA were essentially incorporated unchanged into tissue TAG of all fish in the present study. PUFA composition of hepatic phospholipids was significantly influenced by that contained in the diets. However both 18:2 (n-6) and 18:3 (n-3) in the test diets were extensively elongated and desaturated prior to incorporation into PL. The (n-9) PUFA content was always higher in liver of fish fed the test diets. When 18:2 (n-6) and 18:3 (n-3) were supplied together, the level of (n-3) PUFA exceeded those of (n-6) PUFA. Muscle PL were less influenced by diet than liver. In muscle (n-3) PUFA were always the predominant PUFA irrespective of diet. Only low amounts of (n-9) PUFA were found. It is suggested that (n-3) PUFA are the prime essential fatty acids for Arctic charr, and that they are used in preference to (n-6) PUFA for elongation, desaturation and incorporation into PL. The results suggest that the quantitative requirement of Arctic charr for EFA is may be higher than that of other salmonids.

The uptake and esterification of radiolabelled fatty acids by enterocytes isolated from rainbow trout (Oncorhynchus mykiss)

To study the intestinal fatty acid absorption in fish in vitro, enterocytes were isolated from the intestine of rainbow trout and incubated with an equimolar mixture of seven fatty acids [16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3] in which the component carrying a radioactive label was varied. The fatty acid mixture was presented in the form of micelles formed by sonication with sodium taurocholate. Control studies showed that the presence of sodium taurocholate in the incubation medium caused an immediate 23% increase in the mortality of the cells but the remaining cells were viable. The effect of the bile salt on cellular permeability was evident at longer exposure periods. The proportions of 14C-labelled 20:5n-3 and 22:6n-3 present as monomeric fatty acids in the micellar solutions were higher than those of 16:0, 18:1n-9, 18:2n-6, 18:3n-3 and 20:4n-6. The rates of uptake of 14C-labelled 20:4n-6, 20:5n-3 and 22:6n-3 by enterocytes were significantly lower than those of the other fatty acid substrates over the first minute of incubation but no significant differences in uptake rate between fatty acids were obvious over a 15 min incubation period. Notable differences were observed between substrates in their distribution pattern in enterocyte lipid classes. Although most of the radioactivity from all radiolabelled substrates was recovered in triacylglycerols, the amounts of 14C- labelled 16:0, 20:4n-6, 20:5n-3 and 22:6n-3 recovered in the polar lipid fraction were higher than those of 14C-labelled C18 substrates, particularly after 15 min. Conversely, the initial esterification rates of 18:1n-9, 18:2n-6 and 18:3n-3 into triacylglycerols were significantly higher than those of 20:4n-6, 20:5n-3 and 22:6n-3. It is concluded that isolated enterocytes can be used for the study of the mechanism of intestinal fatty acid absorption in fish.

Lipid digestion in turbot (Scophthalmus maximus): in-vivo and in-vitro studies of the lipolytic activity in various segments of the digestive tract

In-vivo and in-vitro studies were carried out to evaluate lipolytic activity in the gut of juvenile turbot. The in-vivo study examined total lipid (% DW digesta), neutral and phospholipid classes (% total lipid, mg g−1 DW digesta) and the fatty acid compostion of the neutral lipid classes (% wt. of lipid class), as a function of hydrolysis, in the digesta of the stomach, foregut, hindgut and rectum of 50–60 g juvenile turbot. In contrast with earlier investigations, this study found the highest lipolytic activity and absorption occurring in the hindgut and rectum. In addition, various fatty acid groups showed differential uptake in this region which can be expressed, in order of increasing absorption, as saturates < monoenes < polyunsaturates. The in-vitro study investigated the lipolytic ability of digesta from different segments of the turbot digestive tract to hydrolyse the 14C-labelled lipid substrates: triacylglycerol (TAG), cholesterol ester (CE) and phosphatidylcholine (PC). Consistent with findings in the in-vivo analysis, the pattern of hydrolysis was repeated in all substrates tested and can be expressed in order of increasing hydrolytic activity as stomach < foregut < hindgut < rectum. Moreover, both studies suggested a non-specific lipolysis and phospholipid hydrolysis occurring in the posterior region of the gut particularly the rectum. The combined results of these studies concluded that the major site for lipolysis and absorption in juvenile turbot resides in the hindgut-rectum region. Future research should focus on the potential contribution of highly lipolytic bacteria in the posterior gut, such as Vibrio spp., which have been shown to effectively hydrolyse fish oils.

Lipid composition of the pineal organ from rainbow trout (Oncorhynchus mykiss).

The lipid composition of the pineal organ from the rainbow trout (Oncorhynchus mykiss) was determined to establish whether the involvement of this organ in the control of circadian rhythms is reflected by specific adaptations of lipid composition. Lipid comprised 4.9% of the tissue wet weight and triacylglycerols were the major lipid class present (47% of total lipid). Phosphatidylcholine (PC) was the principal polar lipid, and smaller proportions of other phospholipids and cholesterol were also present. Plasmalogens contributed 11% of the ethanolamine glycerophospholipids (EGP). No cerebrosides were detected. The fatty acid composition of triacylglycerols was generally similar to that of total lipids in which saturated, monounsaturated and polyunsaturated fatty acids (PUFA) were present in almost equal proportions. Each of the polar lipid classes had a specific fatty acid composition. With the exception of phosphatidylinositol (PI), in which 20∶4n−6 comprised 27.4% of the total fatty acids, 22∶6n−3 was the principal PUFA in all lipid classes. The proportion of 20∶5n−3 never exceeded 6.0% of the fatty acids in any lipid class. The predominant molecular species of PC were 16∶0/22∶6n−3 and 16∶0/18∶1, which accounted for 33.2 and 28.5%, respectively, of the total molecular species of this phospholipid. Phosphatidylethanolamine (PE) contained the highest level of di-22∶6n−3 (13.0%) of any phospholipid. There was also 4.9% of this molecular species in phosphatidylserine (PS) and 4.1% in PC. In PE, the species 16∶0/22∶6, 18∶1/22∶6 and 18∶0/22∶6 totalled 45.1%, while in PS 18∶0/22∶6 accounted for 43.9% of the total molecular species. The most abundant molecular species of PI was 18∶0/20∶4n−6 (37.8%). The lipid composition of the pineal organ of trout, and particularly the molecular species composition of PI, is more similar to the composition of the retina than that of the brain.

Modification of odd-chain length unsaturated fatty acids by hepatocytes of rainbow trout (Oncorhynchus mykiss) fed diets containing fish oil or olive oil

Hepatocytes isolated from rainbow trout fed on diets containing either fish oil or olive oil were incubated with individual odd-chain length unsaturated fatty acids (19∶1n−9, 19∶2n−6, 19∶3n−3, 21∶2n−6, 21∶3n−6, 21∶4n−6, 21∶3n−3, and 21∶5n−3) to examine whether these fatty acids were substrates for modification by desaturation and elongation. All odd-chain length fatty acids were readily assimilated into the lipids of hepatocytes from both dietary groups of fish, but their conversion to longer-chain, more unsaturated derivatives was more pronounced with cells from trout fed olive oil. Thus, the conversion of 19∶2n−6 and 21∶2n−6 to 21∶3n−6 and 21∶4n−6, and of 19∶3n−3 to 21∶4n−3 and 21∶5n−3, was most obvious in cells from the olive oil group, as was the conversion of 21∶3n−6 and 21∶3n−3 to 21∶4n−6 and 21∶4n−3, respectively. Elongation of 19∶1n−9 to 21∶1n−9 and 23∶1n−9 occurred in cells from both groups. No 23∶6n−3 was detectable as a product of 19∶3n−3 or 21∶3n−3. However, this fatty acid was a major product formed by cells from fish fed olive oil presented with 21∶5n−3. Cells from both groups of fish incorporated 21∶4n−6 and 21∶5n−3 into their lipids largely without modification but chain-shortened around 40, 23, and 19% of the incorporated 21∶2n−6, 21∶3n−3, and 19∶1n−9, respectively. The results demonstrate that odd-chain length unsaturated fatty acids can act as substrates for the desaturation, elongation, and chain-shortening systems of trout hepatocytes.

Lipid digestion in turbot (Scopthalmus maximus) 11: Lipolysis in vitro of 14C-labelled triacylglycerol, cholesterol ester and phosphatidylcholine by digesta from different segments of the digestive tract

Preparations of digesta from the stomach, foregut, hindgut and rectum of turbot (Scophthalmus maximus) were assayed for the ability to hydrolyse glycerol tri[1-14C]oleate (14C-TAG), 1,2-di[1-14C]palmitoyl L-3-phosphatidylcholine (14C-PC) and cholesterol [1-14]oleate (14C-CE) over 1,2,3 and 17h. In the assay of foregut digesta with 14C-TAG substrate, 37.8% of the total radioactivity was found in the FFA class after the first hour of incubation. This value increased to a maximum of 68.5% of the available label after 17h incubation. Over the same time the proportion of radioactivity in diacylglycerols (DAG, 31.6%–7.4%) decreased while that in the monoacylglycerols increased (MAG, 14.0%–22.3%). In assays of digesta from the hindgut and rectum, after 1 h of incubation, the proportion of radioactivity recovered in FFA represented 64.9% and 74.8%, respectively, whereas the proportions in both DAG and MAG decreased with incubation time. Similarly to 14C-TAG, the highest rate of lipolytic hydrolysis of 14C-CE occurred in digesta from the posterior digestive tract where the proportions of radioactivity recovered in FFA of the hindgut (50.0%) and rectum (81.9%) preparations were substantially higher than those of the stomach (3.5%) and the foregut (14.4%) after 1h. With 14C-PC as substrate the levels of radiolabelled FFA in both the foregut and the hindgut (2.4% and 7.6%, respectively) were markedly lower than the 37.5% in the rectum. The results suggest that the posterior digestive tract is very active in non-specific and phospholipid lipolysis and a region where the major part of lipid digestion takes place.