Lipid Untrition And Metabolism In Common Carp (Cyprinus Carpio L.) And Atlantic Salmon (Salmo Salar L.)

Common carp (Cyprinus carpio L.) is omninorous fish and is traditionally edible fresh water fish in China. Atlantic salmon (Salmo salar L.) is carnivorous fish living in sea water. They are all high quality protein and essential fatty acid resources for human being. With the finding that fish oil, being rich in highly unsaturated fatty acid (HUFA), is useful in preventing human being from cardiovascular and cerebrovascular disease, which induced the production of health food of fish oil. Oil is also necessary for growth and reproduction of fish and fish oil traditionally was added in their feeds in providing essential fatty acid and energy. However the effect of fish oil and HUFA on the nutrition and healthy aquculture (e.g. Anti-oxidation system), of fish, especially fresh water fish, has not been sufficiently elucidated. In the same time, the fact that fish stocks in the world are limited induces the limited supplement of fish oil and stimulates the price of fish oil, which further influence the sustainable development of the aqua-feeds, especially of the feeds of Atlantic salmon which requires 30 % of adding of fish oil. Vegetable oil is cheaper and more richful than fish oil then the substitution of fish oil with vegetable oil has been emphasized in aquaculture feeds. However the fatty acid composition of fish oil and vegetable oil is different, for example fish oil is rich in HUFA but vegetable oil is scarce of it, which probably influences the nutrion and metabolism of oil in fish and limited dataes were given to shed light on it. The present experiments were carried out to understand the nutrition and metabolism of fish oil and HUFA, especiall in common carp and also to clarify the possibility of replacing dietary fish oil with vegetable oil in Atlantic salmon feeds.1. Influence of fish oil on growth and lipid metabolism in Common carp (Cyprinus carpio L.)In order to examine the influence of fish oil and the HUFA (Highly unsaturated fatty acid) on growth and lipid metabolism in common carp (Cyprinus carpio) a dietary trial and a HUFA orally administration trial were conducted in the present study. In the front trial, semi-purified diets supplemented with fish oil (FO) and soybean oil (VO) were used and the indexes of growth and the activity of lipid metabolic enzymes in hepatopancreas were assayed. In second trial, hepatopancreas were sampled after fish being orally administered with HUFA and the activity of malate dehydrogenase (MDH) was assayed. Dietary oil sources had no impact on feed conversion ratio, absolute growth rate and relative growth rate. Common carp in FO group had significantly higher intraperitoneal fat body (IPF) ratio than that in VO group. Dietary oil sources had no significant effect on condition factor, gutted body ratio, carcass ratio, muscle ratio, hepatosomatic index and relative intestine length. Common carp in FO group than that in VO group had lower activity of MDH, lipoprotein lipase (LPL) and glucose-6-phosphate dehydrogenase (G-6-P-DH). This was further certified by the lower activity of MDH in HUFA group at 2 h and 4 h in HUFA administration study. This suggested that common carp can utilize fish oil and vegetable oil for growth equally well although the lipid metabolism was affected by dietary oil sources.2. Effect of DHA on anti-oxidation capacity in Common carp (Cyprinus carpio.L)To study the effect of DHA (22:6n-3, Docosahexaenoic acid) on anti-oxidation capacity in Common carp, DHA orally administration study, DHA long term (74 d) dietary study and incubation of hepatocytes with DHA study were carried on. The total anti-oxidation capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione S-transferase (GST), catalase (CAT), maleic dialdehyde (MDA) of hepatopancreas or hepatocytes were determined respectively. T-AOC in both DHA and control group increased before 4 h of post-administration then declined till 32 h. T-AOC in DHA group was higher than that in control group especially at 32 h time point of post-administration indicating that common carp endured oxidative stress and the anti-oxidation capacity of hepatopancreas was induced to increase. MDA, CAT, SOD and T-AOC of the hepatopancreas and SOD as well as T-AOC of the serum were not significantly different between DHA and control group except that GST of serum in DHA group was significantly lower than that in control group in 12 % lipid level but not in 6 % lipid level. The dietary trial suggests that common carp is acclimatized to the oxidative stress caused by the DHA in long term feeding of DHA. MDA in both DHA and control group increased in the beginning of incubation with hepatocytes then decreased swiftly. MDA decreased not so fast in DHA group as that in control group, therefore MDA in DHA group was significantly higher when the cells were incubated with DHA for 2, 4 and 8 h but not for 0.5 or 1 h. T-SOD was also decreased with longer incubation time in both of the two groups and there was no significant difference between them except at 4 h incubation where T-SOD was significantly higher in DHA group compared with control. The in vitro trial suggests that hepatocytes incubated with DHA experience higher oxidative stress and the anti-oxidation capacity of hepatocytes is therefore induced to increase.3. Uptake and intracellular metabolism of long chain fatty acids in Atlantic salmon (Salmo salar L.) hepatocytes To elucidate if the trans-membrane uptake of fatty acid was protein-mediated, the uptake of oleic acid (OA; 18:1n-9) was investigated in vitro in Atlantic salmon (Salmo salar L.) hepatocytes. Firstly, to find the optimal time of OA incubation hepatocytes cultured overnight was incubated with 61μM OA for 0.5, 1, 2, 4, 6, 12 and 24 h respectively (0.2μCi/flask). Secondly, to establish the OA incubation concentration for optimal OA uptake the hepatocytes were incubated with 37.5, 75, 150, 300 and 600μM OA for 2 h with a fixed molar ratio between unlabelled and radiolabelled OA of 30:1. OA uptake revealed saturation kinetics with Km values of 47μM and Vmax values of 2.3 nmol h-1 million cells-1. To identify whether trans-membrane OA uptake in hepatocytes was mainly passive or protein mediated, hepatocytes were pre-incubated with membrane protein inhibitors, cyclodextrin, phloretin, diisothiocyanodisulfonic acid (DIDS), sulfo-N-succinimidyl 4-maleimido-oleic acid ester (SSO) and sulfo-N-succinimidyl 4-maleimido-butyric acid ester (SSMB). The OA uptake was significantly reduced by inhibitors with phloretin giving the highest inhibition (28%) and SSO the lowest (8%). While the other inhibitors, cyclodextrin, DIDS and SSMB, showed the intermediate inhibition between 9.8 and 14.5 %. Our results suggest that trans-membrane OA uptake in Atlantic salmon hepatocytes is due to both saturable and inhibitable protein mediated uptake, as well as passive uptake processes. To investigate the uptake and intracellular metabolism of 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3, Atlantic salmon (Salmo salar L.) hepatocytes incubated overnight were firstly pretreated with known membrane protein inhibitors, phloretin, diisothiocyanodisulfonic acid (DIDS), sulfo-N-succinimidyl 4-maleimido-oleic acid ester (SSO), for 0.5h, then 37.5μM of these fatty acids were added respectively for another 2 hours incubation (0.24- 0.36μCi/flask). Hepatocytes (controls) incubated with fatty acids but without inhibitors were run concurrently. Radioactivity recovered in cells, cellular lipid classes andβ-oxidation products were measured. 20:5n-3 was highest taken up (15 %), 22:6n-3 being the intermediate (13 %), then 18:1n-9, 18:2n-6 and 18:3n-3 was lowest taken up (10%~11 %). Phloretin had the highest inhibition on uptake of 20:5n-3 (67 %) and lowest inhibition on 18:1n-9 (13 %), having the intermediate inhibition on 18:3n-3, 22:6n-3 and 18:2n-6 (46 %, 35 %, and 25 %, respectively). Uptake of 18:2n-6 and 18:3n-3 but not of the other fatty acids was significantly decreased in the presence of DIDS. SSO showed no inhibition on these fatty acids uptake. 18:3n-3 was highestβ-oxidised (7 % of uptake), 20:5n-3, 22:6n-3 and 18:2n-6 being the intermediate (about 3 %), and 18:1n-9 was leastβ-oxidised (<1 %). The intracellular free fatty acid (FFA) distribution was highest with 18:3n-3 (68 % of cellular lipids), followed with 18:1n-9, 18:2n-6 and 20:5n-3 (61-67 %), and it was lowest with 22:6n-3 (50 %). The order of fatty acids incorporation into phospholipids (PL) was reverse to that of FFA, indicating that 22:6n-3 was most incorporated into PL (38 %), followed by 20:5n-3 (27 %), then lowest by 18:2n-6, 18:1n-9 and 18:3n-3 (20 %~23 %). No difference was found in the incorporation of these FAs into neutral lipids.4. Replacing dietary fish oil with vegetable oil affect oleic acid uptake and metabolism in hepatocytes of Atlantic salmon (Salmo salar L.)The aim was to investigate how uptake and metabolism of 18:1n-9 (oleic acid; OA) in Atlantic salmon (Salmo salar L.) hepatocyte is affected by dietary oil sources. Atlantic salmon post smolt was fed diets containing either 100% fish oil (FO) or vegetable oil (VO) for 5 months to produce hepatocytes with typical FO and VO fatty acid composition. Then OA uptake and metabolism in isolated hepatocytes were studied by incubating with 37.5μM OA for 2 h (0.3μCi/flask) with and without (control) membrane protein inhibitors; phloretin, sulfo-N-succinimidyl 4-maleimido-oleic acid ester, diisothiocyanodisulfonic acid, and sulfo-N-succinimidyl 4-maleimido-butyric acid ester. OA uptake was not different in control cells between FO and VO fed fish. The effect of adding membrane protein inhibitors, however, was significantly different with OA uptake being inhibited in VO but not in FO hepatocytes. OA incorporation into cellular neutral lipids was significantly lower and the recovery in cellularβ-oxidation products was significantly higher in VO compared to FO hepatocytes. Intracellular free OA and OA incorporation into phospholipids was not different between the two groups. Inhibitors did not affect OA cellular distribution. The mRNA expression of FAT/CD36, fatty acid binding protein 3 and 10 (FABP3 and FABP10),Δ6 desaturase andΔ5 desaturase were significantly higher in VO than in FO hepatocytes, whereas the expression of FATP, FABP10 and carnitine palmitoyl transferase I (CPT I) was not different between FO and VO hepatocytes. The expression of genes related to stress responses and antioxidant defence; glutathione peroxidase 4, heat shock protein 70, metallothionein and ubiquitin were significantly higher in VO than in FO hepatocytes, however the incubation with inhibitors did not affect the expression these genes. Overall, the results indicate that hepatocyte fatty acid composition is involved in the regulation of fatty acid uptake and intracellular fatty acid metabolism whereas inhibition of OA uptake did not influence intracellular metabolism.