Aquaculture nutrition, tập 17, số 1, 2011

Aquaculture Nutrition 2011 17; Aquaculture Nutrition wishes all contributors a prosperous new year! We have experienced a positive year with further growth of the journal, but also challenges that need to be met adequately to keep up the positive trend Aquaculture Nutrition strengthened the editorial sta with Dr Brett Glencross (CSIRO Marine Research, Australia) in 2008 and Genevieve Corraze (INRA, Pole dếhydrobiologie, France) in 2009, replacing Dr Kim Jauncey (University of Stirling, UK) who stepped down as editor in 2009 Kim has been editor since the journal was launched in 1995 We would like to acknowledge him for his great eort and work for the journal during all these years, during which our journal grew from a quarterly to a bimonthly publication and from a cumbersome analog editorial handling to a fully electronic editorial treatment online Together with our publisher Wiley-Blackwell; thank you very much, Kim In 2011 Aquaculture Nutrition has renewed the editorial board We want to thank the retiring board members for their assistance in their extended period The editors welcome the new board members and look forward to a good and fruitful collaboration Each member has signed an agreement which state their duties and rights, illustrating that it is a demanding task adding to all their other daily working activities We truly appreciate their willingness to work for the journal in the coming years The editors of Aquaculture Nutrition realize that we are evaluated for all parts of our production, from review treatment times, overall time to publication, the product quality and nally the impact factor To fulll our aims and goals we are completely dependent on willing and scientic qualied referees among the aquaculture nutrition fraternity, as well as from other specialists The latter, is important since many scientists are taking to using modern tools with novel approaches and analytical techniques and sometimes this expands our need for technical support for the journalếs reviewing process Even though we experience the competition with specialized journals covering such specialized research, we want to remind aquaculture nutrition scientists that it is important to communicate your research to your major readerships and users, like in Aquaculture Nutrition With the introduction of a web based submission system, Aquaculture Nutrition has experienced a great increase in the ể 2011 Blackwell Publishing Ltd doi: 10.1111/j.1365-2095.2010.00844.x number of paper submissions, and also of varying scientic quality We realize that much of our capacity in the editorial oce goes to ltering and resubmission of manuscripts that not fulll the journals guidelines to authors We have introduced a system of warnings to authors that neglect the instructions and will exclude manuscripts from a third resubmission at this early stage To maintain our high scientic standard, and not at least prevent misuse of our reviewers with low quality manuscripts, we will be endeavoring to introduce even more strict submission procedures and standards in 2011 By following the guidelines, we envisage that our contributing authors will experience shorter production times and the standard of the journal elevate further and with that its impact factor increase too For unknown reasons, publications in Aquaculture Nutrition today have generally increased in their number of pages compared to earlier shorter and more condensed papers This seems not to reect any changes in amounts of presented data To counteract on this ễword richế trend and reduce the pressure on printed pages, Aquaculture Nutrition has in 2011 introduced a page charge on original research articles (not reviews) exceeding pages when in proof (an average 8-page article will have approximately 6200 words in text, gures or tables and 50 references) This reects the mean page number of the journal over a time period Due to the cost-pressures of publishing, Aquaculture Nutrition has not had the opportunity to increase the annual page allocation as planned As it stands, the authors publishing in Aquaculture Nutrition currently experience a long delay in the printing time of their papers Despite the earlyonline facility, where the papers are available in their nal form and with a DOI number on the journals home page on the internet, we recognize that this is not an acceptable situation for the journal We are, however pleased to inform our authors and readers that we will be taking action to get through this backlog of papers in very near future, and that the printing time will be normalized as soon as possible Rune Waagbứ Gro-Ingunn Hemre Genevieve Corraze Brett Glencross Aquaculture Nutrition 2011 17; 212 doi: 10.1111/j.1365-2095.2009.00698.x 1 1 1 Nutrition Laboratory, Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-sen University, Guangzhou, P.R China; Laboratory of Aquaculture Nutrition, College of Fisheries, Ocean University of China, Qingdao, P.R China KEY WORDS: grass carp, growth, herbivorous sh, ominivorous sh, protein-sparing eect, tilapia Two, 8-week feeding trials were conducted to compare protein-sparing capability of dietary lipid in herbivorous grass carp (Ctenopharyngodon idella) and omnivorous tilapia (Oreochomis niloticus ã O aureus) Utilizing a ã factorial design, experimental diets containing two levels of crude protein (380 and 250 g kg)1) and three levels of lipid (0, 40 and 100 g kg)1) were formulated for use in both feeding trials Growth performances showed better response of both sh fed 380 g kg)1 protein diet than those fed 250 g kg)1 protein diet Despite the dietary protein level, weight gain (WG), specic growth ratio (SGR), feed conversion ratio (FCR) and protein eciency ratio were much higher (P < 0.05) for grass carp fed 40 g kg)1 lipid diet than those fed 100 g kg)1 lipid diet; however, there were no signicant dierences in tilapia fed the two diets The feed intake of grass carp fed lipid-free diet was the lowest, but it tended to decrease with increase in dietary lipids in tilapia Lipid retention (LR) was negatively correlated with dietary lipid concentration of both sh Viscerosomatic index (VSI), hepatosomatic index (HSI), intraperitoneal fat ratio (IPF) and whole-body and liver lipid content positively correlated with dietary lipid concentration of both sh Plasma parameters and liver enzymes activities were also positively correlated with dietary lipid concentration of both sh Liver lipid contents were higher and enzymes activities were lower in grass carp when compared with tilapia These data suggested that there was no evidence of a protein-sparing eect of dietary lipids in grass carp Tilapia has relatively higher capacity to endure high dietary lipid level compared to grass carp Received 23 November 2008, accepted 25 February 2009 Correspondence: Yong-Jian Liu, Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-sen University, No 135 Xinếgang Xi Road, Guangzhou P.R China, 510275 Tel.: +86 20 8411 0789; fax: +86 20 8411 5896; E-mail: edls@mail.sysu.edu.cn A recent trend in the manufacture of aquafeed is to increase the lipid concentration in the diet Dietary lipids play a prominent role in sh nutrition as primary energy source and to provide essential fatty acids to maintain biological structures and normal functions of cell membranes (Sargent et al 1999) Increasing dietary lipid levels improves the diet eciency (Johnsen et al 1993; Peres & Ol va-Teles 1999a) by minimizing protein degradation (Beamish & Medland 1986) within certain limits, especially in carnivorous sh species However, there are also some drawbacks to increasing the lipid concentration in sh feed Viscera somatic index (VSI) (Jobling et al 1998; Rasmussen 2001) and viscera lipid concentration (Hillestad et al 1998; Rasmussen et al 2000) have been shown to increase with increasing dietary lipid concentration At present, the viscera portion of the sh is discarded as by-product during processing Therefore, the increase in VSI and viscera lipid concentration presents an economical concern for sh processors due to the increased volume of by-product that is usually discarded at a cost, and the waste of digestible energy from feed Another disadvantage is the potential for increased rate of lipid oxidation in feed and in sh, which could aect the health status of the ể 2009 Blackwell Publishing Ltd sh and the avours and nutritional qualities of sh llets (Chaiyapechara et al 2003) As protein represents the most expensive component of aquafeeds (Cho et al 2005; Craig & McLean 2005; Miller et al 2005), from an economic standpoint it is vitally important that protein be utilized for the synthesis of muscle tissue and not for metabolic energy (Williams et al 2003; Ozorio et al 2006) Protein sparing by non-protein energy sources has been documented in a wide range of species (Thoman et al 1999; Azevedo et al 2002), but protein sparing by lipid has been best documented in the salmonids (Refstie et al 2001; Azevedo et al 2002) The ability to utilize lipid rather than protein as an energy source can lead to a decreased loss of ingested protein by catabolism (Refstie et al 2001; Williams et al 2003), thereby potentially reducing nitrogenous waste input into culture systems (Miller et al 2005) As a typical herbivorous nsh without stomach, one of the major natural food organisms for grass carp (Ctenopharyngodon idella) is grass, for example, Hydrilla verticillata (Linn f.), Potamogeton malaianus Miq and so on (Ni & W 1999) Takeuchi et al (1991) reviewed the essential fatty acid requirement of grass carp and pointed out that the requirement of dietary n-6 and n-3 fatty acids was between and 10 g kg)1of the diet Lin (1991) also reviewed the nutrient requirements of grass carp and underlined from primary experiments that grass carp does not require large amounts of lipids Recently, re-evaluations of optimal dietary lipid level for grass carp showed that the energy requirement of grass carp was relatively lower than that of most carnivorous sh species, and the protein-sparing eect was eective up to 40 g kg)1 of lipid in the diet, while at 60 g kg)1 of dietary lipid was already sucient to negatively aect growth performance and body composition (Du et al 2005) This result highlights the peculiar nutritional requirements of grass carp, as most sh species, especially carnivorous sh, can easily utilize above 100 g kg)1 dietary lipid without any negative eects on growth (Arzel et al 1994; Luzzana et al 1994; Peres & Ol va-Teles 1999b) On the contrary, in tilapia, it has been suggested that protein sparing occurs when lipid levels are increased up to 60100 g kg)1 (Jauncey & Ross 1982) or even 180 g kg)1 (De Silva et al 1991) The aim of the present comparative studies was therefore to compare, from a better insight, the growth, feed utilization, body composition and digestive enzymes activities of herbivorous grass carp and omnivorous tilapia, both fed three graded levels of dietary lipid and two levels of dietary protein to estimate the possible protein-sparing capability of the two species Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Six diets were formulated to provide a ã factorial of crude protein (CP) (250 and 380 g kg)1) and lipid (0, 40 and 100 g kg)1), with three tank replicates of sh per treatment Changes in the dietary concentrations of CP and lipid were achieved by serial adjustment of casein (for protein) or a mixture of sh oil and corn oil (for lipid) at the expense of cellulose (Table 1) Diet ingredients were ground through 60-mesh size Distilled water and oil were added to the premixed dry ingredients and thoroughly mixed until homogenous in a Hobart-type mixer The 2-mm and 3-mm diameter pellets were wetextruded, dried overnight at 60 C in a forced draught oven, sealed in plastic bags and frozen stored ()18 C) until used The initial weight of grass carp and tilapia were 2.36 and 2.47 g, respectively Juvenile grass carp and tilapia were obtained from a local hatchery Prior to the present study, the sh were acclimated to a commercial diet for weeks After the acclimatization, sh were sorted by weight and absence of physical abnormalities into a uniform group The sh were randomly distributed to the experimental 200-l breglass tanks at an equal stocking rate of 25 sh per tank connected to a recirculation system Five of the remaining sh were sacriced to provide an estimate of initial whole-body chemical composition Photoperiod was held to a constant 12 : 12 h light dark cycle The sh were fed for 56 days with a daily ration of 50 g kg)1 of body weight divided into meals day)1 The culture tanks were cleaned weekly Water quality parameters were monitored daily between 10:00 and 17:00 h During the experimental period, temperature ranged 2830 C; dissolved oxygen was 7.77.8 mg L)1; total ammonianitrogen was 0.095 0.05 mg L)1; and pH was 7.9 0.09 At the termination of the 8-week feeding trail, sh in each tank were weighed and sampled for tissue analysis 24 h after the last feeding Nine sh from each tank were randomly collected for proximate analysis, three for analysis of whole-body composition and six for blood collection and to obtain weights of individual whole body, viscera, liver and mesenteric fat The liver were dissected and frozen immediately in liquid nitrogen and stored at )70 C until used and the plasma was separated by centrifugation and stored at )70 C until analysed Gross energy content was calculated using 23.6 kJ g)1 for protein, 39.5 kJ g)1 for lipid and 17.2 kJ g)1 for carbohydrate (NRC 1993) Crude protein, crude lipid and moisture in diets, liver and whole body were determined by standard methods (AOAC 1995) Moisture was determined by oven-drying at 105 C for 24 h; crude protein (N ã 6.25) was analysed by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Auto-analyser, Tecator, Sweden); crude lipid was determined by the etherextraction method using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden) The concentrations of plasma triacyglyceride and total cholesterol (CHO) were assayed by enzymatic procedure using automatic biochemical analyser and attached kit (Hitachi 7170; DAICHI, Tokyo, Japan) Lipase activity was determined by the method of Pan & Wang (1997), while the activity of alkaline phosphatase (AKP) was measured following the method of Bessey et al (1946) Enzyme-specic activities were expressed as micromoles of substrate hydrolysed per minute, per mg of protein (i.e., U mg protein)1) 30 C for lipase and 37 C for AKP The protein concentration of the supernatant solutions was determined by the biuret method, using bovine serum albumin as the standard The data were subjected to one- and two-way ANOVA to test the eects of dietary protein and lipid Dierences for all analyses were considered signicant at P < 0.05 The data are presented as mean SD of the replicate groups All analyses were conducted using SPSS Version 10.0 Grass carp Grass carp fed dierent protein and lipid levels showed the best response in weight gain (WG) (P < 0.05), specic growth ratio (SGR) (P < 0.05), feed conversion ratio (FCR) (P < 0.05), protein eciency ratio (PER) (P < 0.05), protein retention eciency (PR) (P < 0.05) and lipid retention eciency (LR) (P < 0.05) when fed diet with 40 g kg)1 lipid in each dietary protein level (Table 2) WG, SGR and FCR were improved for each dietary lipid level when dietary protein increased from 250 to 380 g kg)1, while the trend of PER and PR was opposite to them (Table 2) The feed intake of grass carp fed lipid-free diet was lower than those fed and 40 g kg)1 dietary lipids in each dietary protein level VSI of grass carp fed 100 g kg)1 lipid in the diet was signicantly higher (P < 0.05) than that fed and 40 g kg)1 dietary lipids in each dietary protein level Intraperitoneal fat ratio (IPF) signicantly (P < 0.05) increased with the increasing dietary lipid level in each dietary protein level There is also a positive correlation between hepatosomatic index (HSI) and dietary lipid in each dietary protein level Feed intake, WG, SGR, FCR, PER, PR, LR, HSI and IPF were signicantly (P < 0.05) impacted by dietary protein and lipid There was no signicant impact of dietary protein on VSI Tilapia No signicant dierences were found in WG, SGR, FCR and PER between dietary 40 g kg)1 lipid and 100 g kg)1 lipid level in each dietary protein level, and the values of them were much higher (P < 0.05) than those fed lipid-free diet (Table 3) PR signicantly (P < 0.05) increased with the increasing dietary lipid level in each dietary protein level LR of tilapia fed 40 g kg)1 lipid in the diet was signicantly higher (P < 0.05) than that fed 100 g kg)1 dietary lipids in each dietary protein level The feed intake tended to decrease with increase in dietary lipids VSI, HSI and IPF of tilapia fed 40 g kg)1 and 100 g kg)1 dietary lipids were signicant higher (P < 0.05) than that fed lipid-free diet in each dietary protein level WG, SGR, FCR, PER, PR, VSI, HSI and IPF were signicantly (P < 0.05) impacted by dietary protein and lipid Feed intake of tilapia was only signicantly (P < 0.05) impacted by dietary protein There was no signicant impact of dietary protein on LR Grass carp Whole-body and liver tissue compositions are shown in Table No signicant dierences were found in moisture of whole body between dierent treatments Higher moisture of liver was found in the sh fed lipid-free diet in each dietary protein Whole-body protein content was signicantly (P < 0.05) impacted by dietary protein Wholebody and liver lipid concentrations were signicantly (P < 0.05) impacted by dietary lipid, with increasing lipid levels with increasing dietary lipid Fish fed the diets containing 380 g kg)1 protein level had higher (P < 0.05) values than those fed 250 g kg)1 protein level for whole body lipid (66 and 58.7 g kg)1 wet weight, respectively) and liver lipid (241 and 207 g kg)1 wet weight, respectively) Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Table Formulation and composition of experimental diets (g kg)1 dry diet) Diet protein/lipid (g kg)1) 380/0 380/40 380/100 250/0 250/40 250/100 Casein Gelatin Cellulose Corn starch Fish oil Corn oil Vitamin mix1 Mineral mix2 others3 Proximate composition Moisture (g kg)1) Crude protein (g kg)1) Protein (E%)4 Crude lipid (g kg)1) Lipid (E%)5 Ash (g kg)1) Gross energy (KJ g)1) P:E (g MJ)1) 380 40 240 200 20 80 40 380 40 200 200 10 30 20 80 40 380 40 140 200 25 75 20 80 40 250 40 370 200 20 80 40 250 40 330 200 10 30 20 80 40 250 40 270 200 25 75 20 80 40 104 391 72.83 59 12.67 30.87 115 395 64.56 43 11.78 58 14.44 27.35 122 393 55.50 101 23.91 58 16.71 23.49 115 269 64.78 57 9.80 27.50 125 271 55.66 42 14.46 57 11.49 23.61 127 269 46.10 101 29.02 56 13.77 19.50 Vitamin mix (mg kg)1 of diet): thiamine, 50; riboflavin, 50; vitamin A, 25000 IU; vitamin E, 400; vitamin D3, 24000 IU; menadione, 40; pyridoxine HCl, 40; cyanocobalamin, 0.1; biotin, 6; calcium pantothenate, 100; folic acid, 15; niacin, 200; inositol, 2000; and cellulose was used as a carrier Mineral mix (g kg)1 diet): calcium biphosphate, 9.8; calcium lactate, 37.9; sodium chloride, 2.6; potassium sulfate, 13.1; potassium chloride, 5.3; ferrous sulfate, 0.9; ferric citrate, 3.1; magnesium sulfate, 3.5; zinc sulfate, 0.04; manganese sulfate, 0.03; cupric sulfate, 0.02; cobalt chloride, 0.03; potassium iodide, 0.002; and cellulose 42 Others (g kg)1 diet): Taurine, 5; Choline chloride (50%), 6; betaine, 4; Carboxymethy cellulose (CMC), 20; vitamin C, Percent of total energy derived by protein Percent of total energy derived by lipid Plasma CHO and TG of grass carp fed 100 g kg)1 lipid diet were higher than those fed and 40 g kg)1 lipid diets (Table 5) Plasma CHO and TG were not signicantly impacted by dietary protein level, but signicantly (P < 0.05) aected by dietary lipid Tilapia Whole-body and liver tissue compositions are shown in Table No signicant dierences were found in moisture of whole body and liver between dierent treatments Wholebody and liver protein levels were not signicantly impacted by dietary protein Whole-body and liver lipid concentrations were signicantly (P < 0.05) impacted by dietary lipid, with increasing lipid levels with increasing dietary lipid Fish fed the diets containing 380 g kg)1 protein level had higher (P < 0.05) values than those fed 250 g kg)1 protein level for whole-body lipid (68.4 and 65.8 g kg)1 wet weight, respectively) and liver lipid (108.6 and 90.8 g kg)1 wet weight, respectively) Plasma CHO and TG of sh fed 100 g kg)1 lipid diet were higher than those fed and 40 g kg)1 lipid diets (Table 5) Plasma CHO and TG were signicantly (P < 0.05) aected by dietary protein and lipid Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Grass carp The activities of lipase and AKP were signicantly (P < 0.05) increased with the increasing dietary lipid level in each dietary protein (Table 6) The activities of them were signicantly (P < 0.05) aected by dietary protein and lipid Tilapia The activities of lipase and AKP were signicantly (P < 0.05) increased with the increasing dietary lipid level in each dietary protein (Table 6) The activities of them were signicantly (P < 0.05) aected by dietary protein and lipid Growth rates of both tilapia and grass carp (around 2.4 g of initial weight) as found here are in the high range of values, comparable to those reported in tilapia (around 1.34 g of initial weight) by Chou & Shiau (1996) and in grass carp (around 6.52 g of initial weight) by Du et al (2005) It is noticeable that better growth rates were shown of sh fed diet containing 100 g kg)1 lipids than those fed a lipid-free diet Table Growth, feed utilization and biometric parameters of grass carp fed with experimental diets varying in protein and lipid concentration Two-way ANOVA (P < 0.05) Protein/lipid (g kg)1) IBW FBW2 Survival Feed intake (g fish)1) WG3 SGR4 FCR5 PER6 PR7 LR8 VSI9 HSI10 IPF11 380/0 2.37 9.46 100 13.2 300 2.48 1.86 1.38 19.9 8.92 1.96 1.13 0.03 0.29c 0.44b 16.2c 0.07c 0.03b 0.02a 0.22a 0.71a 0.34ab 0.14a 380/40 380/100 250/0 2.35 12.3 98.7 14.8 423 2.95 1.50 1.69 24.1 112 9.40 2.36 1.89 2.36 10.5 100 14.2 343 2.66 1.76 1.45 20.7 49.1 10.3 2.47 2.75 2.37 7.90 100 12.4 233 2.15 2.24 1.66 22.9 9.49 1.79 1.39 0.02 0.18e 0.20d 8.59e 0.03e 0.01a 0.02b 0.54c 7.78d 0.58a 0.25c 0.32b 0.02 0.27d 0.28cd 10.5d 0.04d 0.03b 0.03a 0.60ab 2.80b 1.14b 0.25c 0.55d 0.01 0.27a 0.15a 10.9a 0.06a 0.09c 0.07b 1.46bc 1.51a 0.34a 0.22a 250/40 250/100 Protein Lipid PãL 2.37 9.81 96 13.8 313 2.53 1.89 1.96 27.2 76.9 9.56 1.84 2.25 2.36 8.66 97.3 13.3 267 2.32 2.15 1.72 21.7 36.8 10.4 2.05 2.94 0.000 0.306 0.000 0.000 0.000 0.000 0.000 0.003 0.000 0.171 0.000 0.000 0.000 0.449 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 0.000 0.062 0.756 0.861 0.046 0.384 0.999 0.99 0.4 0.008 0.591 0.037 0.008 0.01 0.48c 0.41bc 18.8c 0.08c 0.12b 0.13c 1.50d 6.43c 1.19a 0.16a 0.31c 0.01 0.30b 0.56b 13.5b 0.07b 0.23c 0.18b 2.59abc 4.09a 0.86b 0.37b 0.61d Values represents means SD of three replicates and values with the same column with different letters were significantly different (P < 0.05, one-way ANOVA) IBW, initial mean body weight (g) FBW, final mean body weight (g) Weight gain = 100 ã (final body weight ) initial body weight)/initial body weight Specific growth ratio = 100 ã ln (final weight /initial weight)/days of the experiment Feed conversion ratio = g dry feed consumed/g wet weight gain Protein efficiency ratio = fish wet weight gain/protein intake Protein retention efficiency = 100 ã (final body protein initial body protein)/total protein fed Lipid retention efficiency = 100 ã (final body lipid initial body lipid)/total lipid fed Viscerosomatic index = 100 ã (viscera weight /whole body weight) 10 Hepatosomatic index = 100 ã (liver weight /whole body weight) 11 Intraperitoneal fat ratio = 100 ã (IPF weight /whole body weight) both in herbivorous grass carp and omnivorous tilapia Du et al (2005) reported that grass carp fed a lipid-free diet showed higher growth than those fed diets of 100 and 120 g kg)1 lipids Chou & Shiau (1996) reported that WG of hybrid tilapia fed the lipid-free diet was comparable to those fed the lipid containing diets for the rst weeks followed by a reduction for the rest of the study period, suggesting that the decrease in growth may be due to a deciency in essential fatty acids But in this experiment, from the high survival and low HSI values of sh fed lipid-free diets, there were no obvious essential fatty acid deciency symptoms in both sh fed a lipid-free diet, although the growth rates are comparatively lower than others Chou & Shiau (1996) reported that the optimal dietary lipid for maximal growth of hybrid tilapia is about 120 g kg)1, but WG of the sh in the 50 g kg)1 lipid group was not signicantly dierent from those in the 10 and 150 g kg)1 lipid groups, suggesting that 50 g kg)1 dietary lipid may meet the minimum lipid requirement for the sh In the present study, the growth performance and FCR of grass carp fed diet containing 40 g kg)1 lipid were better than those in 100 g kg)1 lipid group, but there were no signicant dierences between the two groups in tilapia Similar results as grass carp that the decline in growth performance and feed utilization with increasing dietary lipids above 40 g kg)1 lipid have been reported in trout (Regost et al 2001), salmon (Silverstein et al 1999) and carp (Murai et al 1985) Physiological mechanisms related to maintaining overall energy status and control of body weight are often invoked in sh (Cho & Kaushik 1990) as in mammals (Forbes 1988) Kaushik et al found that in rainbow trout, voluntary feed intake was controlled by the availability of energy (Kaushik et al 1981; Kaushik & Luquet 1984) De Silva & Gunasekera (1989) showed that the net intake of protein and energy in Oreochromis niloticus fry was aected by the dietary protein content Inuence of dietary protein level on feed intake has also been shown for channel catsh (Page & Andrews 1973) The results of the present study indicated that feed intake of sh fed 380 g kg)1 dietary protein were higher than those of sh fed 250 g kg)1 dietary protein both in grass carp and tilapia De Silva et al (1991) also reported that feed intake was higher when red tilapia was fed higher dietary protein In the present study, feed intake of tilapia fed lipid-free diet was higher than the lipid-containing diets at each dietary protein Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Table Growth, feed utilization and biometric parameters of tilapia fed with experimental diets varying in protein and lipid concentration Two-way ANOVA (P < 0.05) Protein/lipid (g kg)1) IBW FBW2 Survival Feed intake (g fish)1) WG3 SGR4 FCR5 PER6 PR7 LR8 VSI9 HSI10 IPF11 380/0 2.48 28.9 96.0 50.8 1068 4.38 1.92 1.35 26.7 9.04 1.72 0.45 0.04 2.13c 10.9c 96.3c 0.15c 0.26c 0.20a 4.50a 0.50b 0.21b 0.18a 380/40 380/100 250/0 2.47 35.5 97.3 43.2 1338 4.76 1.31 1.93 37.1 142 9.20 2.08 1.10 2.48 33.0 94.7 40.2 1233 4.62 1.39 2.18 41.3 71.4 11.15 2.16 2.65 2.46 21.9 96.0 29.3 790 3.90 1.51 2.48 46.0 8.32 1.53 0.25 0.02 2.43e 1.67bc 106d 0.13d 0.04ab 0.07b 3.02b 7.06c 0.13b 0.26d 0.32b 0.02 0.92d 10.9b 44.4d 0.06d 0.03ab 0.15bc 0.58bc 4.33b 0.52c 0.25d 0.90c 0.03 0.27a 3.50a 9.53a 0.02a 0.17b 0.28c 7.75c 0.38a 0.11a 0.13a 250/40 250/100 Protein Lipid PãL 2.47 25.8 97.3 28.5 944 4.19 1.23 3.03 56.0 143 8.97 1.74 1.02 2.45 24.9 96.0 27.7 917 4.14 1.41 3.16 57.8 55.7 9.46 1.93 1.30 0.000 0.801 0.000 0.000 0.000 0.036 0.000 0.000 0.087 0.000 0.000 0.000 0.000 0.638 0.126 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.277 0.936 0.289 0.289 0.636 0.057 0.94 0.808 0.045 0.000 0.280 0.000 0.02 0.40b 2.40a 15.5b 0.03b 0.11a 0.28d 3.29d 9.42c 0.87b 0.10b 0.33b 0.02 0.47b 1.72a 17.0b 0.03b 0.08ab 0.16d 1.26d 1.10a 0.91b 0.11c 0.31b Values represents means SD of three replicates and values with the same column with different letters were significantly different (P < 0.05, one-way ANOVA) IBW, initial mean body weight (g) FBW, final mean body weight (g) Weight gain = 100 ã (final body weight ) initial body weight)/initial body weight Specific growth ratio = 100 ã ln(final weight /initial weight)/days of the experiment Feed conversion ratio = g dry feed consumed/g wet weight gain Protein efficiency ratio = fish wet weight gain/protein intake Protein retention efficiency = 100 ã (final body protein initial body protein)/total protein fed Lipid retention efficiency = 100 ã (final body lipid initial body lipid)/total lipid fed Viscerosomatic index = 100 ã (viscera weight /whole body weight) 10 Hepatosomatic index = 100 ã (liver weight /whole body weight) 11 Intraperitoneal fat ratio = 100 ã (IPF weight /whole body weight) level, which is opposite to grass carp The similar results to tilapia that feed intake tended to decrease with increase in dietary lipids for each protein level were also reported in gilthead seabream (Santinha et al 1999) and red porgy ngerlings (Schuchardt et al 2008) Gelineau et al (2001) reported that in hand-fed rainbow trout, the compensation of low dietary lipid levels (17.1 kJ g)1) by an increase in feed intake was not sucient to reach the same amount of energy intake as with a high dietary lipid content (21.4 kJ g)1), and this resulted in a dierence in growth performance A lack of signicant relation between dietary energy content and demand-feeding activity was found by Alanaăraă (1994) in rainbow trout and Alanaăraă & Kiessling (1996) in Arctic charr Gelineau et al (2001) observed that signicant changes in feeding activity were only detected when the dierence between dietary digestible energy contents was at least 2.2 kJ g)1, and in the present study there was a dierence of ca 4.04 kJ g)1 and 3.97 kJ g)1 between g kg)1 lipid diets and 100 g kg)1 lipid diets at each dietary protein level, respectively So, maybe in the present study, the compensation of lipid-free diets (12.67 and 9.80 kJ g)1) by a little increase in feed intake in tilapia was not sucient to reach Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd the same amount of energy intake as with 100 g kg)1 lipid diets (16.71 and 13.77 kJ g)1) The P/E ratios for optimum growth of several sh species ranged from 19 to 27 g MJ)1 (NRC 1993) The maximum growth rate was obtained from the diets containing 380 g kg)1 protein with 40 g kg)1 lipid and 380 g kg)1 protein with 100 g kg)1 lipid of tilapia in this study, corresponding to a P/E ratio of 27.35 and 23.49 g MJ)1, respectively and the maximum growth rate was obtained from the diets containing 380 g kg)1 protein with 40 g kg)1 lipid of grass carp, corresponding to a P/E ratio of 27.35 g MJ)1 Garling & Wilson (1976) reported that adequate levels of protein and energy in the diets must be carefully considered when optimum P/E ratio was estimated From the growth performances of the present study, higher dietary protein level in spite of 100 g kg)1 lipid level was good for the growth of both grass carp and tilapia when compared to dietary 250 g kg)1 protein The results in the present study showed that the increase of percentage of total energy derived by lipid from 11.78 to 23.91 and from 14.46 to 29.02 in each dietary protein level did not harm to growth and feed utilization of tilapia but did harm to those of grass carp After the initial works of Lee & Table Body and tissue proximate composition in juvenile grass carp and tilapia fed with experimental diets varying in protein and lipid concentration (g kg)1 of wet weight basis) Two-way ANOVA (P < 0.05) Protein/lipid (g kg)1) Grass carp Whole body Moisture Protein Lipid Liver Moisture Protein Lipid Tilapia Whole body Moisture Protein Lipid Liver Moisture Protein Lipid 380/0 380/40 380/100 250/0 250/40 250/100 Protein Lipid PãL 773 1.5 141 0.2b 54.5 1.9b 762 9.1 140 3.0b 65.0 3.9c 764 7.4 140 3.8b 78.5 3.5e 774 13.6 135 5.9b 49.5 2.6a 774 12.2 137 6.9b 56.3 2.1b 767 6.8 127 4.6a 70.3 1.3d 0.279 0.008 0.000 0.343 0.139 0.000 0.549 0.247 0.438 627 8.6bc 118 9.7c 196 9.2b 594 25.7b 95.9 12.4a 244 12.0c 548 26.6a 105 11.4abc 293 5.7d 635 10.3c 116 2.4bc 169 12.8a 607 10.8bc 111 7.1abc 210 5.4b 599 17.3b 100 2.2ab 241 20.7c 0.016 0.485 0.000 0.001 0.023 0.000 0.127 0.135 0.218 724 7.5 186 7.6 53.9 3.4a 725 8.8 186 7.8 72.3 3.3bc 725 4.2 184 10.3 80.3 0.4d 721 4.7 178 10.0 52.4 5.2a 723 3.1 180 4.7 68.6 3.7b 729 5.3 174 3.2 76.3 2.3cd 0.926 0.050 0.065 0.464 0.693 0.000 0.487 0.878 0.766 699 13.5 124 5.4ab 88.0 5.7b 683 7.9 132 2.3b 107 2.2c 682 1.2 123 2.4ab 131 8.9d 699 6.4 125 7.3ab 66.9 3.4a 701 4.0 125 4.8ab 93.2 5.9b 693 19.6 121 6.8a 112 9.9c 0.074 0.281 0.000 0.237 0.124 0.000 0.358 0.367 0.600 Values represents means SD of three replicates and values with the same column with different letters were significantly different (P < 0.05, one-way ANOVA) Table Biochemical compositions of plasma from grass carp and tilapia fed with experimental diets varying in protein and lipid concentration (mmol L)1) Two-way ANOVA (P < 0.05) Protein/lipid (g kg)1) Grass carp Cholesterol (CHO) Triacylglyceride (TG) Tilapia CHO TG 380/0 380/40 380/100 250/0 250/40 250/100 Protein Lipid PãL 4.43 0.37a 1.68 0.08a 4.86 0.25ab 1.76 016ab 5.28 0.35c 2.34 0.53c 4.39 0.33a 1.71 0.03ab 4.83 0.13ab 1.78 0.08ab 5.13 0.07c 2.16 0.11bc 0.594 0.710 0.001 0.003 0.919 0.711 3.32 0.18ab 0.71 0.02b 3.34 0.11b 0.88 0.03cd 3.79 0.06c 1.38 0.13e 3.05 0.20a 0.58 0.07a 3.14 0.15ab 0.81 0.05bc 3.73 0.21c 0.95 0.05d 0.031 0.000 0.000 0.000 0.497 0.002 Values represents means SD of three replicates and values with the same column with different letters were significantly different (P < 0.05, one-way ANOVA) Putnam (1973), several authors have shown that increasing the dietary non-protein energy levels leads to a better utilization of ingested protein, because of an increased contribution of the non-protein energy sources to energy expenditure (Cho & Kaushik 1985, 1990) In grass carp, no studies have conrmed the existence of a protein-sparing eect of lipid or digestible carbohydrate and at least in the present study, it did not appear to improve protein utilization with no obvious protein-sparing eect of dietary lipids in this species The lack of protein-sparing eect in the present study could be due to the fact that the 40 g kg)1 dietary lipid level used was already providing sucient amount of metabolizable energy for growth of grass carp and a further 60 g kg)1 increase in dietary lipid level would have a negative eect of the growth response Peres & Ol va-Teles (1999b) believed that this lack of protein-sparing eect by dietary lipids may be related to the high protein levels of the diet According to Dias et al (1998), the benecial eects of an increase in lipid level from 100 to 180 g kg)1 in sea bass diets were signicant only with a low-protein diet, but not with a high protein diet But in the present study, similar results were observed in both high and low protein levels which further demonstrated that no protein-sparing eect of lipid occurred in grass carp De Silva et al (1991) reported that the Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Table Liver enzyme activities of grass carp and tilapia fed with experimental diets varying in protein and lipid concentration Two-way ANOVA (P < 0.05) Protein/lipid (g kg)1) 380/0 Grass carp Lipase (LPS) 0.82 Alkaline phosphatase (AKP) 96.1 Tilapia LPS 1.55 AKP 120 380/40 0.12a 2.55 0.15c 4.45a 127 3.78c 380/100 250/0 250/40 250/100 3.09 0.13d 0.75 0.10a 2.16 0.10b 3.11 0.07d 140 5.31d 96.5 7.71a 111 4.75b 124 4.71c Protein Lipid PãL 0.020 0.001 0.000 0.025 0.000 0.026 0.11a 3.44 0.10b 4.08 0.17c 1.46 0.06a 3.27 0.11b 3.93 0.11c 0.024 4.90a 143 3.59bc 158 6.48d 109 8.00a 133 8.48b 149 6.23cd 0.006 0.000 0.808 0.000 0.952 Values of LPS are mean Umg)1 protein SD (P < 0.05, one-way ANOVA) Values of AKP are mean mU mg)1 protein SD (P < 0.05, one-way ANOVA) protein-sparing capability increased with increasing dietary lipid content up to 180 g kg)1 and the dietary lipid level had a greater inuence on growth, FCR and PER However, Hanley (1991) reported that Nile tilapia were not able to utilize this energy source to improve growth or food utilization eciency, at least in diets containing adequate levels of protein Chou & Shiau (1996) reported that growth, FCR and PER were better for hybrid tilapia fed the lipid diets than those fed the control diet, but there were no signicant dierences in 50, 100 and 150 g kg)1 lipid diets So, from the former studies and the present study, there was no exact information about the existence of a protein-sparing eect of lipid in tilapia, but such high lipid levels as 100 and 150 g kg)1 would be little harmful to the growth performance compared to 40 or 50 g kg)1 lipids diet As to the role of carbohydrate in protein-sparing eect, Shiau & Peng (1993) suggested that starch or dextrin could spare some protein when the dietary protein level was low in juvenile tilapia In contrast with the protein utilization of sh, the increased dietary lipids levels resulted in a signicant decrease in lipid retention (LR) in both sh This agrees with Cho & Watanabe (1985) who observed in rainbow trout, that the highest lipid diet did not promote the highest LR Peres & Ol va-Teles (1999b) also reported a decreased LR when the dietary lipids increased from 120 to 300 g kg)1 The positive correlation between VSI, HSI, IPF, body and liver lipid content and dietary lipid may indicate that when dietary lipid is supplied in excess, a proportion of this lipid is deposited as body fat, both in grass carp and tilapia Similar results were observed in former studies of grass carp (Du et al 2005) and hybrid tilapia (Chou & Shiau 1996) This is also in agreement with the results on other sh species such as rainbow trout (Lee & Putnam 1973), channel catsh (Garling & Wilson 1977), common carp (Takeuchi et al 1979), red drum (Ellis & Reigh 1991) and hybrid Clarias catsh (Jantrarotai et al 1994) The lipid accumulation in the liver could indicate potential for serious health issues for cultured sh with the Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd possibility of decreased resistance to disease Increased hepatic lipid stores can negatively impact the health status of sh, leading to higher levels of oxidative stress (Craig et al 1999; Kiron et al 2004) However, in the present study, the lipid levels of all liver samples were very high (about 200 g kg)1), which is not only abnormal compared to wild grass carp (below 100 g kg)1) and commercially fed sh with liver lipids below 150 g kg)1, but also abnormal compared to tilapia in this study (about 100 g kg)1) It has been demonstrated that excess lipid in diet induces fatty liver in grass carp more easily than in tilapia Irrespective of dietary lipid level, whole-body and liver lipid content increased with increasing protein level in both sh It has been demonstrated that high dietary protein level will induce high lipid content of sh very easily Dierent dietary protein and lipid levels did not have signicant eects on the whole body and liver moisture Tissue CHO concentrations are known to vary depending on the nutritional status of sh (Regost et al 2001) Plasma CHO and triacylglyceride (TG) concentrations were reported to increase as dietary lipid levels increased (Regost et al 2001; Du et al 2005), in agreement with our ndings in both sh, indicating a more active lipid transport, in response to the higher dietary lipid level However, the concentrations of plasma TG and CHO in grass carp were much higher than the values in tilapia, which demonstrated that tilapia might have better lipid processing capability than grass carp To take into account the feeding strategy of shes, one must consider the enzymatic ability to digest the many kinds of food (Chakrabarti et al 1995) The digestive structure can actually reect their food habits in terms of the use of natural resources The homogeneous enzyme distribution in the sh gut has been considered as an adaptation to the variety of the composition of the diet from the wild Therefore, it is more reasonable to consider it as a consequence of the shesế history and their position in the course of evolution In the present study, lipase activity of tilapia was higher than that of grass carp and increased with increasing lipid content in diets in both sh Positive correlation between lipase activity and lipid digestibility in mahseer (Tor khudree) (Bazaz & Keshavanath 1993), rohu (L rohita) (Gangadhar et al 1997) and the European sea bass (Dicentrarchus labrax) (Peres & Ol va-Teles 1999b) has been reported AKP is considered to be involved in absorption of nutrients such as lipid, glucose, calcium and inorganic phosphate (Dupuis et al 1991; Mahmood et al 1994) and its activity has been related to food intake (Fraisse et al 1981) Ribeiro et al (1999) has found a trend for higher AKP activity in Senegalese sole that were seen to feed more actively Nevertheless, in the present study, the eect of feed intake on enzymes activities was not considered and even if the possibility exists that a higher dietary lipid level might potentially lead to a lower food intake in sh, this can only be speculated at the moment and further studies are necessary The activity of AKP had the same trend as lipase in the present study Several studies revealed that the relative activity of the digestive enzymes can be correlated with the nature and composition of the food consumed (Cockson & Bourne 1972; Kawai & Ikeda 1972; Olatunde & Ogunbiyi 1977) Apparently, the dierence in the relative strength of lipase activity between tilapia and grass carp was directly related to the relative amount of lipids in their nature food Tilapia is omnivorous, and micro-organisms, zooplankton and detritus are usual in its natural diet (Bowen 1982) and grass carp is herbivorous, natural food of which is water plant containing very low amounts of usable lipids The results indicate that tilapia can eectively digest lipids better than grass carp and that the digestive tract of both sh is able to adapt and respond to changes in the levels of dietary lipids Digestibility is associated with some factors, as for example, the level of digestive enzymes From the moment the amount of protein and lipid were established, the bre content, as cellulose, is necessarily changed Fibre is the other factor directly linked to digestibility, in addition to the food transit time In the present formulations, the variation of bre was unavoidable However, Silva et al (2003) suggested that in tambaqui, the levels of bre not interfere in the permanence time of the food in the digestive tract This should be an adaptive trait of the species from the feeding habit in the wild In other studies, cellulose was used at higher levels up to 400 g kg)1 for channel catsh and Tilapia zillii without aecting growth rates (Garling & Wilson 1977; El-Sayed & Garling 1988) The growth performances of both sh were improved by raising the dietary protein level over 250 g kg)1 These data suggested that there was no evidence of a protein-sparing eect of dietary lipids in grass carp Dietary lipid levels above 40 g kg)1 did not harm to growth of omnivorous tilapia but did harm to that of herbivorous grass carp A straight correlation between digestive enzymes activities and dietary lipid was observed in both sh, but the values of tilapia were higher than those of grass carp Tilapia has relatively higher capacity to endure high dietary lipid level compared to grass carp Alanaăraă, A (1994) The eect of temperature, dietary energy content and reward level on the demand feeding activity of rainbow trout (Oncorhynchus mykiss) Aquaculture, 126, 349359 Alanaăraă, A & Kiessling, A (1996) Changes in demand feeding behaviour in Arctic charr, Salvelinus alpinus L., caused by dierences in dietary energy content and reward level Aquac Res., 27, 479486 AOAC (1995) Ocial Methods of Analysis Association of Ocial Analytical Chemists, Arlington Arzel, J., Martinez Lopez, F.X., Metailler, R., Stephan, G., Viau, M., Gandemer, G & Guillaume, J (1994) Eect of dietary lipid on growth performance and body composition of brown trout (Salmo trutta) reared in seawater Aquaculture, 123, 361375 Azevedo, P.A., Bureau, D.P., Leeson, S & Cho, C.Y (2002) Growth and eciency of feed usage by Atlantic salmon (Salmo salar) fed diets with dierent dietary protein: energy ratios at two feeding levels Fish Sci., 68, 878888 Bazaz, M.M & Keshavanath, P (1993) Eect of feeding dierent levels of sardine oil on growth, muscle composition and digestive enzyme activities of mahseer, Tor Khudree Aquaculture, 115, 111119 Beamish, F.W.H & Medland, T.E (1986) Protein sparing eects in large rainbow trout, Salmo gairdnen Aquaculture, 55, 3542 Bessey, O.A., Lowry, O.H & Brock, M.J (1946) A method for the rapid determination of alkaline phosphatase with ve cubic millimeters of serum J Biol Chem., 164, 321329 Bowen, S.H (1982) Feeding, digestion and growth-qualitative considerations In: The Biology and Culture of Tilapia (Pullin, R.S.V & Lowe-McConnel, R.H eds), pp 141156, Proceedings of the International Conference on the Biology and Culture of Tilapias, 25 September 1980, Rockefeller Foundation, Bellagio, Italy Chaiyapechara, S., Casten, M.T., Hardy, R.W & Dong, F.W (2003) Fish performance, llet characteristics, and health assessment index of rainbow trout (Oncorhynchus mykiss) fed diets containing adequate and high concentrations of lipid and vitamin E Aquaculture, 219, 715738 Chakrabarti, I., Gani, M.D.A., Chaki, K.K., Sur, R & Misra, K.K (1995) Digestive enzymes in 11 freshwater teleost sh species in relation to food habit and niche segregation Comp Biochem Physiol A, 112, 167177 Cho, C.Y & Kaushik, S.J (1985) Eects of protein intake on metabolizable and net energy values of sh diets In: Nutrition and Feeding in Fish (Cowey, C.B., Mackie, A.M & Bell, J.G eds), pp 95117, Proceedings of the International Symposium on Fish Feeding and Nutrition, Aberdeen, London Cho, C.Y & Kaushik, S.J (1990) Nutritional energetics in sh: protein and energy utilization in rainbow trout In: Aspects of Food Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Retained -TOH (tissue wet wt./feed dry wt.) 1.0 (a) 0.5 Plasma Gonads Intestine Stomach Gills Adipose Red muscle Wh muscle (b) -TOH -TOH -TOH Figure (a) Retained levels of a-tocopherol (TOH) in those tissues of Atlantic salmon where tissue concentrations of all the tocopherols were directly proportional to the feed concentrations (model y = ax) Retention is calculated as the ratio of tissue concentration (wet wt.)/feed concentration (dry wt.) (b) Retained levels of a, c and d-tocopherol (TOH) expressed as % of a-TOH retention (From Hamre & Lie 1997, with permission from Aquaculture Nutrition) Retained TOH (% of retained -TOH) 100 50 Plasma Gonads Intestine Stomach exponentially in response to dietary supplementation, such a relationship cannot be given for liver However, at a supplementation level of 100 mg kg)1 for all three tocopherols, the retention of c-TOH in the liver would be in the range of 22% and d-TOH in the range of 2%, compared to a-TOH The linear model for a-TOH retention in muscle and plasma, and the exponential model for liver are supported in principle by studies with Tilapia (Satoh et al 1987), rainbow trout (Hung et al 1980; Frigg et al 1990; Puangkaew et al 2005) and Atlantic salmon (Hardie et al 1990) In grouper, a-TOH in liver and llet appeared to increase linearly up to a dietary supplementation rate of 200 mg kg)1 and then adopted a lower increase rate (Lin & Shiau 2005) The absolute concentrations were also lower than in salmon, and they were lowered by increased dietary lipid Red drum Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd Gills Adipose Red Wh muscle muscle showed a linear increase in liver and plasma a-TOH in response to dietary a-TOAc supplementation from to 80 mg kg)1 (Peng & Gatlin 2009) Free radicals are characterized by having an unpaired electron, a feature that makes them extremely reactive towards most biomolecules Small amounts of free radicals derived from oxygen and nitrogen are generated through normal metabolism, by electron transport, phagocytic activity and by certain enzymes (oxygenases, cytochrome P450) Low levels of radicals are necessary for regulation of cell growth and development (Rice-Evans & Burdon 1993; Nordberg & Arner 2001; Zingg 2007) The defence against in vivo oxidation includes enzymes (e.g superoxide dismutase, catalase, glutathione peroxidase), endogenously synthesized antioxidants (glutathione, ubiquinone) and antioxidant nutrients (vitamins C, E and carotenoids) The antioxidant enzymes require metal ions (Cu, Mn, Zn, Fe and Se) to be active Oxidative challenge is encountered when the formation of radicals exceeds the capacity of the antioxidant defence Such a situation may develop after tissue injury or inammation, as a result of exposure to pollution or oxidative drugs, or during deciency of antioxidant nutrients Ions of transition metals such as iron and copper catalyse one-electron transfer reactions that may give rise to dierent free radical products Metal ions in the free form are therefore very toxic In vivo such ions are normally bound to proteins, i.e ferritin, transferrin, metallothionine, and only traces of free ions are found in tissues of healthy animals The hydroxyl radical is considered the most reactive of the oxygen derived radicals It may be formed from hydrogen peroxide catalysed by iron (Fenton reaction, Hứlmer 1993; Frankel 1998) Further, the iron bound to haemoglobin and myoglobin may under certain conditions be oxidized to form a ferryl haem protein radical, which is also a potent prooxidant (Rice-Evans & Burdon 1993) Auto-oxidation of lipids is initiated when a free radical reacts with unsaturated lipids by abstracting a hydrogen atom at one of the double bonds, thereby creating a carbon centred lipid radical Uncontrolled lipid oxidation (Fig 5) is normally low in vivo, but the rate may increase if the animal is subjected to oxidative stress When auto-oxidation has been initiated, a self-sustaining reaction cycle is established, where the lipid peroxyl radical formed by one turn of the cycle reacts with a new polyunsaturated fatty acid (PUFA) In the absence of antioxidants, lipid oxidation may proceed as long as PUFAs are available for oxidation Termination is accomplished when two lipid radicals combine to form a non-radical species (Hứlmer 1993; Frankel 1998) The primary products of lipid auto-oxidation are the conjugated dienes and lipid hydroperoxides, which may undergo cleavage to form dierent secondary products of low molecular weight, i.e aldehydes, alkanes, alkenes, alcohols and acids (Horton & Fairhurst 1987; Hứlmer 1993; Frankel 1998) Secondary lipid oxidation products readily diuse to the aqueous compartments of the cell and to the extracellular space The bifunctional aldehydes may crosslink PUFA-OOH XH PUFA-H X PUFA-OO PUFA O2 Figure Auto-oxidation of lipids Initiation: A free radical (Xặ:OHặ, O2)ặ or others) abstracts a hydrogen atom from a polyunsaturated fatty acid (PUFA) The PUFA radical formed reacts with oxygen to form a peroxyl radical Propagation: The lipid peroxyl radical abstracts a hydrogen atom from a new PUFA and enters a new turn in the reaction cycle (Hứlmer 1993; Frankel 1998) proteins, thereby forming Schis bases, while the 4-hydroxyalkenals bind to and inactivate glutathione (GSH)- and SHcontaining proteins (Horton & Fairhurst 1987) The low molecular secondary residues can also reside on the glycerol or phospholipid molecules giving rise to core aldehydes that may interfere with membrane structure and function (Ahmed et al 2003) Further, the F2-isoprostanes have been identied as products of lipid oxidation (Awad et al 1994) It is assumed that a-TOH is positioned with the phytyl chain buried in the hydrophobic inner part of the membrane, while the chromanol ring, which carries the reactive and polar OHgroup, resides at or near the membrane surface (Kagan et al 1993; Wang & Quinn 2000; Quinn 2004) a-TOH competes with PUFA in donating a hydrogen atom to the lipid peroxyl radical (Fig 6), thereby breaking the chain of reactions involved in lipid auto-oxidation In non-polar homogenous solutions, lipid peroxyl radicals react approximately 105 times faster with a-TOH than with PUFA (Table 1) The tocopheroxyl radical (Fig 6) is resonance stabilized and reacts slowly with PUFA (Table 1) The reaction rates and the dierences between them decline in more polar solutions and in lipid dispersions, but apparently not enough to change the reaction directions (Ingold et al 1993) From measurements with red blood cell ghosts and linoleate micelles, Buettner (1993) calculated that one molecule of a-TOH can protect approximately 1000 molecules of PUFA against oxidation, which is in accordance with ratios of a-TOH to PUFA found in animal tissues (Poukka Evarts & Bieri 1974) This gure must be corrected for the peroxibility of the PUFA, which increases dramatically as the Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd Table Standard reduction potentials of redox couples at pH = (Buettner 1993) The overall potential difference (DE) is described by the Nernst equation and is determined by the standard potential difference and the reactant and product concentrations If DE > 0, the reaction is exergonic and thermodynamically feasible Redox couple E0Â mV, pH = a-Tocopheroxylặ, H+/a-Tocopherol Ascorbateặ), H+/Ascorbate Dehydroascorbate/Ascorbateặ) GSSG/GSSGặ) GSặ/GS) ROOặ, H+/ROOH ROặ, H+/ROH PUFAặ, H+/PUFA-H 500 282 )174 )1500 920 7701440 1600 600 PUFA, polyunsaturated fatty acid Figure Proposed mechanism for the reaction of a-tocopherol with oxidising lipids The peroxyl radical group formed during lipid oxidation is polar and oats to the surface of the membrane where it can react with a-TOH, rendering a lipid hydroperoxyde and the tocopheroxyl radical (Buettner 1993) number of double bonds increases (Table 1, Bieri & Puokka 1970) The kinetic properties of the reactions between vitamin E and oxidizing lipids are important for the eciency of a-TOH as an antioxidant However, under certain conditions, for example at high vitamin E combined with low vitamin C concentrations, vitamin E may be present mainly as tocopheroxyl radicals, which abstract hydrogen atoms from PUFA at low rates, thereby initiating lipid oxidation (Bowry et al 1992; Ingold et al 1993) Under these circumstances, a-TOH may act as a prooxidant Rate constants for lipid oxidation and vitamin E function have also been reviewed by Brigelius-Flohe (2009) and Traber & Atkinson (2007) It is well documented that increased dietary PUFA increases the requirement of vitamin E in sh (Woodall et al 1964; Watanabe et al 1981a,b; Satoh et al 1987; Schwarz et al 1988; Roem et al 1990; Lin & Shiau 2005) This may be modulated by at least two dierent mechanisms, rstly by decreased absorption of a-TOH because of increased oxidation and degradation of vitamin E in the feed and in the digestive tract, and secondly by a decreased ratio of a-TOH/ PUFA in the sh body which would be the case even with no extra degradation of vitamin E (Bieri & Puokka 1970; Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd Poukka Evarts & Bieri 1974) Further, two dierent strategies of lipid supplementation should be considered, i.e supplementation of dierent levels of PUFA at a constant level of dietary lipid, and variation of the dietary lipid level at a constant relative content of PUFA The whole body concentration of PUFA in Atlantic salmon increases in response to increased dietary supplementation (Fig 7) Schwarz et al (1988) demonstrated that increased PUFA at a constant level of dietary lipid increases the vitamin E requirement in carp (Cyprinus carpio, L.) Unfortunately, tissue a-TOH levels were not given in their article, and it is not known whether the dietary PUFA had any eect on the retention of a-TOH In our own studies, increasing n-3 PUFA from 200 to 300 g kg)1 lipid (Hamre & Lie 1995b) had no inuence on the retention of a-TOH when vitamin E was supplemented in excess, and reduced retention by only 15% in unsupplemented sh On the other hand, a-TOH retention was clearly reduced by increased dietary lipid levels in studies with rainbow trout, tilapia and grouper (Watanabe et al 1981a; Satoh et al 1987; Lin & Shiau 2005) Pollock liver oil, supplemented as native oil or methyl esters, was added at 50150 g kg)1 to diets containing 500 mg kg)1 allrac-a-TOAc In rainbow trout, there was a 10-fold dierence in liver a-TOH, and a twofold dierence in muscle a-TOH, whereas tilapia showed a fourfold dierence in whole body a-TOH concentration (Watanabe et al 1981a; Satoh et al 1987) Similar results were found in studies with carp (Watanabe et al 1981b) It is not clear to what extent this eect depended on the unsaturation of the lipids, and whether it was caused by changes in the eciency of absorption, or by other mechanisms Retention of vitamin E in rainbow trout supplemented with a-TOAc was also lowered in sh fed oxidized lipids (Hung et al 1981) n-3 PUFA body (% of TL) 40 Lie, unp a 35 30 Paper 25 Lie, unp b Paper 20 15 20 25 30 n-3 PUFA feed (% of TL) Oxidized oil combined with low vitamin E causes reduced growth and increased mortality in a large number of animal species These eects are reversed when the vitamin E supplementation is increased, and to a lesser extent with increased ethoxyquin supplementation Further, oxidized oil often leads to reduced vitamin E levels in the tissues as shown in rainbow trout and African catsh (Hung et al 1981; Tacon 1996; Baker & Davies 1997) However, based on these experiments it is dicult to say whether the oxidized oil leads to decreased available vitamin E in the gut and therefore causes a secondary vitamin E deciency, or whether oxidation products are taken up into the body where they initiate lipid oxidation and thereby an increased consumption of vitamin E in vivo The hypothesis that the tocopheroxyl radical is reduced by ascorbate, thereby regenerating a-TOH (Fig 8), was rst proposed by Tappel (1962) and has been found valid in several in vitro studies with liposome model membranes (Niki 1987), organelle preparations (Wefers & Sies 1988) and LDL (Kagan et al 1992a) Transient appearance of the ascorbyl and tocopheryl radicals after pulse radiolysis of a homogenous solution of ascorbate and tocopherol was shown by Packer et al (1979) Electron spin resonance techniques give similar results for liposomes and natural membrane preparations (Packer & Kagan 1993), and standard reduction potentials show that this recycling reaction is exergonic, and therefore thermodynamically feasible (Buettner 1993) Other water-soluble compounds with antioxidant properties, i.e GSH, cysteine and urate, regenerate a-TOH in vitro 35 40 Figure The relationship between dietary n-3 polyunsaturated fatty acids (PUFA) and the relative amount of n-3 PUFA in the whole body lipids of Atlantic salmon juveniles fed sh oil from different sources Data are given as % of total lipids (Niki 1987; Miki et al 1988) GSH recycles vitamin E in homogenous solutions (Niki et al 1982), but experiments aiming to show that this reaction occurs in biological membranes have given conicting results (McCay et al 1987; Ho & Chan 1992; Murphy et al 1992) The results of Mrtensson & Meister (1991) demonstrated that GSH reduces dehydroascorbate in newborn rats It is not clear whether this reaction proceeds spontaneously or by enzyme catalysis (Meister 1994; Winkler et al 1994) Thus, GSH may indirectly stimulate recycling of tocopherols by regenerating ascorbate (Fig 8, Packer & Kagan 1993) Further, a NADH-dependent reductase system, which catalyses the one-electron reduction of the ascorbyl radical, has been partly characterized (Meister 1994) Alpha-tocopheroxyl radicals present in rat liver mitochondrial and microsomal membranes may also be reduced by the electron transport chains, using succinate and NADPH, respectively, as electron donors (Kagan et al 1992b; Maguire et al 1992) In mitochondria, this process relies on the presence of ubiquinone, but ubiquinone alone was less eective than electron transport from succinate, to regenerate vitamin E (Maguire et al 1992) This is in line with experiments showing that reduced, but not oxidized ubiquinone protects a-TOH from oxidation (Noack et al 1994) Both these routes of recycling of vitamin E indicate that reduced vitamin E is maintained in membranes using reducing equivalents from energy metabolism (Fig 8) Increasing the vitamin C supplementation from to 60 mg kg)1 did not inuence the retention of a-TOH in Atlantic salmon as long as the sh were not vitamin C decient (Hamre et al 1997) Similar results were found in yellow perch and channel catsh (Lee & Dabrowski 2003; Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd had developed the well-known pathologies of vitamin C deciency, such as deformed vertebrae and lowered vertebra hydroxyproline concentration (Sandnes et al 1992) Liver concentration of vitamin E was also increased by dietary vitamin C in vitamin E-decient yellow perch and channel catsh (Lee & Dabrowski 2003; Yildirim-Aksoy et al 2008) Figure shows the close similarity between the normalized concentrations of liver a-TOH and vertebra hydroxyproline in response to supplementation of vitamin C in Atlantic salmon The parallel development in vitamin E-supplemented sh, of liver vitamin E, vertebrae hydroxyproline, growth and mortality in response to dietary vitamin C (Hamre et al 1997) suggests that the decrease in liver a-TOH was of metabolic origin, and that a vitamin C status above deciency was necessary to maintain the body stores of vitamin E The results may be taken as support of the hypothesis that vitamin C regenerates vitamin E in vivo (Tappel 1962; Fig 8) Thus, a secondary vitamin E deciency may develop in vitamin C-decient sh, possibly explaining why anaemia and increased lipid oxidation are observed in vitamin C deciency in some experiments, but not in others (Sandnes et al 1990; Frischknecht et al 1994) Further, high dietary vitamin E appears to have a prooxidant eect in sh decient in vitamin C, because mortality, growth depression, backbone hydroxyproline and liver vitamin E (Fig 9) (and C) concentration all indicated a faster development of vitamin C deciency in sh fed 300 mg kg)1 all-rac-a-TOAc, than in those fed 150 mg kg)1 (Hamre et al 1997) According to the hypothesis that vitamin E is recycled by vitamin C (Tappel 1962), tocopheroxyl radicals accumulate in the membranes when vitamin C drops below a critical Figure Proposed mechanism for regeneration of a-tocopherol from the tocopheroxyl radical (Tappel 1962) Ascorbic acid is oxidized in the process but can be regenerated by glutathione (Mrtensson & Meister 1991) either chemically or enzymatically (Meister 1994; Winkler et al 1994) or enzymatically by NADH (Meister 1994) Oxidized glutathione (GSSG) is reduced by glutathione reductase at the expense of NADPH generated in the pentose phosphate shunt (Meister 1994) Yildirim-Aksoy et al 2008) and White et al (1993) found that tissue vitamin E levels in Atlantic salmon were independent of vitamin C supplementation between 50 and 2750 mg kg)1 On the other hand, there was a large drop in liver vitamin E concentration in Atlantic salmon that became vitamin C decient (Fig 9; Hamre et al 1997) The salmon 1.4 0.8 0.6 1.4 0.4 1.2 0.2 0 10 Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd 20 30 -TOH (fraction of max) Figure Vertebra hydroxyproline (HPRO) and liver a-TOH concentration in response to dietary vitamin C Results are given as the fraction of average concentrations of HPRO and a-TOH when vitamin C was adequate Each point represents a pooled sample from 10 sh, and there are ve samples per group Vertebrae and livers were dissected from the same sh HPRO (fraction of max) 1.2 40 50 0.8 60 150 300 70 Dietary vitamin C (mg/kg dry diet) 0.6 0.4 Dietary vitamin E: (mg kg1 dry diet) 0.2 0 10 20 30 40 50 Dieaty vitamin C (mg kg1 dry diet) 60 70 150 300 level, in amounts proportional to the original concentration of a-TOH The tocopheroxyl radicals abstract hydrogen atoms from surrounding molecules (Bowry et al 1992) and may promote irreversible oxidation of the remaining vitamin C The apparent prooxidant eect of vitamin E in vitamin Cdecient sh therefore supports the hypothesis of Tappel (1962) The antioxidant eect of selenium is accounted for by its incorporation in glutathione peroxidases (GPx) (Ursini 1993) which reduce hydroperoxides at the expense of reduced GSH Four GPx have been described in mammals; a cytosolic cGPx, which is the classical enzyme that neutralizes watersoluble and fatty acid hydroperoxides, plasma pGPx, which has similar properties as cGPx, gastro-intestinal GI-GPx, which is exclusively expressed in the gastro-intestinal tract, and phospholipid hydroperoxide glutathione peroxidase (PHGPx), which is active in biological membranes and reduces lipid hydroperoxides (Brigelius-Flohe 1999; Arteel & Sies 2001) The active site of these enzymes contains selenocysteine residues In addition, selenoprotein P (SeP), present in the plasma of mammals and expressed in cellular membranes, contains 10 selenocysteine residues and is regarded as a transport protein for selenium but also has antioxidative properties (Steinbrenner et al 2006) Apparently PHGPx prevents the formation of lipid alkoxyl radicals (LOặ) in microsomal membranes, which mediate a ễsecondaryế initiation of lipid oxidation (Fig 10) LOmediated lipid oxidation is thought to be ineciently inhibited by vitamin E, because the alkoxyl radicals react with PUFA at a similar rate as they are scavenged by vitamin E (Maiorino et al 1989) The selenium-dependent peroxidase for lipid hydroperoxides therefore provides an explanation for the signicant in vivo interactions between a-TOH and selenium demonstrated in sh (Poston et al 1976; Bell et al 1985) as well as in other animals In sh, studies of the interactions between selenium and vitamin E have shown that deciency of selenium may lead to reduced levels of tissue a-tocopherol Combined selenium and vitamin E deciency signs are muscular dystrophy, muscle specic proteins in plasma and anaemia (Poston et al 1976; Bell et al 1985, 1986, 1987; Gatlin et al 1986a) The rst indications that vitamin E had functions beyond the antioxidant eects were reported by Mahoney & Azzi (1988) and Boscoboinic et al (1991) who showed that a-TOH inhibited proliferation of smooth muscle cells through inhibition of protein kinase C Later research has shown that tocopherols may have multiple signalling functions at the post-translational level such as activation of protein phosphatase 2A, diacylglycerol phosphatase and protein tyrosine phosphatase and inhibition of protein kinase A2, Figure 10 Model of the proposed role of phospholipid hydroperoxyde glutathione peroxidase (PHGPX) in protecting membranes against lipid oxidation PHGPX decomposes lipid hydroperoxides to alcohol and water, oxidising glutathione in the process This prevents the formation of the lipid alkoxyl radical which will stimulate lipid oxidation even in the presence of a-TOH (Maiorino et al 1989; Ursini 1993) Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd phospholipase A2, cyclooxygenase, lipoxygenases and mitogen-activated protein kinase (Zingg & Azzi 2004; Zingg 2007) The expression of certain genes is modulated by vitamin E, and vitamin E has been shown to inhibit platelet aggregation and monocyte adhesion, in addition to cell proliferation (Quinn 2004) There is, however, a controversy as to whether these eects are mediated directly by vitamin E or by lipid oxidation products, which will vary in concentration within the cell in response to vitamin E concentration (Azzi 2007; Traber & Atkinson 2007; Brigelius-Flohe 2009) Many of the proteins modulated by vitamin E are translocated to the membrane and activated there but the mechanism of vitamin E function remains to be elucidated Zingg (2007) suggests several modes of action of vitamin E Vitamin E binds directly to enzymes involved in signal transduction, such as phospholipase A2, cyclooxygenase-2 and lipoxygenases Vitamin E also modulates the translocation of enzymes to the plasma membrane It may compete with lipophilic substrates for the active site of enzymes and modulate synthesis and transport of messenger molecules The activity of many enzymes is regulated by reversible oxidation and reduction, processes that may be aected by vitamin E A recent discovery of the naturally occurring tocopheryl phosphate may indicate that the vitamin participates in intermediary metabolism and has strengthened the view that vitamin E has non-antioxidant functions Furthermore, in model membranes of phosphatidyl choline and phosphatidyl ethanolamine, vitamin E tends to form clusters with phosphatidyl choline at a molar ratio of vitamin E per 10 phosphatidyl choline molecules (Wang & Quinn 2000; Quinn 2004) This is also consistent with the vitamin having other roles than being an antioxidant The only example of regulatory functions of vitamin E in sh is the nding that vitamin E downregulates activity of the enzyme gulonolactone oxidase, involved in vitamin C synthesis, in sturgeon (Moreau & Dabrowski 2003) Because the notion of non-antioxidant functions of vitamin E is quite new, most experiments determining vitamin E requirements in sh have assumed that the primary eect of vitamin E deciency is increased lipid oxidation (Figs & 6) and most other deciency signs are secondary to this Oxidation of lipids in cell membranes leads to loss of membrane integrity, leaky cells and eventually cell lysis A deciency will aect all cells and organs However, some cell types may be more susceptible to oxidation than others, for example because of a high rate of oxidative metabolism, low a-TOH Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd to PUFA ratio, high turnover rate of vitamin E or the presence of prooxidants Taking into account the interactions with other nutrients, and dierent body levels and organ distribution of vitamin E in dierent animal species (Hamre & Lie 1995b), it is not surprising that the vitamin E deciency signs vary with the experimental conditions and from one animal species to the other In our studies with Atlantic salmon, anaemia and degeneration of the liver were the most dominant deciency signs We did not nd muscular degeneration, which is common in vitamin E-decient terrestrial animals (Nelson 1980) and in sh (Poston et al 1976; Cowey et al 1984; Frischknecht et al 1994) Susceptibility of erythrocytes to vitamin E deciency is a common feature in most, if not all, animal species (Nelson 1980) The events leading to anaemia are complex and probably include components of the water phase in addition to the lipid compartments Haemoglobin may act as a prooxidant in itself or by liberation of trace amounts of iron (Horton & Fairhurst 1987; Hứlmer 1993; Rice-Evans & Burdon 1993) Erythrocytes also have high concentrations of oxygen available for the formation of reactive oxygen species Further, salmon erythrocytes incorporate high levels of PUFA (Lie, unpublished) but are relatively low in a-TOH (Hamre & Lie 1995a,b; Hamre et al 1997) One would expect that these features make them highly susceptible to oxidation Anaemia has been shown in most studies with sh where vitamin E deciency was observed and haematological parameters measured (Woodall et al 1964; Whitmore 1965; Murai & Andrews 1974; Poston et al 1976; Hung et al 1980; Cowey et al 1981, 1983, 1984; Moccia et al 1984; Frischknecht et al 1994; Hamre et al 1994, 1997; Bai & Lee 1998; Kocabas & Gatlin 1999) A signicant negative correlation between mortality and whole blood haemoglobin concentration (r = )0.59, week 22) was found by Hamre et al 1997; and the anaemia was characterized by a combination of reduced cell number, cell volume and cell haemoglobin concentration, while only lowered cell number and volume have been found by others (Woodall et al 1964; Whitmore 1965; Moccia et al 1984; Frischknecht et al 1994) The lowered cell haemoglobin concentration could have been caused either by degeneration of haemoglobin or decreased haemoglobin synthesis In normal human erythrocytes, about 3% per day of the total haemoglobin is spontaneously oxidized to methaemoglobin and is recycled to haemoglobin by methaemoglobin reductase located in the plasma membrane In old, abnormal erythrocytes, methaemoglobin is not reduced but instead transformed into haemichromes which undergo crosslinking reactions with other proteins This process eventually leads to death of the erythrocyte (Giardina et al 1991) The uorescent granules found in erythrocytes by Hamre et al (1994) are probably similar to the cross-linked haemichromes (Giardina et al 1991) and Heinz bodies (Wintrobe 1967; Nagel 1988; Andersen et al 1994), indicating that degeneration of haemoglobin contributed signicantly to the lowered cell haemoglobin concentration Hamre et al (1997) found that the secondary lipid oxidation products, thiobarbituric acid reactive substances (TBARS), accumulated in the liver of vitamin E-decient Atlantic salmon, in accordance with the concept that vitamin E deciency causes lipid oxidation Further, accumulation of TBARS was negatively correlated with dietary vitamin C, suggesting that vitamin C protected the sh against vitamin E deciency by preventing lipid oxidation In vitamin E-sucient sh, liver TBARS was unaected by dietary vitamin C and E Haem-containing enzymes, which, like haemoglobin may be susceptible to oxidation, are abundant in the liver (Andersen et al 1994) Therefore, the autouorescent material found in liver vacuoles of vitamin E-decient sh (Hamre et al 1994) may have been similar to the autouorescent granules found in the red blood cells In grouper fed 40 and 90 g kg)1 lipid, respectively, hepatic TBARS decreased with increasing levels of vitamin E up to an anticipated requirement of 68 and 115 mg kg)1, after which there was a constant TBARS (Lin & Shiau 2005) Vitamin E-decient sh may have yellowish coloured livers, probably attributable to the accumulation of yellow autouorescent material in vacuoles in the liver cells (Hamre et al 1994) Histological examination revealed that the vacuoles also contained ceroid as indicated by Ziel Nielsen and PAS positive staining properties Liver discolouration and accumulation of ceroid, accompanied by hepatocyte necrosis, are major eects of vitamin E deciency in sh (Murai & Andrews 1974; Poston et al 1976; Lovell et al 1984; Moccia et al 1984; Roem et al 1990; Hamre et al 1994) This condition seems to be more prevalent when the vitamin E-decient diets are rancid (Murai & Andrews 1974; Moccia et al 1984) It was not found in vitamin E-decient Chinook salmon and rainbow trout by Woodall et al (1964), Cowey et al (1984) or Frischknecht et al (1994), but all these experiments were performed with less than 120 g kg)1 dietary lipid Although muscular degeneration appears to be a major pathology of vitamin E deciency, it was not identied by Hamre et al (1994) Upon examination of the literature, we found that when muscular degeneration occurred in vitamin E-decient salmonids, the supplementation of selenium was often low, i.e at or under the requirement for maximal GPx activity in rainbow trout (0.150.38 mg kg)1, Hilton et al 1980; Poston et al 1976; Cowey et al 1984; Frischknecht et al 1994) When the diets were supplemented with higher selenium levels, muscular degeneration was not detected (Cowey et al 1981, 1983; Moccia et al 1984; Bell et al 1985; Hamre et al 1994) This point was also addressed by Gatlin et al (1986a,b), in a discussion on the variability of gross vitamin E deciency signs in channel catsh (Ictalurus punctatus) The high number of interactions of vitamin E with other nutrients shows that when measuring the vitamin E requirement, it is important to consider the interactions with other feed components Vitamin C (Hamre et al 1997; Lee & Dabrowski 2003, 2004; Yildirim-Aksoy et al 2008), astaxanthine (Christiansen et al 1995), selenium (Poston et al 1976; Bell et al 1985) and probably other minerals necessary for activity of the antioxidant enzymes must be supplied in appropriate amounts Lipid-soluble antioxidants should be added to prevent the oxidation of dietary lipid (Hung et al 1981), and the inuence of dietary lipid on the vitamin E requirement (Watanabe et al 1981a; Schwarz et al 1988) should be considered To illustrate the potential magnitude of the eect of dietary lipid and PUFA on the vitamin E requirement, one can compare the studies of Cowey et al (1981) and Watanabe et al (1981a) Cowey et al (1981) fed a diet with 10 g kg)1 linolenic acid as only PUFA source to rainbow trout for 16 weeks and did not nd any pathologies in sh supplemented with mg kg)1 all-rac-a-TOAc In sh fed the unsupplemented diet with less than mg kg)1 a-TOH, the only deciency sign was a lowered haematocrit Watanabe et al (1981a) found that 50 mg kg)1 all-rac-a-TOAc was insucient to prevent vitamin E deciency in rainbow trout fed a diet with 150 g kg)1 sh oil for 12 weeks Table Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd Table Requirements of vitamin E measured in different sh species Species Atlantic salmon Rainbow trout Channel catfish Common carp Yellowtail Blue tilapia Red drum Grouper Mrigal Hybrid striped bass Korean rockfish shows that the vitamin E requirement measured in dierent sh species ranges from 25 to 120 mg all-rac-a-TOAc kg)1dry diet and the requirement measurements within the same species may vary considerably As discussed previously, this may be attributed to variation in experimental conditions, including levels of interacting nutrients in the experimental diets, in addition to possible dierences between sh species A third factor aecting the measured requirement is the response variable used in the determination An example is mrigal, where the requirement based on weight gain was estimated to 99 mg kg)1 by broken line regression, while osmotic erythrocyte fragility showed a stepwise reduction in sh fed up to 216 mg kg)1 vitamin E Also, in a study with red drum, Peng & Gatlin (2009) found dierent requirement estimates when monitoring liver TBARS (31 mg kg)1) and superoxide anion production (60 mg kg)1) In this study, there was no clear eect on weight gain The discussion on what responses actually determine the requirement is complex and will not be further developed here When supplementing sh feed with vitamin E, one should consider including a safety margin, in view, rstly of the potential eects of interactions between vitamin E and other feed components, and secondly of diseases that may aect the absorption and metabolism of a-TOH Examples are pancreas disease (PD) and infectious pancreatic necrosis, which give signicant economical losses in the salmon industry Aected sh show extensive necrosis of exocrine pancreatic tissue and have lowered plasma and liver vitamin E levels (Taksdal et al 1995), possibly because of lowered absorption (Traber et al 1993; Taksdal et al 1995) Infectious diseases may also cause increased utilisation of a-TOH Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd Requirement mg kg)1 dry diet all-rac-a-TOAc 35 60 30 100 50 25 50 100 119 25 31 61115 99 28 45 Comment Dep on dietary lipid References Lall et al (1988) Hamre & Lie (1995a) Woodall et al (1964) Watanabe et al (1981a) Cowey et al (1983) Murai & Andrews (1974) Wilson et al (1984) Watanabe et al (1970) Shimeno (1991) Roem et al (1990) Peng & Gatlin (2009) Lin & Shiau (2005) Paul et al (2004) Kocabas & Gatlin (1999) Bai & Lee (1998) in the tissues, secondary to activated immune responses, haemolysis and tissue injury (Rice-Evans & Burdon 1993; Taksdal et al 1995) Vitamin E improves spawning quality in a wide range of sh species (reviewed by Fernandez-Palacios et al submitted) In gilthead sea bream, diets decient in vitamin E decreased the percentage of fertilized egg (Fernandez-Palacios et al 2005) This may have been related to the decrease in the number and motility of the spermatozoids, as has been described for other vertebrates (Donnelly et al 1999; Danikowski et al 2002) and in sh such as ayu (Hsiao & Mak 1978) Lee & Dabrowski (2004) found that the level of sperm plasma tocopherol decreased signicantly and sperm viability was seriously compromised in American perch broodstock fed with diets decient in vitamin E Insucient vitamin E also decreased the percentage of viable eggs with normal morphology in several species, and larval survival was signicantly improved with vitamin E inclusion in broodstock diets (Fernandez-Palacios et al in press) The vitamin E requirement is dependent on the dietary content of PUFAs An increase in n-3 PUFA levels at a xed level of vitamin E improved the spawning quality in gilthead sea bream but also caused a higher percentage of deformed larvae with hypertrophy of the yolk sac (Fernandez-Palacios et al 1995) Elevation of both n-3 PUFA and vitamin E prevented deformities in the larvae (Fernandez-Palacios et al 2005) Similar interactions between DHA and vitamin E have been found in cod (Takeuchi et al 1994) The optimal level of dietary vitamin E for broodstock has been investigated for several sh species and is reported between 150 and 190 mg a-TOH kg)1, in some cases more (Fernandez-Palacios et al in press) The vitamin E content is generally high in sh eggs and low in broodstock tissues after the spawning period (Mukhopadhyay et al 2003) This may be a result of mobilization of vitamin E from peripheral tissues to the ovary during vitellogenesis as it has been shown in turbot and Atlantic salmon (Hemre et al 1994; Lie et al 1994) Marine sh larvae are probably subjected to high levels of oxidative stress Live feed production and enrichment is performed under highly pro-oxidative conditions, with high levels of n-3 polyunsaturated acids, air or oxygen addition to the culture water, high temperature and bright light Formulated diets for marine sh larvae also contain high levels of PUFAs and pro-oxidants, for example in the form of minerals The high surface to volume ratio of the feed particles also favours oxidation It is therefore important to supplement marine sh larval diets with vitamin E, but vitamin E at high levels, in the absence of sucient amount of vitamin C, may work as a pro-oxidant, as discussed previously (Hamre et al 1997) The concentration ratio of the two vitamins in copepods, which is the main natural diet of marine sh larvae (110 mg kg)1 vitamin E and 500 mg kg)1 vitamin C, Hamre et al 2008), may give a guideline for supplementation of larval diets An interaction between vitamin E and the dietary level of highly unsaturated fatty acids has been shown in marine sh larvae Betancor et al (2010) fed diets with dierent ratios of DHA (22:6n-3) to vitamin E to sea bass larvae and found that increasing the level of DHA increased the incidence of muscular degeneration, while adding extra vitamin E at the high DHA levels reduced the incidence muscular pathology Furthermore, Atalah et al (2008) found similar variation in mortality, both under normal culture conditions and in response to stress, in sea bass larvae The requirement of vitamin E in marine sh larvae is not known, but Atalah et al (2008) suggested an optimal level of g kg)1 dry diet, because these high levels reduced mortality after stress, but not the mortality during standard rearing conditions This is in line with general opinion that vitamins C and E in larval diets should be at the g kg)1 level, while the requirements given by NRC (1993) for sh are 30 and 25 120 mg kg)1, for vitamins C and E respectively Kolkovski et al (2000) found no positive eects of increasing vitamin E in Artemia from 6.5 to 17 mg kg)1 wet weight (corresponding to approximately 65170 mg kg)1 dry weight) on performance of fresh water walleye larvae Further studies are needed to better understand how the interaction of vitamin E with other nutrients aects the requirement for this vitamin in marine sh larvae An excessive amount of literature exists on eects of nutrition on the immune system and disease resistance in sh, where vitamin E has been found to modulate dierent parts of the immune system and prevent mortalities during disease outbreaks (see reviews by Waagbứ 1994, 2006; Verlhac Trichet 2010) Montero et al (1998) found that the serum alternative complement pathway (ACP) was inhibited in gilthead sea bream decient in a-tocopherol In channel catsh, increasing dietary vitamin E increased the respiratory burst in the form of superoxide anion production but did not aect chemotaxis and phagocytosis of macrophages (Yildirim-Aksoy et al 2008) The interactions between PUFAs and vitamin E and vitamins C and E also seem to aect the responses of the immune system High serum lysozyme activity and lowered ACP caused by crowding stress were normalized by high dietary vitamin C and E in gilthead sea bream (Montero et al 1999) Wahli et al (1998) fed rainbow trout with diets decient, adequate and with excessive levels of vitamins C and E combined and found both increase in the activity of the non-specic immune system and in the protection against infections Protection against virus was even improved with the increase in dietary levels from adequate (30/30 mg kg)1) to excessive (2000/800 mg kg)1) There were also strong and signicant eects of both vitamin E, n-3 PUFA and their interactions on the non-specic immune responses in Japanese ounder, represented by the ACP and lysozyme activities and the respiratory burst The resistance to an infection with Edwardsiella tarda was modulated by the two nutrients in a similar manner as the non-specic immune system and was even improved when dietary vitamin E was increased from 98 to 213 mg kg)1 (Wang et al 2006) In grouper, the non-specic immune response, represented by white blood cell count, respiratory burst, lysozyme activity and ACP activity, showed stepwise increases in activity in sh supplemented by 0800 mg kg)1 vitamin E The requirement based on weight gain was estimated to 60100 mg kg)1, dependent on dietary lipid level (Lin & Shiau 2005) Similar eects on immunity by vitamin E and interacting nutrients are found in in vitro studies Mulero et al (1998) Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd found that migration and phagocytosis of head-kidney leucocytes from gilthead sea bream increased in response to both vitamins C and E concentrations in the media, while the respiratory burst was synergistically enhanced when both vitamins were used together Changes in the ultrastructure of phagocytes in response to the two nutrients were parallel to changes known to occur during activation of the cells Sealey & Gatlin (2002) found no eect of vitamin C or vitamin E concentrations on the extracellular respiratory burst However, intracellular superoxide anion production was modulated by vitamins C and E in interaction The function of vitamin E in the modulation of the immune system could be to scavenge oxidation products, thereby alleviating the eects of oxidation, on both the biochemical and gene expression levels Vitamin E also appears to interact directly with the antioxidant response element on DNA and regulate gene expression of antioxidant enzymes Biological membranes are strengthened by vitamin E through protection of the membrane lipids from oxidation, and prostaglandin synthesis is inhibited through modulation of cyclo-oxygenase gene expression and activity (Meydani et al 2001) According to Waagbứ (2006), it is a common thought that deciency of single nutrients almost always leads to impaired immunity, while supplementation in excess, far above the requirement, leads to improved immunity This line of thought is especially valid for vitamins C and E The high nutrient levels needed for extra protection will, however, limit their use to situations where the sh are exposed to critical conditions, for example during disease outbreaks There have been some trials designed to increase the vitamin E content of sh muscle to improve esh quality and shelf life through protection against lipid oxidation The results of these trials are variable, because induction of lipid oxidation in the llets is dependent on many factors, e.g fatty acid composition of the llet lipid, vitamin E levels, storage conditions and time In some studies, lipid oxidation is evident only after forced oxidation, using iron and ascorbate (Gatlin et al 1992; Gatta et al 2000) However, the resistance to forced oxidation is dependent on the vitamin E contents of the llet Gatlin et al (1992) showed that a-TOH was more ecient in protecting catsh llets against oxidation than synthetic antioxidants, perhaps owing to a better retention in the llet Ru et al (2003) found that vitamin E protected turbot llets stored on ice against lipid oxidation and colour deterioration, while vitamin C had no eect The combina- Aquaculture Nutrition 17; 98115 ể 2010 Blackwell Publishing Ltd tion of low vitamin E and a high level of n-3 polyunsaturated diets gave increased rancid avour and change in colouration in Atlantic salmon llet stored at dierent conditions (Waagbứ et al 1993) The most studied function of vitamin E is its role as a lipid soluble antioxidant, while additional functions in cell signalling, modulation of enzyme activities and gene expression are just in the beginning to be discovered The work in sh is focused on the antioxidant function and has shown that vitamin E interacts with a number of other nutrients Consequently, the requirement of vitamin E increases at high levels of unsaturated lipid and at low levels of vitamin C and selenium Oxidized dietary lipid results in increased consumption of vitamin E and therefore an increased requirement, while pathologies caused by oxidized lipids are partly alleviated by increased supplementation of vitamin E Experiments with feeding of non-a-TOH show that the metabolism of tocopherols in Atlantic salmon is similar to that in mammals, with preferential retention of a-TOH Many thanks to Dr Mari Moren for critical reading of the manuscript Ahmed, Z., Ravandi, A., Maguire, G.F., Kuksis, A & Connelly, P.W (2003) Formation of apolipoprotein AI-phosphatidylcholine core aldehyde Schi base adducts promotes uptake by THP-1 macrophages Cardiovasc Res., 58, 712720 Andersen, H.J., Pellet, L & Tappel, A.L (1994) Hemichrome formation, lipid peroxidation, enzyme inactivation and protein degradation as indexes of oxidative damage 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(Sciaenops ocellatus) Aquaculture, 176, 343353 Williams, K.C., Barlow, C.G., Rodgers, L., Hockings, I., Agcopra, C & Ruscoe, I (2003) Asian seabass Lates calcarifer perform well when fed pelleted diets high in protein and lipid Aquaculture, 225, 191206 Aquaculture Nutrition 17; 212 ể 2009 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2009.00701.x 2011 17; 1323 ... Aquaculture Nutrition 17; 1323 ể 2009 Blackwell Publishing Ltd Lipids (kJ g)1) = )0.005 time2 + 0.1492 time+1.5688 (r2 = 0.77, P < 0.001) Mixture DW (kJ g)1) = )0.0072 time2 + 0.2183 time+4.4689 (r2 = 0. 91, P < 0.001) )1 OW (kJ g ) = )0.0034 time2 + 0.0614 time+16.626 (r2 = 0.52, P < 0.001) Lipids (kJ g)1) = )0.043 time2 + 0.1336 time+1.5857 (r2 = 0.94, P < 0.001) Aquaculture Nutrition 2011 17;... thermophilum Aquaculture, 164, 277288 Jiang, G.J., Yu, R.C & Zhou, M.J (2004) Modulatory eects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus Aquaculture, 2 41, 6175 Karupiah, G., Xie, Q.W., Buller, R.M.L., Nathan, C., Duarte, C & Macmicking, J.D (1993) Inhibition of viral replication by interferon-c-induced nitric oxide synthase Science, 2 61, 14451448... parameters levels in Oreochromis aureus Chin J Vet Sci., 26, 183185 Aquaculture Nutrition 17; 2432 ể 2009 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2009.00704.x 2011 17; 3343 1 1 2 1 3 1 1 NIFES, National Institute of Nutrition and Seafood Research, Bergen, Norway; 2 Department of Aquaculture, Faculty of Fisheries, Cáukurova University, 01330, Adana, Turkey;... Aquaculture Nutrition 17; 1323 ể 2009 Blackwell Publishing Ltd energy equivalents (kJ g)1) during larval development for the three experimental diets T suecica DW (kJ g)1) = 4.5523e0.024 time (r2 = 0. 91, P < 0.001) OW (kJ g)1) = 15.168e0.018 time (r2 = 0.63, P < 0.001) Lipids (kJ g)1) = 0.1305 time+1.2443 (r2 = 0.76, P < 0.01) I galbana DW (kJ g)1) = )0.0089 time2 + 0.2659 time + 4.3537 (r2 = 0. 91,. .. dietary protein and lipid levels in young red tilapia: evidence of protein sparing Aquaculture, 95, 305318 Dias, J., Alvarez, M.J., Diez, A., Arzel, J., Corraze, G., Bautista, J.M & Kaushik, S.J (1998) Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus Labrax) Aquaculture, 1 61, 169186 Du, Z.Y., Liu, Y.J., Tian, L.X., Wang, J.T., Wang, Y & Liang, G.Y (2005)... added to at-bottomed 96 well microtiter plates with 50 lL poly-L-lysine (2 mg mL )1, Sigma, Louis, MO, USA) and centrifuged at 300g for 10 min at 4 C After removing the supernatant, 100 lL of phorbol myristate acetate (PMA) (1 lg mL )1, Sigma, Louis, MO, USA) was added and allowed to react for 30 min at 37 C NBT (100 lL) (3 mg mL )1, Sigma, Louis, MO, USA) was added to the mixture and incubated for 30 min... optimum salinity and temperature conditions Aquac Nutr., 11, 235240 Chou, B.-S & Shiau, S.-Y (1996) Optimal dietary lipid level for growth of juvenile hybrid tilapia Oreochromis niloticus_Oreochromis aureus Aquaculture, 143, 185195 Cockson, A & Bourne, D (1972) Enzymes in the digestive tract of two species of euryhaline sh Comp Biochem Physiol A, 41, 715718 Craig, S.R & McLean, E (2005) The organic movement:... on the growth and fatty acid composition of Tapes philippinarum Aquaculture, 162, 287299 Caers, M., Couteau, P., Sorgellos, P & Gajardo, G (2003) Impact of algal diets and emulsions on the fatty acid composition and content of selected tissues of adult broodstock of the Chilean scallop Argopecten purpuratus (Lamarck, 1819) Aquaculture, 217, 437452 Christie, E.E (1982) Lipid Analysis: Isolation, Separation,... salar) fed diets with partial replacement of sh meal by soy proteins products at medium or high lipid level Aquaculture, 193, 91106 Regost, C., Arzel, J., Cardinal, M., Robin, J., Laroche, M & Kaushik, S.J (2001) Dietary lipid level, hepatic lipogenesis and esh quality in turbot (Psetta maxima) Aquaculture, 193, 291 309 Ribeiro, L., Zambonino-Infante, J.L., Cahu, C & Dinis, M.T (1999) Digestive enzymes