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1 Available online at Aquaculture 273 (2007) Parr smolt transformation and dietary vegetable lipids affect intestinal nutrient uptake, barrier function and plasma cortisol levels in Atlantic salmon Fredrik Jutfelt a,, Rolf Erik Olsen b, Björn Thrandur Björnsson a, Kristina Sundell a a Department of Zoology/Zoophysiology, Göteborg University, PO Box 463, S , Göteborg, Sweden b Institute of Marine Research, Matre Aquaculture Research Station, N-5984 Matredal, Norway Abstract For Atlantic salmon, the gastrointestinal tract is the site of food digestion and nutrient uptake, a regulatory site for ion and water balance as well as a barrier against invading pathogens. During the parr smolt transformation and subsequent seawater (SW) transfer, major changes occur in the intestine. A global shortage of fish oils (FO) for feed production is estimated to appear within a few years, and vegetable oils (VO) are being considered as alternatives for FO in fish feed production. However, VO influences the fatty acid composition of the polar lipids of cell membranes in the intestine which can disturb intestinal functions. A VO-based diet during the parr smolt transformation, which is a sensitive developmental period, may cause adverse effects. Therefore, Atlantic salmon parr were fed either sunflower oil (SO) or FO as the major lipid source during out-of-season light controlled parr smolt transformation. At three time points gill Na +,K + -ATPase activity and plasma levels of cortisol and growth hormone were assessed. Intestinal epithelia were sampled for assessment of nutrient absorption and bacterial translocation using an Ussing chamber in vitro system. While both dietary groups showed plasma hormone profiles indicative of successful parr smolt transformation, the SO-fed fish had consistently increased cortisol levels compared to the FO-fed fish. Translocation of pathogenic bacteria increased, probably due to disturbed barrier functions, during the parr smolt transformation. However, the fish fed the SO-diet maintained a higher barrier function compared to FO-fed fish, an effect that may be beneficial to these fish. Nutrient uptake was less affected by smoltification. Fish fed the SO-diet had higher uptake rates of amino acids and free fatty acids during mid-smoltification than fish fed the FO-diet. The combined effects of barrier function and nutrient uptake may suggest a positive effect of including vegetable lipids in the diet during the parr smolt transformation. However, the vegetable lipid diet also seemed to act as a stressor and elevated the basal cortisol levels, which may be of concern in the context of general fish health and welfare Elsevier B.V. All rights reserved. Keywords: Ussing chamber; Smoltification; Growth hormone; Osmoregulation; Aeromonas salmonicida; Salmo salar; Bacterial translocation; Intestine; Amino acid uptake; Fatty acid uptake 1. Introduction Corresponding author. Tel.: ; fax: address: fredrik.jutfelt@zool.gu.se (F. Jutfelt). The Atlantic salmon parr smolt transformation is a complex process when the fish prepare for downstream migration and a marine life while still in fresh water (FW) (McCormick and Saunders, 1987). A fundamental physiological change which occurs during this developmental /$ - see front matter 2007 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 F. Jutfelt et al. / Aquaculture 273 (2007) period is the increased ability of the fish to osmoregulate in seawater (SW). Being a major organ for maintenance of water and ionic balance, the intestine needs to pre-adapt for its role in SW, i.e. to actively be able to absorb ions and water. During the parr smolt transformation, the intestinal fluid transport increases (Veillette et al., 1995) due to increased Na +,K + -ATPase activity and a slight increase in the epithelial paracellular permeability (Sundell et al., 2003). After adaptation to SW, drinking rate increases (Nielsen et al., 1999; Fuentes et al., 1996) and the intestinal Na +,K + -ATPase activity is further increased concomitant with a decrease in paracellular permeability. This suggests a redirection of the water flow from a paracellular to a more transcellular pathway after a period in SW (Sundell et al., 2003). The increased strength of the physical intestinal barrier is likely to help restrict the passive passage of potentially hazardous substances, including pathogens, from the ingested water. The barrier against pathogens consists of several layers of defense mechanisms including, endogenous microbiota, mucus and antibody secretions, the epithelial immune system and the physical barrier consisting of the epithelial monolayer and the tight junctions (Jutfelt, 2006). Disturbances in any of these components can result in increased translocation of live pathogenic bacteria from the intestinal lumen to the systemic circulation (Berg, 1995). Thus, during the parr smolt transformation and the period immediately after seawater transfer, the changes occurring in intestinal permeability may disturb the barrier function. This is supported by the fact that disease resistance is known to decrease during smoltification and for a short period after seawater transfer (Cipriano and Bullock, 2001). In fish, as in other vertebrates, the gastrointestinal tract is also the site of food digestion and nutrient uptake, and the migration from FW to SW also includes diet changes. In the parr stage, the diet is mainly freshwater crustaceans and insects, while at sea, crustaceans and fish are the main prey (Bell et al., 1994). Thus, the limnic diet is rich in 18:2n-6(n-6) and 18:3(n-3) fatty acids compared with the marine diet which is rich in the longer chain highly unsaturated fatty acids (HUFA) such as 20:5(n-3) and 22:6(n-3) (Sargent et al., 1999). Pre-adaptative changes in both fatty acid metabolism and tissue fatty acid composition have been found to occur during the parr smolt transformation. This includes an increase in fatty acid elongase and desaturase activity of isolated hepatocytes as well as increases in the ratio of C20 and C22:C18 and n-3:n-6 fatty acids in salmonid tissue (Tocher et al., 2000, 2004). In salmon aquaculture, the main source for feed production is wild-caught fish, but there is a need for replacing this with more sustainable feed sources, such as vegetable lipids (Naylor et al., 2000). Fish oil can be replaced with vegetable oils without detrimental effects on fish growth and mortality (Hardy et al., 1987; Dosanjh et al., 1998; Bendiksen et al., 2003). However, the fatty acid profiles of vegetable lipids differ from that of fish oils, in a similar fashion as the natural diet in FW differs from that in SW, and these differences are to a large extent reflected in the fatty acid profiles of muscle and liver (Bell et al., 2001; Dosanjh et al., 1998; Tocher et al., 2000). As the first tissue exposed to, and utilizing, the diet is the gastrointestinal tract, the enterocyte cell membranes may be particularly prone to alterations in dietary lipid composition. Recent studies have found a change in the fatty acid composition of polar lipids of the intestinal mucosa in response to dietary vegetable lipids in both rainbow trout and Atlantic salmon (Olsen et al., 2003; Björnsson et al., 2004). Changes in the fatty acid composition can lead to a range of physiological changes such as altered membrane fluidity (Leray et al., 1984), changes in epithelial enzyme activities (Di Costanzo et al., 1983; Cahu et al., 2000) and decreased barrier function (Barton et al., 1992). The aim of the present study was to investigate the effects of a SO-based diet, expected to alter the membrane lipid composition, on intestinal nutrient absorption and barrier function during the sensitive period of parr smolt transformation. 2. Materials and methods 2.1. Fish and experimental design In this study, Atlantic salmon were monitored through three samplings during out-of-season parr smolt transformation. The experiment started at the Matre Aquaculture station, Matredal, Norway, using juvenile Atlantic salmon of the NLAstrain. The salmon were hatched in mid-january 2003, and reared under continuous light from first feeding (standard commercial diet; Nor-Aqua Innovation Ltd.) in late February. In August 2003, 900 Atlantic salmon parr with an average weight of 42 g were transferred into six 60 L fiber glass tanks supplied with aerated fresh water. The fish were allowed to acclimate for two weeks, during which they were fed a standard commercial diet (Nor-Aqua Innovation Ltd.), at 2% body weight per day, by automatic feeders. On September 15th, the fish (average weight 44 g) were divided into two groups (in triplicates) and the feeding of the two experimental diets was initiated (see below). At the same time, the fish were subjected to a transient, square-wave change in light regime, from continuous light (LL) to short day (12:12LD) for six weeks, and then back to LL for six more weeks. This photoperiod treatment has been proven successful in inducing outof-season smoltification (Hansen, 1998; Björnsson et al., 2000; Sundell et al., 2003).

3 300 F. Jutfelt et al. / Aquaculture 273 (2007) Two weeks before first sampling, the fish were transported to the Institute of Marine Research in Bergen, Norway. Light as well as feeding regimes was maintained throughout the whole experimental period and the temperature was kept at 10 C. Sampling was first carried out just prior to the termination of the 12:12LD period (October sampling), then after three weeks on LL (November sampling), and finally after six weeks on LL regime (December sampling), when parr smolt transformation has been shown to be completed (Berge et al., 1995; Björnsson et al., 2000; Sundell et al., 2003). Each sampling lasted for nine days. Mortality was zero throughout the experiment for both dietary groups Experimental diets Two different experimental diets were used in this study, one was based on fish oil (FO) and the other on sunflower oil (SO). Sunflower oil was chosen because it is a vegetable oil that is readily available for large scale feed production, and is thus a candidate for replacing fish oil. The major fatty acid in SO is 18:2. This fatty acid has previously been shown to affect the fatty acid composition of polar lipids in the intestine (Olsen et al., 2003; Björnsson et al., 2004). Both diets were prepared by the Norwegian Herring Oil and Meal Industry Research Institute in Bergen, Norway using standard commercial recipes. The major ingredients were low-temperature (LT) fish meal (FM 291/02) 61.3 g kg 1, maize suprex 15 g kg 1, vitamin premix 1 g kg 1, mineral premix 0.5 g kg 1, inositol 0.03 g kg 1, betain 0.1 g kg 1, carophyll pink 0.08 g kg 1 and 21.5 g of either FO or SO kg 1. This mixture yielded a calculated composition by dry weight of 28.7% lipid, 50.2% protein, 12.3% carbohydrate and 8.2% ash, and a total energy content of 23.8 MJ kg 1. The pellet size was 3 mm. The fatty acid composition was determined by gas chromatography according to the method of Olsen et al. (2005). The diets differed in fatty acid composition and the major differences are given in Table 1. As expected, the FO-diet contained high concentrations of long chain PUFA while the SO-diet contained high levels of 18:2n-6 and 18:1n-9 (Table 1) Sampling procedures Fish were collected randomly by netting and killed with a blow to the head. Fork length and wet weight were measured. Heparinized syringes were used to collect blood samples from the caudal vessels. After centrifugation at 3000 g for 5 min, the obtained plasma was frozen on dry ice and stored at 80 C until further analyses. Two gill arches from each fish were removed. The filaments were separated from the gill arch and placed into ice-cold SEI buffer (150 mm sucrose, 10 mm Na 2 - EDTA, 50 mm imidazole at ph 7.3). The samples were frozen in liquid nitrogen and stored at 80 C until analyses of Na +, K + -ATPase activity. Intestinal sampling for studies of nutrient uptake and barrier functions, using an Ussing chamber methodology, was done according to Sundell et al. (2003). In brief, the peritoneal cavity of the fish was opened laterally, and the mesenteries and adipose tissue were removed. The Table 1 Fatty acid Fish oil Sunflower oil 16: :1n :2n :1n :5n :1n :6n SAT MONO PUFA n n n-3/n The table shows the fatty acid profiles of total lipid extract from the two experimental diets, the sunflower oil and the fish oil based diet, as measured by gas chromatography. The fatty acids presented are those showing the greatest difference between the diets. Also shown are calculated total saturated fatty acids, total monounsaturated fatty acids, total polyunsaturated fatty acids, n-3, n-6 and the ratio of n-3/n-3. All values are shown as the average percent of total fatty acids. intestine from just posterior to the last pyloric ceca to the anus, was carefully removed, opened longitudinally and placed in an ice-cold, aerated salmon Ringer solution (140 mm NaCl, 2.5 mm KCl, 15 mm NaHCO 3, 1.5 mm CaCl 2, 1 mm KH 2 PO 4, 0.8 mm MgSO 4, 10 mm glucose, 20 mm glutamine and 5 mm HEPES buffer and ph was set to 7.8 with 1.5 mm TRISbase). The intestine was then separated at the ileorectal junction into an anterior and a posterior region, and the proximal half of each region was mounted into the Ussing chambers Plasma cortisol and growth hormone levels Plasma cortisol levels were measured in duplicate, unextracted plasma using radioimmunoassay (RIA) according to Young (1986) and validated by Bisbal and Specker (1991). The cortisol antibodies were purchased from Endocrine Sciences, CA, USA (lot ). Growth hormone levels were analyzed by a RIA specific for Atlantic salmon (Björnsson et al., 1994) Gill Na +,K + -ATPase activity After thawing of the gill samples, the gill tissue was homogenized in 1 ml of SEI buffer containing 0.1% Nadeoxycholate with a glass/glass tissue homogenizer (Contes Glass, Vineland, NJ). The homogenate was centrifuged at 3000 rtf for 30 s, and the supernatant was treated as described by the microassay protocol of McCormick (1993). In short, 10 μl supernatant was added to 200 μl assay medium, with and without 0.5 mm ouabain, in a 96 well microplate. Plates were read at 340 nm using a kinetic protocol for 10 min at room temperature. Protein content was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL).

4 F. Jutfelt et al. / Aquaculture 273 (2007) Ussing chamber protocol To ensure sufficient oxygenation of the tissues and to remove unwanted diffusion barriers, the serosal layer and parts of the longitudinal muscle layer were removed. The procedure was conducted in ice-cold Ringer under magnification, using fine forceps. The intestinal segments were mounted in Ussing chambers and 4 ml of Ringer solution was added to each side of the intestinal epithelium. The area of exposure was 0.75 cm 2. Oxygenation and stirring were ensured by an airlift on both sides of the intestinal segments. The temperature in the Ussing chambers was kept at 10 C, the same as the fish holding temperature, by the use of cooling mantles. The electrical parameters; transepithelial resistance (TER), transepithelial potential (TEP) and short-circuit current (SCC) were measured every 5 min throughout the experimental period (150 min) for continuous monitoring of preparation viability. After mounting, the intestinal preparations were allowed a period of 60 min for stabilization of the electrical parameters before the start of the experiment. The experiment was started (t=0) by renewing the Ringer solution on the serosal side, while the Ringer solution on the mucosal side was substituted with one of the experimental solutions (see below). In order to study the transfer rate of the experimental substances across the epithelium, 50 μl of serosal Ringer solution was sampled at t=10, 15, 20, 50, 80, 85 and 90 min. Samples from the mucosal compartment were withdrawn at t=0 and 90 min. To assess the radioactivity, the samples were added to 4.6 ml of scintillation fluid (Optiphase Hisafe II, Wallac, Finland), in 5 ml plastic scintillation vials, and counted using a liquid scintillation counter (Beckman LS 1801, Sweden) Amino acid uptake The two amino acids L-proline and L-leucine were selected in order to study the uptake of an imino acid and a neutral amino acid, respectively. The uptake of both amino acids was studied simultaneously by using 3 H-labeled L-proline and 14 C- labeled L-leucine. The experimental solution contained 10 μm [2, 3-3 H]-labeled L-proline (Amersham, St. Louis, USA), 10 μm [U- 14 C]-labeled L-leucine (Amersham, St. Louis, USA), 0.1 mm non-labeled L-proline, and 0.1 mm non-labeled L-leucine (Sigma). The specific activity for L-proline was 10 mci mmol 1, and for L-leucine 6 mci mmol Fatty acid uptake The uptake rate of two selected free fatty acids (FFAs) was measured. The fatty acid 18:2n-6 was used as a representative for polyunsaturated fatty acids (PUFAs), due to the high content of 18:2n-6 in the sunflower oil diet, and the fatty acid 16:0 was selected as the unsaturated component for the resynthesis of polar lipids. The experimental solution was a normal Ringer solution devoid of Ca 2+, containing 0.1 mm taurocholate (TC), 70 nm [9,10-3 H]-labeled 16:0 (Amersham, St. Louis, USA) and 15 μm [1-14 C]-labeled 18:2n-6 (Amersham, St. Louis, USA). Non-labeled 16:0 and 18:2n-6 were included at a concentration of 0.01 mm yielding specific activities of 360 mci mmol 1 for 16:0 and 36 mci mmol 1 for 18:2n-6. Normal Ringer solution was used during the stabilization period and at the serosal side of the Ussing chamber also during the experimental period. This approach maintains tissue viability while it avoids Ca 2+ - complex formation with FFAs in Ussing chamber experiments on Atlantic salmon (Björnsson et al., 2004) as well as in rats (Zakelj et al., 2004). In order to form TC-FFA micelles, the experimental solution was sonicated in a water bath at room temperature for 20 min. The solution was thereafter cooled to 10 C before it was added to the mucosal side of the Ussing chambers at t= Translocation of live Aeromonas salmonicida and transport of fluorescent microspheres Fluorescence labeling of the pathogenic bacteria A. salmonicida using fluorescein isothiocyanate (FITC) was performed according to Sjursen et al. (1989). The validation for FITClabeling of A. salmonicida is described by Jutfelt et al. (2006). In short, A. salmonicida ssp. salmonicida were cultured in brain heart broth (Merck, Germany). When the culture reached the optical density 3.7 at 520 nm, it was collected by centrifugation (6000 g, 10 min). The bacteria were incubated in room temperature for 45 min with 0.25 mg ml 1 of FITC (F7250, Sigma) in 10 ml of phosphate buffered saline (PBS). Four cycles of washing with PBS (6000 g, 10 min) were applied to remove non-bound FITC. The fluorescent bacteria were re-suspended in Ringer at a concentration of CFU ml 1 and 4 ml of this experimental solution was introduced to each mucosal compartment of the Ussing chambers (t=0).att=90, 3 ml of the serosal Ringer was collected, centrifuged at 8000 g for 10 min and the pellet was stored at 20 C until fluorescence measurements. Fluorescent microspheres (Molecular Probes, Fluospheres, carboxylate-modified 1.0 μm 505/515 nm) were used at mucosal concentrations equal to the bacteria. Ussing chamber procedures and collection of transported microspheres followed the same protocol as described for the bacteria. Fluorescence for both bacteria and microspheres was measured using a microplate reader (Victor 1420 Multilabel Counter) at 480/535 nm for 1 s. Detection limits were 50 bacteria or 10 microspheres in the serosal compartment after 90 min of exposure Paracellular permeability For assessment of paracellular permeability, the hydrophilic marker molecule 14 C-labeled mannitol (MW: 184, Amersham, St. Louis, USA) was used. Apparent permeability of mannitol was calculated according to Eq. (1), where dq/dt is the steadystate appearance rate of radioactivity in the serosal compartment, A is the area of the epithelium exposed, and C 0 is the concentration of 14 C-labeled mannitol in the experimental solution in the mucosal chamber. P app ¼ dq=dt 1=AC 0 : ð1þ

5 302 F. Jutfelt et al. / Aquaculture 273 (2007) Statistics All data are presented as means±sem and have been subjected to the Levene test for homogeneity of variance. In the case of homoscedasity, the data were subjected to two-factorial analyses of variance. To obtain detailed information about significant differences between sampling points, the Student Neuman Keuls post-hoc test was used. Data sets not showing homoscedasity were subjected to square root, ln and log 10 transformations, and the data sets showing the highest p-values in Levene's test were chosen for further analysis. For data sets resulting in equal variances after transformation, the transformed data were subjected to statistical treatment as described above. For data sets not resulting in equal variances despite of the transformation, a non-parametric statistical analysis is usually employed. However, non-parametric tests for two-way analysis are not available, or require custom-made software. Therefore, these transformed data sets were also analyzed using the parametric two-factorial analysis of variance. The possible risk in this procedure is an increased incidence of Type I error (increased risk for falsifying the null-hypothesis by chance). However, in large experiments with several treatments, sample sizes lager than 6 and similar sample sizes, heterogeneous variances do not noticeably increase the risk for Type I error (Underwood, 1997). These prerequisites are all met in the present study and a two-factorial analysis of variance was therefore the most preferable statistical test available also for data sets not resulting in homoscedasity. The following data sets showed non-homogenous variances after transformation ( p- values for Levene's homogeneity test in brackets): condition factor ( p =0.029), L-leucine uptake rate in the proximal intestine ( p =0.021), L-proline uptake rate in the proximal intestine ( pb0.001), bacterial translocation in the proximal and in the distal intestine ( p=0.008 and p b respectively). All statistical analyses were performed using SPSS 13.0 for Windows and the significance level was set to pb Results 3.1. Growth and smolt status Body weight (Fig. 1A) increased significantly from October to November, and further in December. The same results were obtained for fork length (Fig. 1B). No significant differences were obtained in either weight or length between the FO group and the SO group, and the specific growth rate (SGR=ln(W2/ W1)*100/d) for both weight and length did not differ between the groups during the two growth periods (October November, November December) of the experiment. Condition factor (CF) decreased continuously in both groups. In addition, there was a significant interaction in CF between time and diet ( p=0.003). Thus, the SO group had a faster initial decline in CF, but at the last sampling, the FO group had the lowest CF (Fig. 1C). Samples for gill Na +,K + -ATPase activity were obtained for all sampling points, but samples from the October sampling were Fig. 1. Body weight (A), fork length (B), and condition factor (C) of 0+ age Atlantic salmon fed a fish oil based diet (FO; open symbols) or a sunflower oil based diet (SO; filled symbols). Sampling was performed during photoperiod-manipulated parr smolt transformation under ambient temperature conditions. First sampling point was after five weeks on 12L:12D light regime (20/10). After six weeks on 12L:12D, the fish were subjected to an abrupt change to continuous light (24L). Second sampling was performed after three weeks on 24L (19/11) and third sampling was performed after 6 weeks on 24L (10/12). Data are shown as means±sem (n=45). Data were analyzed using two-way ANOVA and a subsequent SNK post-hoc test when appropriate. Different letters above data points indicate significant differences in time ( pb0.05) and diamonds denote significant interaction between diet and time.

6 F. Jutfelt et al. / Aquaculture 273 (2007) Fig. 2. Plasma levels of growth hormone (A) and cortisol (B) of 0+ age Atlantic salmon fed a fish oil based diet (FO; open symbols) or a sunflower oil based diet (SO; filled symbols). Sampling was performed during photoperiod-manipulated parr smolt transformation under ambient temperature conditions. First sampling point was after five weeks on 12L:12D light regime (20/10). After six weeks on 12L:12D, the fish were subjected to an abrupt change to continuous light (24L). Second sampling was performed after three weeks on 24L (19/11) and third sampling was performed after 6 weeks on 24L (10/12). Data are shown as means±sem (n=9). Different letters above data points indicate significant differences in time ( pb0.05). Asterisks denote significant differences ( pb0.05) between FO and SO-fed fish. Fig. 3. Intestinal uptake rate of A) L-leucine (n=12), B) L-proline (n=12), C) D-glucose (n=12) and D) the fatty acid 16:0 (n=12) in Atlantic salmon fed a fish oil based diet. The examples presented are from the October sampling. All graphs represent anterior intestine (circles) and posterior intestine (squares). Data are presented as means±sem.

7 304 F. Jutfelt et al. / Aquaculture 273 (2007) lost in the transport. For the other time points, gill Na +,K + - ATPase activity increased significantly ( p =0.002) in both dietary groups, from 1.83±0.29 and 1.35±0.13 μmol ADP h 1 mg prot 1 in November to 2.68±0.40 and 2.48±0.28 μmol ADP h 1 mg prot 1, for the FO and SO-fed fish, respectively, in December. No differences in gill Na +,K + -ATPase activity were seen between the two dietary groups Plasma hormone levels TheplasmaGHlevelswererelativelylow,0.28±0.033ng ml 1, in October, but increased substantially thereafter (Fig. 2A). There was no difference in GH levels between the two experimental diets, and no significant interaction between time and diet. Plasma cortisol levels increased from October to November, to reach the highest levels of 21.87±6.81 and 43.79±8.01 ng ml 1 for FO and SO-fed fish, respectively. In December, the cortisol levels declined again (Fig. 2B). The plasma cortisol levels differed significantly between the diet groups at each sampling point, with the SO-fed fish having higher cortisol levels than FO-fed fish at all sampling points. No significant interaction between time and diet was obtained Ussing chamber experiments Viability data and paracellular permeability For viability control of intestinal preparations in the Ussing chamber, the electrical parameters PD, TER and SCC were used. In all Ussing chamber preparations, the intestinal segments displayed stable PD, TER and SCC values, indicating good viability. TER and the apparent permeability (P app ) for the hydrophilic molecule 14 C-labeled mannitol further function as internal standards for preparation integrity, and can be used to compare the status of the in vitro preparations with previous experiments. The two parameters showed the same range as previously reported for smoltifying Atlantic salmon (Sundell et al., 2003). None of the experimental mucosal solutions used, significantly altered the TER or P app for mannitol, indicating that viability and integrity of the intestinal preparations were unaffected (data not shown) Amino acid uptake There was a large difference in the uptake rate of amino acids and fatty acids between the anterior and the posterior regions of the intestine, with accumulation in the serosal solution being approximately an order of magnitude greater in the anterior than in the posterior intestine (Fig. 3A, and B). The uptake rates by the posterior intestine followed a linear relationship and were close to the limit of detection. This suggests the predominant component of the uptake to be due to passive diffusion, which is of limited physiological significance. Therefore, only data from the anterior intestine are presented and discussed. The intestinal uptake rate of the amino acid L-leucine during the parr smolt transformation is presented in Fig. 4A. The experimental diet had a significant (p=0.001) effect on the uptake rate, with an overall increased uptake rate in the fish fed SO and a significant ( pb0.0005) interaction between time and Fig. 4. Anterior intestinal uptake rates of L-leucine (A) and L-proline (B) of 0+ age Atlantic salmon fed FO (open bars) or SO (filled bars). Sampling was performed during photoperiod-manipulated parr smolt transformation under ambient temperature conditions. First sampling point was after five weeks on 12L:12D light regime (20/10). After six weeks on 12L:12D, the fish were subjected to an abrupt change to continuous light (24L). Second sampling was performed after three weeks on 24L (19/11) and third sampling was performed after 6 weeks on 24L (10/12). Data are shown as means± SEM (n=12). Asterisks denote significant differences (pb0.05) between FO and SO-fed fish and diamonds denote significant interaction between diet and time.

8 F. Jutfelt et al. / Aquaculture 273 (2007) diet. This interaction indicates different responses to the experimental diet during the different stages of the parr smolt transformation. The largest diet effect was found in November, where the FO-fed fish had less than half of the uptake rate of the SO-fed fish (Fig. 4A). In December, the effect of the diet was reversed. A similar pattern of uptake rate was also found for L-proline (Fig. 4B). However, the effects were less pronounced, and only the interaction between time and diet was significant ( p b ). L-proline uptake rate was affected by diet primarily in November, where the SO-fed group had twice the uptake rate of the FO group Fatty acid uptake The uptake rates of fatty acids across the epithelium were clearly divergent in the two intestinal regions. The posterior intestine showed negligible uptake rates compared with the anterior intestine (Fig. 3D) and data from the posterior intestine will thus not be presented further. A difference in uptake rate was found between the fatty acids 16:0 and 18:2n-6, with the uptake of 18:2n-6 being approximately four times faster than the uptake of 16:0. This difference was consistent throughout the experiment. The uptake rate of linoleic acid (18:2), the most abundant fatty acid in the SO-based diet, as well as palmitic acid (16:0) was not affected by smoltification or diet per se (Fig. 5A and B). There was, however, a significant interaction between time and diet for 18:2n-6 (p=0.032) and a similar trend for 16:0, although not statistically significant ( p = 0.080) Bacterial translocation and microsphere transport There was a clear difference in rate of bacterial translocation compared with the transfer of microspheres, with bacteria generally being transported at higher rates than microspheres (Figs. 6 and 7). Bacterial translocation differed between the two intestinal regions sampled (Fig. 6A and B). In the posterior intestine, on average 0.38% of the bacteria was translocated during 90 min, while the anterior intestine translocated on average 0.12%. Apart from having the higher translocation rate, the posterior intestine also showed a higher sensitivity to both developmental and dietary changes. The bacterial translocation rate in October was significantly ( p=0.003) lower than in November and December (Fig. 6B). Furthermore, the posterior intestine had overall a significantly ( p = 0.046) lower translocation rate in SO-fed fish compared with FO-fed fish. No significant interaction between time and diet was observed (Fig. 6B). In the anterior intestine, no significant effects were present for either factor (Fig. 6A). The transport rate of inert fluorescent microspheres increased significantly in the posterior intestine from October to November as well as from November to December ( pb0.0005; Fig. 7B). Diet did not significantly affect the transport rate, but there was a Fig. 5. Anterior intestinal uptake rates of the free fatty acids 16:0 (A) and 18:2n-6 (B) of 0+ age Atlantic salmon fed FO (open bars) or SO (filled bars). Sampling was performed during photoperiod-manipulated parr smolt transformation under ambient temperature conditions. First sampling point was after five weeks on 12L:12D light regime (20/10). After six weeks on 12L:12D, the fish were subjected to an abrupt change to continuous light (24L). Second sampling was performed after three weeks on 24L (19/11) and third sampling was performed after 6 weeks on 24L (10/12). Data are shown as means±sem (n=12). Diamonds denote significant interaction between diet and time.

9 306 F. Jutfelt et al. / Aquaculture 273 (2007) Fig. 6. Translocation of Aeromonas salmonicida in anterior intestine (A) posterior intestine (B) of 0+ age Atlantic salmon fed FO (open bars) or SO (filled bars). Sampling was performed during photoperiod-manipulated parr smolt transformation under ambient temperature conditions. First sampling point was after five weeks on 12L:12D light regime (20/10). After six weeks on 12L:12D, the fish were subjected to an abrupt change to continuous light (24L). Second sampling was performed after three weeks on 24L (19/11) and third sampling was performed after 6 weeks on 24L (10/12). Data are presented as means±sem (n=12). Different letters above data points indicate significant differences in time (pb0.05). Asterisks denote significant differences (pb0.05) between FO and SO-fed fish and diamonds denote significant interaction between diet and time. tendency towards decreased transport rates in the SO-fed group compared with the FO-fed group in October and November. The same tendency was also seen in the anterior intestine, where the SO-diet appeared to decrease transport rates at all sampling points, but the effect was not statistically significant ( p=0.15; Fig. 7A). Fig. 7. Uptake rates of microspheres in anterior intestine (A) posterior intestine (B) of 0+ age Atlantic salmon fed FO (open bars) or SO (filled bars). Sampling was performed during photoperiod-manipulated parr smolt transformation under ambient temperature conditions. First sampling point was after five weeks on 12L:12D light regime (20/10). After six weeks on 12L:12D, the fish were subjected to an abrupt change to continuous light (24L). Second sampling was performed after three weeks on 24L (19/11) and third sampling was performed after 6 weeks on 24L (10/12). Data are presented as means±sem (n=12). Different letters above data points indicate significant differences in time (pb0.05).

10 F. Jutfelt et al. / Aquaculture 273 (2007) Discussion A recent major development in intensive Atlantic salmon aquaculture is the induction of an out-of-season parr smolt transformation of large underyearling (0+ age) fish by photoperiod manipulation, as has been used in the present study. This procedure has been shown to induce physiological and endocrine changes comparable to natural smoltification (Berge et al., 1995; Björnsson et al., 2000; Sundell et al., 2003). In the present study, the low plasma GH levels observed at the end of the photoperiod simulated winter, together with the rapid and protracted increase in GH levels during the subsequent return to LL, are well in line with previous data from 0+ smolts (Björnsson et al., 2000). The transient increase in plasma cortisol levels, the increased gill Na +, K + -ATPase activity, and the general reduction in condition factor after return to LL, are in line with previous reports on both natural (McCormick, 1996; Sundell et al., 2003) and photoperiod-induced smoltification (Berge et al., 1995; Björnsson et al., 2000; Sundell et al., 2003). Thus, the underyearling fish in the present study completed a successful parr smolt transformation during the period covered by the three sampling points. A major new finding of the present study is the consistently elevated plasma cortisol levels of fish fed sunflower oil, compared with fish fed a traditional fish oil diet. Thus, in spite of the similar growth rate observed for both groups, it appears that a diet containing sunflower oil as the main lipid source may act as a chronic stressor for the fish. While this is the first observation of elevated basal cortisol levels in fish fed vegetable oil diet, it is notable that in seabream, fish on a linseed oil based diet react to acute stress with higher plasma cortisol levels than fish on a fish oil based diet (Montero et al., 2003). The mechanisms behind these increased cortisol levels due to vegetable oils are unknown. However, vegetable lipids are known to alter membrane composition and to reduce the availability of precursors for eicosanoids in fish (Bell et al., 1992; Henderson, 1996; Dosanjh et al., 1998; Tocher et al., 2000). Thus, it is possible that changes in membrane functions or decreases in some eicosanoids may act as chronic stressors and lead to the observed increases in cortisol levels. At the level of the intestine, chronic stressors leading to increased corticosteroid levels are known to reduce the intestinal barrier function and lead to increased translocation of pathogens in mammalian studies (Groot et al., 2000; Söderholm and Perdue, 2001; Velin et al., 2004). In fish the effects of corticosteroids on the intestinal barrier function have not been sufficiently investigated. However, there are indications of a cortisol-mediated regulation of intestinal permeability in salmonids. Acute stress increases the paracellular permeability of the Atlantic salmon intestine, an effect that may be mediated through cortisol (Olsen et al., 2005). Further, the paracellular permeability of the intestine has been shown to increase after treatment with slow-release cortisol implants in rainbow trout (K. Sundell, unpublished results). A direct correlation between increased paracellular permeability of the intestinal epithelium and an increased bacterial translocation could not be demonstrated in the present study. Instead, a lowered bacterial translocation rate was demonstrated in the fish fed SO. This may seem contradictory, but there are several possible scenarios that may explain such a pattern. An increased intestinal leakage would enhance antigen presentation to the intraepithelial immune system. This may induce the immune functions and result in a reduced bacterial translocation rate, as has been shown in mammals (Söderholm and Perdue, 2001). The results could also reflect an effect of the vegetable oil diet on epithelial transport functions. Atlantic salmon fed a SO-based diet showed a decreased ratio of 20:5n-3:20:4n-6 of membrane polar lipids (Björnsson et al., 2004). Such changes in membrane phospholipids could alter membrane fluidity and represent a mechanism for disturbance of the epithelial barrier function. Bacteria are thought to translocate across the mammalian intestinal epithelium mainly through transcytosis (Kucharzik et al., 2000). The antigen transporting phagocytotic epithelial cells of mammalian intestines, M-cells (Baumgart and Dignass, 2002), have not been found in fish. However, endocytosis of bacteria by intestinal epithelial cells has been shown in two salmonids; the rainbow trout (Vigneulle and Laurencin, 1991) and the Arctic charr (Ringø et al., 2001). Hence a similar translocation process, as seen in mammalian M-cells, may be present also in enterocytes of the Atlantic salmon. The mechanisms behind transcellular transports, endocytosis and exocytosis are processes involving several specific membrane-bound enzymes as well as membrane reorganization. Membrane fluidity and the function of membrane-bound enzymes may be affected by changes in phospholipid fatty acid profiles induced by a vegetable lipid diet as used in this study (Bell et al., 1986). The observed decrease in translocation rates of bacteria may therefore be an indication of disturbed endocytosis and/or exocytosis mechanisms. It would be interesting to investigate if vegetable oil diets may confer some protection against pathogens by any of these mechanisms. Bacterial translocation and transport of microspheres were markedly different between the two intestinal regions. The posterior region had higher translocation

11 308 F. Jutfelt et al. / Aquaculture 273 (2007) rates, which could be explained by higher rates of phagocytosis in the posterior region of salmonids (Buddington et al., 1997; Georgopoulou et al., 1988) assuming that the bacterial translocation in fish mainly occurs transcellularly (Jutfelt et al., 2007; Ringø et al., 2003), though paracellular translocation or disruption of the epithelium has also been suggested (Ringø et al., 2004). Translocation of bacteria and transport of bacterialsized particles in the posterior intestine significantly increased with time. The highest translocation rates were found in December, at the end of the smoltification process. During this period, the transient peak in plasma cortisol levels is still apparent and concomitant with a decrease in intestinal paracellular permeability (Sundell et al., 2003). The increased cortisol levels may also lead to decreased immune competence of the fish (Barton and Iwama, 1991). Sensitivity to infections has indeed been shown to increase during this life stage (Inglis et al., 1993; Smith, 1993). The increased translocation rates of the posterior intestine during the parr smolt transformation may thus be the result of a disturbance in the barrier functions, as well as a decreased immune competence within the epithelium, which may provide a mechanism for the increased disease susceptibility during this period of the Atlantic salmon life cycle. During parr smolt transformation, a difference was found in the uptake of the two amino acids studied, with L- leucine consistently being transported at twice the rate of L-proline. Evolutionary theory predicts that intestinal transporting capacities will match luminal concentrations of the transported substance. The intestinal epithelium should be able to transport nutrients rapidly while not having excess transporting capacities, as there is a cost associated with maintaining transporter enzymes (Diamond, 1991). The amount of brush-border membrane amino acid transporters should thus be modulated to fit with normal luminal concentrations of amino acids. The higher transporting rate of L-leucine in comparison with L- proline fits well with the higher concentrations of L- leucine found in the Atlantic salmon anterior intestinal lumen after feeding a regular commercial feed (Berge et al., 2004). In the present study, the uptake rates of amino acids were significantly reduced in FO-fed fish. Interestingly, the most pronounced effect occurred during midsmoltification, which possibly may be a side-effect of other physiological changes occurring in the epithelium at that stage. The SO-fed fish, however, did not go through the transient decrease in uptake rate, suggesting a positive effect of the SO-diet in maintaining a high uptake rate of amino acids throughout the parr smolt transformation. If this increase in uptake that is beneficial to fish health and growth is unclear, then this warrants further investigation. Both amino acids display the same pattern of uptake rates during smoltification, and both respond to the diets in the same way. The uptake mechanisms for L-leucine and L- proline are thought to be mediated by neutral and imino acid transporters respectively (Collie and F.R.P., 1995). As the two pathways are affected similarly, it can be argued that the diet may induce changes to a part of the uptake which is shared by both amino acids. Another possibility is a general change in the epithelial membrane fluidity caused by the different fatty acid profiles of the diet which may affect membrane-bound amino acid transporter enzymes in a similar manner. There was a significant interaction between smolt status and diet regarding the uptake of both L-leucine and L-proline. The amino acid transport rates in the SOfed group declined at the last sampling point. This may indicate that possible positive effects of vegetable lipids on nutrient uptake seen during the smoltification are reversed towards the end. This coincides with the expected natural change in diet from limnic lipids to marine lipids, suggesting that a shift in the optimal fatty acid composition of the diet occurs during this time. The anterior intestinal region is morphologically and physiologically distinct from the posterior intestine, and the luminal content is also different in the two intestinal regions. Several studies show that the pyloric caeca (not investigated in this study) and the anterior intestine are the intestinal regions responsible for the greatest part of the nutrient absorption in salmonids (Buddington and Diamond, 1987; Collie and F.R.P., 1995). The present study supports these findings as the nutrient (amino acid and FFA) uptake was substantially greater in the anterior intestine. The contribution by the posterior intestine to the total amino acid and FFA uptake is thus suggested to be of negligible physiological importance. This is in contrast with two recent studies assessing L-proline accumulation in everted sleeve gut segments from Atlantic salmon, where no or relatively minor differences between the anterior and posterior regions were found (Nordrum et al., 2000; Bakke et al., 2000). This discrepancy with the present results could be due to methodological differences, as only uptake into the enterocytes, and not absorption across the epithelium, was measured in the everted gut sleeve studies. Further, a relatively high concentration (10 mm) of L-proline was used in the study by Nordrum et al. (2000). At the level of 10 mm, the diffusional component of the uptake is larger than the active component (Berge et al., 2004) whereas at a level of 0.1 mm, as used in the present study, the carrier-mediated transport is thought to be the dominating uptake mechanism.

12 F. Jutfelt et al. / Aquaculture 273 (2007) The uptake rates of the fatty acid 18:2n-6 were consistently higher than the uptake rates of the fatty acid 16:0. This may indicate higher affinities of fatty acid binding proteins (FABP) in brush-border membranes to unsaturated fatty acids (Richieri et al., 2000), or faster diffusion across brush-border membranes by unsaturated fatty acids (Stremmel, 1988; Olsen and Ringø, 1997; Stahl et al., 1999; Ho and Storch, 2001). The observed difference may also have a methodological basis as the solubility of 18:2n-6 is much higher in aqueous solutions, and 18:2n-6 is thus easier to incorporate into TC micelles than 16:0. The availability of the two fatty acids to the epithelium may thus differ in favor of 18:2n-6. Theuptakerateof18:2n-6co-varieswithtimeand thus with developmental stage of the fish. During midsmoltification, the SO-fed fish have higher FFA uptake rates. That implies a transient period during midsmoltification when the availability of vegetable lipids maybebeneficialtothefattyaciduptake,andduring this same period, the amino acid uptake is also increased by the SO-diet. A prior study on Atlantic salmon parr smolt transformation shows enhanced osmoregulatory function by dietary vegetable lipids (Tocher et al., 2000). It has been suggested that vegetable lipids may better reflect the fatty acid profiles of the regular freshwater diets of salmonids, with the implication that the inclusion of vegetable lipids in the diet may be beneficial to the health and growth of salmonids at early life stages (Bell et al., 1994; Tocher et al., 2000). This view is supported by the results on nutrient uptake and barrier function in the present study. In conclusion, the present study demonstrates that many aspects of intestinal functions are modulated during the parr smolt transformation as well as by dietary vegetable lipids. Translocation of pathogenic bacteria increases as barrier function declines throughout the smoltification, with possibly harmful consequences for the fish. A sunflower oil containing diet has positive effects on amino acid transport and uptake of free fatty acids during mid-smoltification. Bacterial translocation is lower in SO-fed fish, indicating a possibly protective role for SO against enteric infections. However, the vegetable lipid diet also acts as a stressor and induces chronically elevated cortisol levels, which is of concern in the context of general fish health and welfare. Acknowledgements We would like to thank: Torgny Bohlin and Henrik Sundh for helpful discussions on statistical analyses, Henrik Sundh and Barbro Egnér for technical assistance and Hari Rudra and Ivar Helge Matre for technical assistance and fish handling. The work was funded by the EU project GUTINTEGRITY (Q5RS ) under the Quality of Life and Management of Living Resources program and by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning to K. Sundell (dnr:22.3/ ). The work has been carried out with financial support from the Commission of the European Communities, but does not represent the opinion of the European Community, which is thus not responsible for any use of the data presented. References Bakke, M.A.M., Nordrum, S., Krogdahl, A., Buddington, R.K., Absorption of glucose, amino acids, and dipeptides by the intestines of Atlantic salmon (Salmo salar L.). Fish Physiol. Biochem. 22, Barton, B.A., Iwama, G.K., Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteriods. Rev. Fish Dis. 1, Barton, R.G., Cerra, F.B., Wells, C.L., Effect of a diet deficient in essential fatty acids on the translocation of intestinal bacteria. JPEN. J. Parenter. Enteral. Nutr. 16, Baumgart, D.C., Dignass, A.U., Intestinal barrier function. Curr. Opin. Clin. Nutr. Metabol. Care 5, Bell, M.V., Henderson, R.J., Sargent, J.R., The role of polyunsaturated fatty acids in fish. Comp. Biochem. Physiol., B 83, Bell, J.G., Sargent, J.R., Raynard, R.S., Effects of increasing dietary linoleic acid on phospholipid fatty acid composition and eicosanoid production in leucocytes and gill cells of Atlantic salmon (Salmo salar). Prostaglandins Leukot. Essent. Fat. Acids 45, Bell, J.G., Ghioni, C., Sargent, J.R., Fatty acid compositions of 10 freshwater invertebrates which are natural food organisms of Atlantic salmon parr (Salmo salar): a comparison with commercial diets. Aquaculture 128, Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R., Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr. 131, Bendiksen, E.A., Berg, O.K., Jobling, M., Arnesen, A.M., Masoval, K., Digestibility, growth and nutrient utilisation of Atlantic salmon parr (Salmo salar L.) in relation to temperature, feed fat content and oil source. Aquaculture 224, Berg, R.D., Bacterial translocation from the gastrointestinal tract. Trends Microbiol. 3, Berge, A.I., Berg, A., Fyhn, H.J., Barnung, T., Hansen, T., Stefansson, S.O., Development of salinity tolerance in underyearling smolts of Atlantic salmon (Salmo salar) reared under different photoperiods. Can. J. Fish Aquat. Sci. 52, Berge, G.E., Goodman, M., Espe, M., Lied, E., Intestinal absorption of amino acids in fish: kinetics and interaction of the in vitro uptake of L-methionine in Atlantic salmon (Salmo salar L.). Aquaculture 229,