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Aquaculture Research, 2012, 1–16

doi:10.1111/j.1365-2109.2012.03170.x

Influence of long term ammonia exposure on Atlantic salmon (Salmo salar L.) parr growth and welfare
Jelena Kolarevic1, Roger Selset1, Olga Felip3, Christopher Good4, Kevin Snekvik5, ˚ ˚ Harald Takle2, Elisabeth Ytteborg2, Grete Bæverfjord1, Torbjørn Asgard1 & Bendik Fyhn Terjesen1
1 2 3 4 5

Nofima AS, Sunndalsøra, Norway ˚ Nofima AS, As, Norway University of Barcelona, Department of Animal Physiology, Barcelona, Spain The Conservation Fund’s Freshwater Institute, Shepherdstown, WV, USA Washington Animal Disease Diagnostic Laboratory, Department of Veterinary, Microbiology and Pathology, College of

Veterinary Medicine, Washington State University, Pullman, WV, USA
Correspondence: J Kolarevic, Nofima AS, NO-6600 Sunndalsøra, Norway. E-mail: jelena.kolarevic@nofima.no

Abstract
The objective of this study was to determine the long-term effects of ambient unionized ammonia nitrogen (NH3-N) combined with different feeding regimes on Atlantic salmon Salmo salar L parr growth, welfare and smoltification. Previous studies on the parr stage of Atlantic salmon have mostly focused on acute exposure, or at low temperatures. Atlantic salmon parr were exposed for 105 days (at 12°C, pH 6.8) to four sublethal ammonia concentrations ranging from 0.1 to 35 lg LÀ1 NH3-N (0.1–25 mg LÀ1 TAN) at two feeding levels: full feed strength (+20% overfeeding) and 1/3 of full feed strength. After 21 days, it was observed that 32 lg LÀ1 NH3-N reduced growth rate of parr fed full ration, but this effect was not evident at the end of the exposure. Feed utilization was not affected by ammonia exposure at any sampling point. Increasing ammonia levels were associated with a higher prevalence and severity of gill damage at 22 days but not at the end of the exposure. The examination of welfare indicators revealed only a few pathologies, not related to ammonia exposure. In addition, higher ammonia concentrations did not appear to influence the development of hypoosmoregulatory ability during parr-smolt transformation.

Introduction Ammonia is the main nitrogenous waste product in most teleosts and it is mainly excreted through gills to the surrounding water (Randall & Wright 1987; ˚ Fivelstad, Kellevik, Iversen, Møretrø, Vage & Binde 1993; Randall & Tsui 2002; Wilkie 2002; Eddy 2005). Ammonia is a toxicant with negative effects on teleosts dependent on e.g. the species, developmental stage, feeding rate, swimming activity and other factors (Wright & Fyhn 2001; Finn 2007; Terjesen 2008). In intensive high-density flow-through systems ammonia build-up can cause reduced growth and mortality (Person-Le Ruyet, Galland, Le Roux & Chartois 1997). In water, total ammonia exists as the sum of the un-ionized (NH3) and ionized (NH4+) forms whose equilibrium varies with pH, temperature and salinity. Toxicity, of total ammonia nitrogen (TAN) in the environment, increases with the concentration of the more toxic NH3-N (Randall & Tsui 2002; Eddy 2005). Recommended safe levels of TAN and NH3-N are given for a number of fish species based on acute toxicity studies in controlled environmental conditions (USEPA 1998; Norwegian Food Safety Authority 2004). Knoph (1995) conducted a series of experiments to determine effects of acute ammonia toxicity on Atlantic salmon parr and post-smolts. However, the effects of chronic exposure to sublethal ammonia levels have not been well documented, especially for Atlantic salmon (Salmo salar) in freshwater at relevant production temperatures (Terjesen, Ulgenes, Fjæra, Summerfelt,

Keywords: ammonia exposure, Atlantic salmon, growth, welfare, gill histology, smoltification

© 2012 Blackwell Publishing Ltd

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Adams. Huntingford. One month prior to the experiment.5°C. Brauthwaite. time of the year and the environment to which fish are exposed to (Huntingford et al. Recently it was shown in two separate experiments that long-term exposure to low levels of exogenous ammonia can promote growth in rainbow trout (Oncorhynchus mykiss) without a change in feed intake (Wood 2004). The objective of this study was to examine the effects of long-term ammonia exposure. The ‘3R’s’ principle (Russell & Burch 1959) was applied and humane end points were established prior to the start of the experiment. K+ 2. unlike feeding to satiation.20 ± 0. 1–16 2 . Atlantic salmon parr (6. The fish were reared in 150 L tanks with fresh water from ground wells (ion concentrations in mg LÀ1: Mg2+ 3.02 (±SEM). Bergen. feeding and combination of these two factors on growth. Ca2+ 4.5) at 12. Influences of different feeding regimes on ammonia tolerance of Atlantic salmon are largely unknown. Aquaculture Research.03 g) were transferred to the experimental facilities at Nofima. Bron & Huntingford 2005. Materials and methods Experimental animals and rearing conditions Atlantic salmon used in this experiment originated from the Salmobreed strain 3. Different water ammonia concentrations were achieved by using silicone and marprene tubes with appropriate diameter sizes delivering on average 26. Aquaculture Research. 1–16 Brunsvik. a system for ammonia dosage was set up and tested. ten fish from each tank were euthanized using 100 mg LÀ1 of tricaine © 2012 Blackwell Publishing Ltd. Johansen et al. The question of fish welfare in aquaculture and scientific research is becoming increasingly important (EFSA 2008). Wicks and Randall (2002b) showed that feeding protects juvenile rainbow trout from ammonia toxicity during the first 24 h of exposure to high environmental ammonia and that fasting exacerbates ammonia toxicity. Johansen. Poppe & Smith 2006). Na+ 48. The welfare concept is complex. SO42À 21. NC. Nawata & Wood 2010). with several definitions that have focus on the animal’s condition. Takle. These conditions were maintained during the whole experiment. medium and low ammonia concentration treatments respectively. The Research Council of Norway 2009). Feeding was done by automatic band feeders for 45 min every 8 h using a commercial pelleted feed that varied in size during the trial according to the fish size (Table 1).Wilmington. Kudri. Watson Marlow Bredel. The use of experimental animals for this trial was approved by the Norwegian Animal Research Authority. 2012. Adams. 21 g (NH4)2SO4 LÀ1 fresh water) into three header tanks at a constant speed of 150 rpm.Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. Nerland. Bæverfjord. Norway) in February 2009 under conditions of continuous light (LD 24:0) and at a water temperature ranging from 13. USA) was used to deliver a stock solution of (NH4)2SO4 from the mixing tank (Iwaki Unit.11°C ± 0.2. Water flow was set to 3 L minÀ1 for each of the experimental units and flow was monitored on a monthly basis until the end of the experiment. Alevins were first fed with a formulated commercial diet (Ewos. Zimmer. Delivered doses of the stock solution were measured five times a week and the tubes were replaced if the observed oscillations exceeded 10% of the desired flow. However. life stage. pH of 6. 12. welfare and smoltification of farmed Atlantic salmon in fresh water. Water from each header tank was supplied to six experimental tanks.5.84 ± 0.0 to 13.2. Norvik & Kittelsen 2008).9. Pottinger. Needham. A number of recommendations have been made regarding the welfare of fish under production conditions. Experimental design and sampling Prior to the start of the experiment. This finding challenges the traditional view that ammonia is always damaging for fish. A peristaltic pump (520DuN. there is still a need to validate the most commonly used welfare indicators in relation to gender. Sandøe & Turnbull 2006). did not induce the growth promoting effect of exogenous ammonia in Wood’s (2004) long-term ammonia exposure study. 2006. Colquhoun. Restricted feeding. The fish were hatched in December 2008 at the Nofima research station in Sunndalsøra (Norway). At the start of exposure.0 mL minÀ1 of (NH4)2SO4 to header tanks for high. Several authors have emphasized the effect of feeding on acute ammonia toxicity (Wicks & Randall 2002a.V-06 MB (L) located ˚ in Mjaneset (Norway). Bell. Sunndalsøra and randomly distributed into 24 similar experimental tanks (55 fish per tank). 2006. on its subjective experience of that condition and/or on whether it can live a natural life (Turnbull.02 and 24 h continuous light.4.0 and 7. and on methods to assess welfare indicators (The Farm Animal Welfare Council 1996.

6 14. *Asx represents Asp and Asn.5–35& salinity.6 Leu 36.7 Met 13. the remaining fish were subjected to a photoperiod manipulation treatment following the exposure experiment. fish were exposed to a photoperiod with 12 h of light and 12 h of darkness (LD 12:12). During 7 weeks. USA) and individually measured for weight (0. 42 and 63 days.9 19.6 4.4 79.5 Crude lipid 207.5 20. Redmond.4 21. containing three or four fish from each of the previous ammonia treatments.8 12. † Glx represents Glu and Gln.9 Ser 20. The same procedure was repeated after week five (SWT2) and week six (SWT3). gill condition and externally visible/palpable deformities.4 His 12. the fish were not fasted (Wood 2001) and calculated biomass was therefore corrected for removed gut content.2).4 22. bulk weight for each tank was noted before. Waste feed was collected and weighed © 2012 Blackwell Publishing Ltd. Aquaculture Research. Atlantic salmon parr (45 fish per tank) with an average individual weight (SEM) of 17.7 Pellet size (mm) 1. 1–16 Effects of ammonia exposure on Atlantic salmon J Kolarevic et al.6 Asx* Cys 5. During the experiment.9 25.1 4. WA. After 105 days of exposure. three tanks in which the fish received full feed strength (+20% overfeeding) at expected growth capacity according to ClubN 2002 (Skretting 2007) and three tanks in which fish received only 1/3 of the above mentioned feed strength.9 34.5 18.7 2.7 11.6 Gly 27.1 82.3 Pro 22. the fish were distributed in three circular tanks.1 4.3 daily and feed intake was determined according to Helland.3 26.5 40.1 70. The static sea water used in this test was made by adding artificial sea-salt to reach 34.9 Ash 90. Grisdale-Helland and Nerland (1996).2 Dry matter (DM) 947.4 926.5 Tyr 15. fish from the first tank were exposed to a 72-h seawater challenge test (SWT1). the fish were inspected for fin condition.2 18. however. fish sampling.5 25. skin lesions.8 11.7 23. pH 7.4 Hydrolized dispensable amino acids Ala 27. After 4 weeks of continuous light.9 33. while ten fish were removed after weighing at 84 and 105 days.3 Glx† 75. were exposed to four different ammonia concentrations ranging from 0.2 478. 2012.9 Val 24.1–25 mg LÀ1 TAN) during 105 days.1 cm accuracy).2 34. with fish from the remaining two tanks.4 77.5 68.4 84. the fish were counted and weighed in bulk each third week.5 Phe 22.1 24.3 25.3 21.4 (0.9 Hydrolized indispensable amino acids Arg 29.3 17.3 17. Prior to weighing. Each ammonia treatment (Table 2) was represented with six tanks. After the initial sampling.5 Crude protein 505. and after.3 Trp 5.9 240.8 Ile 22.1) g.1 to 35 lg LÀ1 NH3-N (0. Gill samples were also collected at day 22 and day 105 from three fish from each tank and subsequently fixed in phosphate-buffered formalin (4%.1 212.0 43.1 45.4 459. followed by 6 weeks of continuous light regime (LD 24:0).0 32. respectively. to reduce the biomass and prevent the oxygen saturation from falling below 85%. Ten fish from the initial sampling and ten fish from each tank after 105 days were sampled for body composition analysis and stored at À20°C.1 20. Ten fish from each experimental tank were used for external assessment of fish welfare at the end of the exposure period.1 g accuracy) and length (0.Aquaculture Research.1 Starch 80.8 13.3 12. In addition. ten fish from each tank were individually PIT-tagged and placed in a circular tank (2 m2) with running freshwater and subjected to a photoperiod treatment to induce smoltification.8 926. Immediately after euthanasia with 100 mg LÀ1 of MS 222.7 4. methane sulphonate. Full randomization of fish tanks was prevented by system infrastructure limitations.2 21.5 Thr 19. Five fish from each tank were individually measured for length and weight after 21. cataract. MS 222 (Argent Chemical Laboratories. 1–16 3 . Table 1 Pellet sizes and analysed chemical composition of the experimental diets (in g kgÀ1) Diet Pellet size 1 Pellet size 2 Pellet size 3 2. At the start of the continuous light period.7 28. Smoltification and seawater challenge test To examine if the long-term ammonia exposure influenced development of hypo-osmoregulatory ability during parr-smolt transformation.5 Lys 37. each of the four blocks of tanks contained two of eight randomly selected treatments.

Aquaculture Research. Lund and Thurson (1975).1 0. haematocrit (Htc) and haemoglobin (Hb). After 72 h.3 16.1 6. IL. Thermo Orion.1 0. USA) was used to measure plasma sodium.1 ± ± ± ± ± ± ± ± 0.7 0. TX.2 ± ± ± ± ± ± ± ± 0. in terms of lg LÀ1 of NH3-N and was therefore disregarded. USA). USA) and crude fat (Soxtec 2055. Beckman) and plasma stored at À20°C for subsequent ion analysis. Redmond. nitrogen (Kjeltec Auto System. Samples were centrifuged for 10 min at 3000 rpm (Allegra 6R centrifuge. IL. potassium and chloride levels.2 0. St.1 14. Daily conductivity values were calculated as the average of two consecutive conductivity measurements taken before and after the day in question. At the end of the ammonia exposure. After the seawater challenge test. MS 222 (Argent Chemical Laboratories. Due to discrepancies between values obtained using the two different pH metres. Hoganas. OI Analytical. Tecator. Tecator. All diets and whole-body samples were analysed for dry matter (105°C until constant weight). a PHC28103 Intellical pH ultra electrode (Hach Lange.9 0. measured total ammonia (TAN) and calculated average un-ionized ammonia (NH3-N) and approximate partial pressure of NH3 (PNH3) for all treatments Feeding level Reduced feeding (1/3 of full feed strength) TAN (mg LÀ1)* 0.1 0. according to measurements using the Intellical pH ultra electrode. ¨ ¨ Sweden). Louis. The oxygen saturation was kept above 85% at all times. In addition. Parr.1 mg LÀ1 TAN) on ionic strength and ammonia pKa represented only up to 2%. USA). blood samples were collected from the caudal vessel using heparinised vacutainers (Terumo Europe. An i-STAT Portable Clinical Analyser with EC8 + disposable cartridges (Abbott Laboratories. MO.1 0. 1–16 4 .4 32. Leuven. based on Emerson. Aquaculture Research. using formulas at the American Fisheries Society website (Colt 2011). The effect of the added (NH4)2SO4 between control and the highest exposure concentration (25. Germany) with HACH Intelli¨ CAL CDC401 Standard Conductivity probe.3 25.6 34. USA) for cross reference. Belgium).7 11. especially for the control group (ground well water with low buffering capacity). and on a daily basis during the last month of the exposure period. The pH values were adjusted for the last one and a half months of the experiment. Germany) with HACH PHC101 IntelliCAL pH electrode.0 0.1 TAN (µmol LÀ1)* 4 465 800 1704 4 478 809 1794 ± ± ± ± ± ± ± ± 2 4 8 44 1 37 8 46 PNH3 (µtorr) 0 13 22 46 0 13 22 48 Full feed strength (+20% overfeeding) *TAN and NH3-N values are given as means ± SEM for all treatments. MA.4 1. College Station. Conductivity was measured ten times throughout the experiment in all fish tanks using a portable HQ40D system (Hach Lange. Germany) designed for fast response in lowionic strength samples was used for verification.1 0. pH and TAN values for the day in question.0 0.6 0. in the effluent water of all tanks with HQ40D system and Intellical LDO outdoor sensor (LDO101-5). 1–16 Table 2 Feeding regimes. The oxygen levels were kept above 85% with continuous aeration. Measurements and analytical procedures Water quality was monitored throughout the experimental period. Blood samples were collected from caudal vessels using 1 mL heparinized syringes (~37 USP units of Li Heparin. plasma ClÀ concentration was measured with an electrolyte © 2012 Blackwell Publishing Ltd.Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. USA).6 NH3-N (µg LÀ1)* 0.1 6. 2012.2 0. WA. Abbott Park.5 11.2 0. Beverly. Moline.1 8. ash (550°C until constant weight). Dusseldorf. energy (Parr 6300 Bomb calorimeter. Hoganas. ten to 11 fish from each treatment group were euthanized using 100 mg LÀ1 of tricaine methane sulphonate. Russo. Values for NH3-N were calculated using the temperature. Sigma–Aldrich. Water samples were collected and analysed daily for total ammonia nitrogen (TAN mg LÀ1) with an automated chemistry analyser (Flow Solution IV. Daily measurements of pH were done in the water outlet of fish tanks using a portable HQ40D system (Hach Lange. Oxygen was measured weekly. dry matter ¨ ¨ was also analysed in the waste feed (105°C until constant weight).1 9.0 0. conductivity. glucose (Glc). Periodic measurements of samples were also done in the laboratory (720A.2 23.4 1. Sweden).

Basel. Each tank received a total score (between 0 and 20) that was the sum of the individual scores for all ten examined fish. were examined at the end of the exposure period by a veterinarian not otherwise involved in this study. Sigma–Aldrich) and stained according to standard haematoxylin-eosin (H&E) histological protocol. The transcription ratios of Hsp70 were tested by using the Relative Expression Software Tool. Grimholt and Gjøen (2007). gill condition and externally visible/palpable deformities. USA) and DNAse1 treated (Invitrogen). Purity and quantity of isolated RNA were measured by spectrophotometry (Nanodrop® ND-1000 Spectrophotometer. Switzerland) at the following thermal cycling conditions: 95°C for 10 min. USA) and sequenced with Big Dye Terminator chemistry and the ABI 3730 automated sequencer. USA). such as fin condition. Specificity was assessed by the melting curves and on EDTA stained agarose gel. BioCare Corporation. Primers for expression analysis were based on known Atlantic salmon sequences. Triplicate real-time qPCR reactions were performed using the Light cycler 480 and SYBR Green chemistry (Roche. including exact PCR efficiency of each amplicon according to Pfaffl. The PCR products from both primers were cloned using pGEM T-easy (Promega. Total gill RNA was isolated from 15 individuals per treatment using TRIzolTM and Micro to Midi Kit (Invitrogen. Relative Hsp70 mRNA was normalized to relative 18S rRNA mRNA levels. Taoyuan County. NanoDrop Technologies. skin lesions and skin coloration. WI. both delivered by Applied Biosystems. AJ632154) primers (forward: TGA CGTGTCCATCCTGACCAT and reverse: CTGAAGA GGTCGGAACACATCTC) were designed using the Vector NTI Advance 10 (Life technologies. Horgan and Dempfle (2002). cut into 5 lm sections after surface decalcification (decalcifying solution light. USA) software. 1–16 Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. Press. Hetland. For all samples. Each of the observed welfare indicators were given a welfare index between 0 and 2. MD. CA. followed by 45 cycles at 95°C for 15 s. Based on the scores for all examined welfare indicators. The Hsp70 (Genbank no. Gill tissues collected from 72 fish after 22 and 105 days of exposure were embedded in paraffin. CA. with 0 indicating good and 2 indicating bad condition. USA). USA). 60°C for 15 s and 72°C for 15 s. 2012. and NetPrimer (PREMIER Biosoft.Aquaculture Research. while primers against the internal standard gene 18S rRNA (forward: TGTGCCGCTAGA GGTGAAATT and reverse: GCAAATGCTTTCGC TTTCG) were found in Jorgensen. Table 3 Scoring criteria for determination of lesion severity used in the histopathological evaluation of Atlantic salmon gills Lesion severity Absent Very minimal Minimal Moderate Marked Severe Score 0 1 2 3 4 5 Description Normal tissue <5% of examined tissue or microanatomical regions affected/corresponding very minimal numbers of inflammatory infiltrates 25% of examined tissue or microanatomical regions affected/corresponding minimal numbers of inflammatory infiltrates 50% of examined tissue or microanatomical regions affected/corresponding moderate numbers of inflammatory infiltrates 75% of examined tissue or microanatomical regions affected/corresponding very marked numbers of inflammatory infiltrates Essentially 100% of examined tissue affected/obliteration of normal architecture/numerous inflammatory infiltrates © 2012 Blackwell Publishing Ltd. NC. All reactions were performed in accordance with the manufacturer’s protocol. analyser (Biolyte 2000. 0. MD. each fish was then given an overall welfare index between 0 and 2 with a low index value indicating good welfare. REST. Several welfare indicators. Taiwan).5 mg total RNA was reverse transcribed to cDNA using a 50:50 mix of random hexamer and oligo(dT) primers and Taqman Gold RT-PCR kit (Applied Biosystems. 1–16 5 . Wilmington DE. H&E stained slides of gill tissues were evaluated for evidence of damage or tissue change and the severity of lesions observed was scored according to criteria stated in Table 3. Aquaculture Research. cataract.

Ammonia exposure and feed regime (plus an interaction term for these variables) were included as categorical dependent variables. 1 a. Condition factors (CF) at the end of the ammonia exposure period and after the three saltwater tests were calculated from the fish body weight (W. 2002) and data are presented as means ± SEM. days) were calculated using measured body weight at the start (BW1) and at the end (BW2): SGR ¼ ðlnBW2 À lnBW1Þ Â 100=t Body weight measurements. Mean individual weight increased five and threefold in groups with full strength feeding and restricted feeding. If significant. At the end of exposure. the whole-body composition analysis revealed significantly higher energy and lipid levels in the group exposed to 14 lg LÀ1 NH3-N compared with the corresponding control group (0.0001).05. an ordinal logistic regression was carried out in SAS using the GENMOD procedure. Among the groups with full feed strength. Specific growth rates (SGR. with an overall gill pathology score (i.0 (SAS 2007. The groups with restricted feeding showed reduced growth without any additional effects of differential ammonia exposure (Fig. there was no effect of Statistical analyses Data were analysed using JMP 7. All data are expressed as means ± standard error of the mean (SEM).e. Square root and angular transformations were performed for all percentage and scoring data to normalize variances prior to analysis. 1 c. respectively. a one-way analysis of variance (ANOVA) was performed to determine differences between treatments. Aquaculture Research. This modelling was repeated using data from each sampling event.d). 6 © 2012 Blackwell Publishing Ltd.1 lg LÀ1 NH3-N) (Table 4). Differences between the transcription ratios of Hsp70 mRNA were tested for significance by the Pair Wise Fixed Reallocation Randomization Test© (Pfaffl et al.b) (P < 0. Apart from significant difference in body mass (P < 0. BW1 and BW2 were also used to calculate feed conversion ratios (FCR) between two consecutive sampling points: FCR ¼ TFI=ðBW2 À BW1Þ where TFI represents the total dry feed intake over the experimental period in question. individual weight. USA) where tank was used as the statistical unit. A multivariate analysis of variance (MANOVA) was used to examine the influence of ammonia treatment and feed regime and their interaction on measured parameters. Normality of distributions was assessed by the Shapiro–Wilk test and homogeneity of variances was tested with Levene’s F-test.05) (Fig. apart from molecular data. due to the difference in feeding regime. 2012. cm) (Handeland. A significantly lower SGR (P < 0. Among the groups that received full feed strength. 1–16 . however. g) and corresponding length (L. 1). the highest environmental ammonia concentration induced a negative effect on growth rate after 22 days of exposure.02) was noted for the group exposed to 32 lg LÀ1 NH3-N compared with the control group. Fish individual weight was calculated by dividing bulk weight (corrected for the estimated gut content weight) with the number of fish in the tanks (g). Results Growth and body composition There were no treatment-related mortalities during the trial. Cary. Among treatments with restricted feeding levels.0001).. at the end of the long-term exposure to sublethal ammonia concentrations (Fig. % dayÀ1) between two sampling points (t.Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. NC. Aquaculture Research. SAS Institute Inc. 2) did not differ among the four groups. Wilkinson. Sveinsbø. 2) and average individual weight (P = 0. and difference between treatments was judged significant if P 0. the sum of scored pathologies observed per tissue specimen) serving as the dependent variable. and Student’s t-test to establish difference between control and each other experimental group. SGR and FCR (Fig. McCormick & Stefansoon 2004): CF ¼ W Â LÀ3 Â 100 To examine statistically whether ammonia concentration and feeding regime were associated with overall gill damage. 1–16 Calculations Cumulative feed intake was calculated by dividing the total amount of feed eaten per tank by the total number of fish present at that moment (g feed individualÀ1). groups with restricted feeding also had a significantly lower cumulative feed intake (Fig.

1–16 Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. Welfare indicators The average overall welfare index for the groups with restricted feeding levels were significantly 7 . © 2012 Blackwell Publishing Ltd. and exposed to 9 lg LÀ1 NH3-N also showed significantly higher Glu values compared with the corresponding control group (0. The highest levels of blood Na+ (142 ± 1 mM) were found for the 14 lg LÀ1 NH3-N treatment. 14 and 32 lg LÀl NH3-N and in (b) groups with restricted feeding exposed to 0. Blood glucose (Glu) was influenced by an interaction between ammonia treatment and feeding regime (MANOVA. This value was significantly higher than blood Na+ of Atlantic salmon parr exposed to 0.1 lg LÀ1 NH3N and 8 lg LÀ1 NH3-N. Individual body weight development over period of 105 days for fully fed and groups with restricted feeding are presented in graphs (c) and (d).1 lg LÀ1 NH3-N). 8. (a) (b) (c) (d) Figure 1 Cumulative feed intake per fish in (a) full fed groups exposed to 0.1. Aquaculture Research. among the groups with full feed strength. Among groups with full feed strength regime. 1–16 exposure to 8 lg LÀ1 NH3-N and 32 lg LÀ1 NH3N significantly increased Glu values in comparison with the control group (0. In addition. 17 and 35 lg LÀl NH3-N. Measured blood parameters At the end of the ammonia exposure Htc and Hb values were higher in the full feed groups compared with fish fed restrictedly (Table 5).1 lg LÀ1 NH3-N). full fed fish exposed to 8 lg LÀ1 NH3-N had significantly higher Htc and Hb compared with the control group (0. Fish fed restrictedly. P < 0. fish exposed to 9 lg LÀ1 NH3-N and 35 lg LÀ1 NH3-N had lower Htc and Hb values than control group fish (0.05).1 lg LÀ1 NH3-N).Aquaculture Research. All values are given as tank means + SEM (n = 3 for each treatment).1.1 lg LÀ1 NH3-N). 2012. Among treatments with restricted feeding levels. 9. ammonia exposure on any examined body composition parameter.

The transcriptional levels of the stress marker Hsp70 in the gills were not significantly up-regulated after 105 days of ammonia exposure (Fig. The most commonly observed abnormalities were short or crippled fins and slightly shortened opercula noted mostly for the restricted feed ration treatments. hypo-osmoregulatory ability of the remaining experimental © 2012 Blackwell Publishing Ltd. i. Histopathological evaluations of gill tissue identified four distinct lesion types: epithelial hypertrophy.1 lg LÀ1 NH3-N. The transcription of Hsp70 in fish of the 35 lg LÀ1 NH3-N group with restricted feeding. Aquaculture Research. 5). The highest overall score of six (lowest welfare) was given to fish of the control group with restricted feeding.06) and 14 lg LÀ1 NH3-N (P = 0. and the severity of observed lesions was never more than minimal.05) associated with increased prevalence and severity of gill damage. secondary lamellae fusion and lymphohistiocytic inflammation (Fig. 4). At the end of the ammonia exposure period. 1–16 Figure 2 Feed conversion ratio (FCR) and specific growth rate (SGR) in full fed groups exposed to 0. 14 and 32 lg LÀl NH3-N at day 22 and at day 105 (tank mean + SEM. different compared with the full feed strength treatments (Fig. followed by a score of five in the group with restricted feeding exposed to 17 lg LÀ1 NH3-N and 35 lg LÀ1 NH3-N and a score of four for the fish exposed to 9 lg LÀ1 NH3N. Feeding regime was not associated statistically with gill damage. 2012. However. Aquaculture Research. feeding did not significantly affect the ratio of Hsp70 transcription at the lower ammonia exposure concentrations. no statistical associations between gill damage and ammonia exposure were identified. increasing ammonia levels were significantly (P < 0. 3).e. epithelial hyperplasia. 1–16 8 . 8. After 22 days of exposure.09). n = 3). 14 lg LÀ1 NH3-N and 32 lg LÀ1 NH3-N (score = one).Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. lesion score = 2. Among these the best scores were given to groups with exposure to 0. down-regulation of the mRNA expression was evident for groups exposed to 17 lg LÀ1 NH3-N (P = 0. was significantly higher compared with the 32 lg LÀ1 NH3-N with full feeding. The prevalence of each lesion type was low among all gills evaluated (Table 6). In contrast.1. The best fish condition was found in the treatment groups with full feed strength. Different letters denote significant difference between treatments on day 22 (P 0. Smoltification indicators After 13 weeks of light manipulation.05). while the group exposed to 8 lg LÀ1 NH3-N received an overall score of two.

4 ± ± ± ± 2. One month long sublethal exposure of Atlantic salmon to ammonia in freshwater and seawater at very low temperature (4°C.4 108. were documented for rainbow trout fed to satiation (Linton.1 107. 1–16 Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. Furthermore.6 0. Positive effects of moderately elevated water ammonia on growth and protein production. The growth promoting effect of ammonia seen in the rainbow trout (Wood 2004).6 ± ± ± ± 2. Schwarz & Strømsnes 1995) did not have any effect on growth parameters.6 ± ± ± ± 9 .3* 0.4 0.1 ± ± ± ± 5. 1993. Plasma chloride levels increased significantly after SWT1 (sea water test 1.7 21.1 106. 3. Schram & Stefansson 2009) were documented after 96 and 64 days of exposure to 60 lg LÀ1 and 130 lg LÀ1 NH3-N respectively.5 8.7 0.1 0.7 0.8 8.9 0.3 178.5 2.5 1.1 180. however. Scophthalmus maximus (Foss. In subsequent tests (SWT2 and SWT3).3 164. however. or due to ammonia exposure.1 115. plasma chloride levels were lower (<150 mM).4 0.4 178.7 21.0 fish was tested. Significantly different values from comparable control group values are marked with an asterisk (P < 0.3 8. 6a) in all treatments. Discussion The results of this study suggest that growth. was not evident for the Atlantic salmon strain used in this study. at the end of the photo manipulation period.3 3. Fivelstad.Aquaculture Research.2 20.2 8.6 0.2 20.3 175.7 170. Imsland. At SWT3 the highest chloride level (146 mM) was observed for fish originating from the group with restricted feeding and exposure to 9 lg LÀ1 NH3-N.4 102.5 179. no visible physical damages or mortalities were attributed to increased environmental ammonia levels at the end of the experiment.2 0.05).3 7.6 9.1 20.1* 111.8 8. Fivelstad et al.4 3.5 170.7 ± ± ± ± 8. Gadus morhua (Foss. 5 and 6 weeks of exposure to continuous daylight (LD 24:0). Significant differences between rationed and full fed treatments exposed to the same ammonia levels are presented with black bullet sign (P < 0.1• Values are given as mean ± SEM for all treatments at the end of ammonia exposure period.0 0. however.1 µg LÀ1 NH3-N Starting point Experimental treatments (feeding regimes/environmental ammonia concentration) Full feed strength (+20% overfeeding) Reduced feeding (1/3 of full feed strength) © 2012 Blackwell Publishing Ltd. 1–16 Crude lipid Crude protein Ash Gross energy 90.0 0.2 0.9 20. 1999.4 ± ± ± ± 7. None of the examined treatments resulted in statistically different plasma chloride levels compared with the control group with full feed strength regime.6 2. P < 0. 2012. Sæther & Evensen 2004) and juvenile turbot. Table 4 Whole-body composition in Atlantic salmon at starting point and at the end of a long-term ammonia exposure (g or MJ kgÀ1 of wet weight) 35 µg LÀ1 NH3-N 101. There was no significant difference in CF between groups fed full feed strength and groups with restricted feeding levels.6 0.6 8. welfare and smoltification of the Atlantic salmon parr used in this study were not affected by long-term exposure to sublethal ammonia levels up to 35 lg LÀ1 NH3-N (25 mg LÀ1 TAN).3 123. 6b) was evident in fish from all original ammonia treatments after 4.7 21.0 176. Aquaculture Research. Roth.05).6 0.8 2. Negative effects of ammonia on growth of juvenile Atlantic cod.05).1 ± ± ± ± 3.5 0. Reid & Wood 1997.3 0. Wood 32 µg LÀ1 NH3-N 14 µg LÀ1 NH3-N 8 µg LÀ1 NH3-N 0.6 20. while fish that were previously exposed to 14 lg LÀ1 NH3-N with full feed strength had significantly lower chloride levels (135 mM. A significant decrease in CF (Fig. Fig. Siikavupio.4 ± ± ± ± 1.1 µg LÀ1 NH3-N 17 µg LÀ1 NH3-N 9 µg LÀ1 NH3-N 0.6 0.3 8.9 21.

Small letters denote identified gill lesions: (a) epithelial hyperplasia.56 4.73 4.03 112. 14 and 32 lg LÀl NH3-N and treatments with restricted feeding exposed to 0.72t 2.81 36.33 33.60† 1.31† 0. Aquaculture Research.83 131.02*† 0.03a 0.22 138.96 ± ± ± ± ± ± ± ± 0.47 129.47 ± ± ± ± ± ± ± ± 0.31* 0. 1–16 Table 5 Measured blood parameters at the end of the 105-day long-term ammonia exposure in Atlantic salmon parr NH3-N (lg LÀ1) 0.70ab K+ (mmol LÀ1) 3.18a 1. Figure 3 Welfare index of full feed treatments exposed to 0.63bc 0.96 ± ± ± ± ± ± ± ± 0.46c 0.03 ± ± ± ± ± ± ± ± 0.95 4.12*† 1.40* 0.22 130.84 4. n = 3). Different letters denote significant differences (Student t-test) between full feed treatments and † and * denote significant differences (Student t-test) between reduced feeding regimes.36 0.88 0.30* 2.58 142.02 0. 9.46 0.65b 1.53 4.36 40.05) between treatments.63 0.83 1.40 0.22b 4.28 35.08 37.64 140.55ab Values are given as tank means ± SEM (n = 3 for each treatment). 10 © 2012 Blackwell Publishing Ltd.11 3. 1–16 .53 140.44 3.59 0. (a) (b) Figure 4 Gill tissue of Atlantic salmon with rationed feeding (a) exposed to 35 lg LÀ1 NH3-N and (b) exposed to 32 lg LÀ1 NH3-N.19 0.22 141.34 3.34 3.1.70 0.67ab Hb (g LÀ1) 126.89 139. 2009 magnifications). 17 and 35 lg LÀl NH3-N at day 105 of exposure (tank mean + SEM. (b) secondary lamellae fusion and (c) general inflamation (H&E stain.92 132.73 0.86 ± ± ± ± ± ± ± ± 0. 2012.17 ClÀ (mmol LÀ1) 130.05b 0.11 0.76a 0.83 125.67 131.86 0.22 141. 8.11a 5.51t 0.18 2.21*† 0.38 3.59 3.46 0.14*† 0.47 4.61 132. P 0. Different letters denote significant difference (Student t-test.47 116.16a Htc (%PCV) 37.1 8 14 32 Feeding level Reduced feeding (1/3 of full feed strength) Full feed strength (†20% overfeeding) Na+ (mmol LÀ1) 141.10b 0.39 138.04 0.11 0.1.61 120.1 9 17 35 0.47 127.Effects of ammonia exposure on Atlantic salmon J Kolarevic et al.39 ± ± ± ± ± ± ± ± 1.89 130. Aquaculture Research.95 0.25 0.28 4.60 Glu (mg dLÀ1) 4.92 38.69 131.52b 1.12b 5.22 34.12† 4.

2012.1 8 14 32 0. The present study suggests that the Atlantic salmon parr used in this study were more tolerant to chronic ammonia exposure than previously assumed for this species. Expression ratios are shown in relative mRNA expression along the y-axis. © 2012 Blackwell Publishing Ltd. 2004). 11 . except for the group exposed to 32 µg LÀ1 NH3-N (n = 8).Aquaculture Research. After an initial significant decrease in specific growth rate at 32 lg LÀ1 NH3-N.1 9 17 35 0. no difference was present at the end of the exposure when compared with the control group. Aquaculture Research. Figure 5 Relative Hsp70 transcription in Atlantic salmon gills after 105 days of exposure to different levels of ammonia and fed either a restricted or full feed strength diet. It was demonstrated that the PNH3 rather than the TAN concentration is critical in initiating the growth response. Data are given as mean values + SEM.1 8 14 32 Day of exposure Day 22 Feeding level Reduced feeding (1/3 of full feed strength) Epithelial hypertrophy 0/9 0/9 0/9 2/9 1/9 1/9 1/9 0/8 1/9 1/3 1/9 1/9 0/9 1/9 1/9 1/9 Epithelial hyperplasia 2/9 4/9 2/3 1/3 2/9 1/3 2/9 1/8 1/9 1/9 2/9 0/9 1/9 2/9 1/9 2/9 Secondary lamellae fusion 1/9 4/9 1/3 2/9 0/9 0/9 0/9 0/8 0/9 1/9 2/9 0/9 1/9 1/9 0/9 1/9 Inflammation 0/9 1/3 0/9 5/9 1/9 1/9 1/3 1/8 1/9 0/9 0/9 2/9 1/9 0/9 1/9 2/9 Full feed strength (+20% overfeeding) Day 105 Reduced feeding (1/3 of full feed strength) Full feed strength (+20% overfeeding) n = 9 in all treatments. and between groups with restricted and full feed strength exposed to the same ammonia level. In two separate experiments at different water temperatures it was concluded that a PNH3 of approximately 23 ltorr significantly increased body mass after 71 days of exposure (Wood 2004). 1–16 The ‘window’ of ammonia’s effectiveness as a growth stimulant may be quite small and it may also depend on the duration of exposure (Wood 2004). Although the calculated average PNH3 of 20 ltorr in the present study (~14 lg LÀ1 NH3-N) did not have any significant effect on growth.05 or P < 0. n = 15 and significant differences (P < 0.10) are indicated by a and b respectively. Table 6 The prevalence of identified gill lesions at day 22 and 105 of exposure NH3-N (µg LÀ1) 0.1 9 17 35 0. 1–16 Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. it significantly increased energy content of salmon parr after 105 days of exposure. genes along the x-axis.

Vollen & Øiestad 2003). Additional investigations of amino acid metabolism will be necessary to elucidate effects of ammonia on this compound class in Atlantic salmon. Coves.1. Esteban & Meeguer 2002. while Fivelstad et al. 9. Ackerman & Nakano 2004. The present results indicate that reared Atlantic salmon are capable of developing an acclimation response to a long-term ambient ammonia exposure. Many authors have. 1–16 12 . © 2012 Blackwell Publishing Ltd. Chen. Different physiological and molecular mechanisms are in place to regulate this acclimation response and maintain acceptable tissue ammonia levels in fish (Wright & Wood 2009). suggesting some connection between sodium uptake and ammonia excretion (Buckley. When chronically exposed to elevated ammonia. however. Todgham. Dutto. However. should be done. Treatments legend: full feed treatments exposed to 0. Liou & Chang 2006. Iwama et al. Aquaculture Research. Dockray. and plasma glucose has been suggested to be a sensitive parameter in detecting a sublethal stress response in Atlantic salmon (Fivelstad et al. Cheng. Dosdat.Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. rainbow trout responded with enhanced liver protein turnover through increased ribosomal translational efficiency (Reid. Dicentrarchus labrax (Lamarie. but a clear dose-response relationship was not evident. Aquaculture Research. This suggests that a higher level of TAN and NH3N than recommended (Norwegian Food Safety Authority 2004) did not induce any adverse effects on growth of Atlantic salmon. 1–16 (a) (b) Figure 6 Plasma chloride levels (a) and condition factor (b) at the end of ammonia exposure (EXP) and after three 72 h seawater challenge tests (SW1. as it’s levels can be affected by several other factors (Ortuno. SW2 and SW3). pointed out the danger of using glucose as the only stress indicator. A significant effect of the ammonia treatments on plasma glucose levels was found in the present study. 14 and 32 lg LÀl NH3-N and treatments with restricted feeding exposed to 0. 2004). Linton. follow-up studies on ammonia exposure of other lifestages of Atlantic salmon. European seabass. 1995). Gasset & PersonLe Ruyet 2004) and spotted wolffish (Foss. 8. McDonald & Wood 1998). Afonso. exposed to 16 lg LÀ1 NH3-N. but at much lower water temperature (4°C).1. 17 and 35 lg LÀl NH3-N. and at different physiological and nutritional conditions. 2004). Oncorhynchus kisutch. 1993. Restricted feeding and increased water ammonia levels can cause stress (Iwama. 2012. Asterisk indicates significant difference in plasma chloride levels between EXP and SWT1 and significant difference in condition factor at EXP and at the seawater tests. All values are given as means + SEM. Foss et al. Similar acclimation effects have been observed in juvenile Atlantic cod (Foss et al. Several studies on marine teleost species have found no effect of long-term sublethal ammonia exposure on plasma glucose levels (Knoph & Thorud 1996. Whitmore & Liming 1979). 2004. A significant rise in plasma Na+ was also observed in coho salmon. Another study demonstrated a stimulative effect of ammonia on net protein synthesis in rainbow trout and an increase in whole-body protein proportion (Wood 2004). Increased Htc values were observed in fish exposed to 8 lg LÀ1 NH3-N in this study. 1993 observed a similar effect for Atlantic salmon smolt only at an exposure to 37–65 lg LÀ1 NH3-N. In the present study full feed strength and exposure to 14 lg LÀ1 NH3-N significantly increased plasma Na+ concentration. Feeding conditions and elevated water ammonia levels had a pronounced effect on several blood parameters in the present study. Peterson & Small 2004).

exposure to 14 lg LÀ1 NH3-N (11 mg LÀ1 TAN). Goldberg & Voellmy 1986. Aquaculture Research. Basu. In conclusion. K+-ATPase activity. 1–16 tissues suggested that higher levels of environmental ammonia. Vries. Vis & Flik 2010). Bæverfjord. Takle. Schulte & Iwama 2002. such as fin condition. Instead. indicating again an acclimatory response to a long-term presence of environmental ammonia. Thomas. Ackerman. Ammonia tolerance of Atlantic salmon is becoming a current issue with increased use of 13 . Benli. Pollock & Dube 2008.Aquaculture Research. In contrast. Hence. subsequent exposure did not seem to further deteriorate gill status of Atlantic salmon. Moyano & Vijayan 2005). was not apparent at 11 weeks after the onset of ammonia exposure. condition factor and plasma chloride levels are the most commonly used smoltification indicators (Wedemeyer et al. hypertrophy and lamellar fusion have been reported to be the most common effects of elevated water ammonia observed in gills of a number of fish species following exposure (Fivelstad et al. we did observe a significantly higher Hsp70 transcription in gill of the group exposed to 35 lg LÀ1 NH3-N compared with the control group. 1993. The examination of welfare indicators. and FCR. Feed restriction has previously been shown to induce HSP70 protein expression in fish (Cara. It’s role during stress is related to a cytoprotective function as it can act to prevent and repair protein damage (Ananthan. revealed only a few pathologies. skin lesions. the prevalence of almost all noted anomalies was reduced. Together with gill Na+. Poor water quality during early development can have an adverse effect on smoltification and early marine survival (Wedemeyer. Hansen. Hyperplasia. The present study indicated no direct relationship between long-term ammonia exposure and an increase in the transcription level of Hsp70 in the gills. Martinez-Cordova & RamosEnriquez 2009). Feed intake is considered to be a reliable criterion to estimate health and welfare in farmed fish ˚ (Sørum. in fish fed restrictedly. the salmon gill hsp70 expression may be upregulated by high ammonia exposure when the fish is kept at a restricted diet. 2003). In fact. This relationship. In the present study. cumulative feed intake per fish. Similarly. resulted in almost 50% decrease in mRNA expression level of Hsp70. In contrast. in fish on full feed strength. It has been suggested that such structural changes may lead to a reduced gas-exchange capability of the fish and their physiological impairment (Lease et al. The histopathological evaluations of gill © 2012 Blackwell Publishing Ltd. the appetite of rainbow trout exposed to 23 ltorr pNH3 (Wood 2004) was identical to their control group throughout the 71 days of exposure. Spencer. ammonia exposure did not appear to have significant negative effects on the welfare parameters in question. Kolstad & Andersen 2005). were not affected by ammonia exposure at the end of the trial. The potential consequences of ammonia exposure on the smoltification capabilities of Atlantic salmon are largely unknown. Contrary to feeding levels. On the other hand. Geething & Sambrook 1992). Lunde. Frances. cataracts. Sparings. Todgham. In the present study. however. Handeland & Stefansson 2001). Toften & Damsgard 2004). This protein plays a critical role in the general stress response in fish (Iwama. Martinez-Porchas. regardless of the feeding level. Aluru. the Atlantic salmon parr used in this study appeared to be more resilient to ammonia than previously thought for this species and were capable of acclimating and developing responses to increased sublethal levels of this compound. These results suggest that the experimental fish smoltified and that previous exposure of up to 35 lg LÀ1 NH3-N does not seem to have negative effects on parr-smolt transformation in Atlantic salmon. Virtanen 1987. were associated with minor gill damage after 3 weeks of continuous exposure. Abbink. Foss et al. Although gill damage was noted in all groups after 22 days of exposure. hypertrophy of the epithelium associated with lamellar fusion has been interpreted as an adaptation to reduce the permeability of the gills and therefore ammonia entry (Schram et al. saltwater challenge tests showed that the plasma chloride levels in all groups were below 150 mM and that the condition factors were significantly reduced. Bierman. increased ammonia reduced total feed intake in juvenile Atlantic cod and turbot and in the latter case it was suggested to be the cause of an observed reduction in growth rate (Person-Le Ruyet et al. Forsythe & Vijayan 1998. Saunders & Clarke 1980). Nakano. Koksal & Ozkul 2008. The lack of a Hsp70 stress response in the full fed fish suggests that the tested ammonia levels did not reduce the welfare of the fish in this group. 1997. gill condition and externally visible/palpable deformities. 1980. Roques. 2004). Bergman & Meyer 2003. Nowak & Allan 2000. compared to the control group. although in this case ammonia had a positive effect on gross food conversion efficiency. Bibeau. 2010). 1–16 Effects of ammonia exposure on Atlantic salmon J Kolarevic et al. 2012. Schram. Lease.

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