Aquaculture 436 (2015) 160–166

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Dietary n-6/n-3 LC-PUFA ratio, temperature and time interactions onnutrients and fatty acids digestibility in Atlantic salmon

Clara Trullàs Huguet a, Fernando Norambuena b,⁎, James A. Emery b, Karen Hermon b, Giovanni M. Turchini b

a Departament de Ciència Animal i dels Aliments, Servei de Nutrició i Benestar Animal (SNiBA), Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spainb School of Life and Environmental Sciences, Deakin University, PO Box 423, Warrnambool, Victoria 3280, Australia

⁎ Corresponding author at: School of Life and EnvUniversity, PO Box 423, Warrnambool, Victoria 3280, Aufax: +61 3 55633462.

E-mail address: f.norambuena@deakin.edu.au (F. Nora

http://dx.doi.org/10.1016/j.aquaculture.2014.11.0110044-8486/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 October 2014Received in revised form 7 November 2014Accepted 10 November 2014Available online 15 November 2014

Keywords:Arachidonic acidDigestibilityEicosapentaenoic acidlong-chain polyunsaturated fatty acidsSalmo salarWater temperature

The objective of the present studywas to evaluate the effects of altered dietary n-3/n-6 LC-PUFA ratio, adaptationto diet over time, different water temperatures, and their interactions on nutrients and fatty acids digestibility injuvenile Atlantic salmon. Three experimental dietswere formulated to be identical, with the only exception of theratio of eicosapentaenoic acid (EPA, 20:5n-3) to arachidonic acid (ARA, 20:4n-6), and fed to triplicate groups ofjuvenile Atlantic salmon (Salmo salar) of 55 g initial body weight. Fish were reared in a fully controlledrecirculating aquaculture system, fed to apparent satiety twice daily and kept at 10 °C and for an initial periodof 100 days, and faeces were collected for digestibility estimation. Then, half of the fish of each experimentaltank were moved to a separate system, where the water temperature was gradually increased up to 20 °C. Fishweremaintained in the two systems for an additional period of 50days, and faeceswere collected for digestibilityestimation from both groups of fish at the twowater temperatures. This study concluded that dietary treatmentsand time had only minor effects, whereas environmental temperature resulted in modified digestibility values,with increased nutrient digestibility with increasing temperature. Varying EPA/ARA ratio in the diet had onlyminor direct effects on digestibility, with no direct effect on overall nutrients digestibility, and fundamentallyonly statistically significant effects in the fatty acid digestibility of EPA and ARA themselves. Because of currentincreasing pressure for more efficient fish oil replacement strategies, increasing interest in dietary ARA inaquafeed and increasing relevance and occurrence of sub-optimal rearing temperature in commercial aquaculture,this study can be considered to be important as it provided a series of fundamental information,which are envisagedto be useful towards addressing these constraints and possible nutritional remedial strategies.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

It is envisaged that the limited supply of fish oil (FO) as a source ofdietary lipid will be a primary factor influencing the economic viabilityof FO in the future (Bostock et al., 2010; Turchini, 2013; Turchini et al.,2009). Consequently, an intensive global research effort is focusing onpossible remedial strategies, and primarily aiming at finding suitablealternative oils to replace dietaryfish oil in aquafeed.Within the contextoffish oil replacement in aquafeed, considerations regarding the possibleeffects on nutrients and fatty acid digestibility are of primary importance(Caballero et al., 2002; Francis et al., 2007; Karalazos et al., 2011;Menoyoet al., 2003).

Fish oil is commonly substituted with alternative lipid sources at highinclusion levels (up to 75% of total added lipid sources) in commerciallymanufactured aquafeeds of many species including Atlantic salmon(Turchini et al., 2009). Digestive system phenotypic adaptation to diets

ironmental Sciences, Deakinstralia. Tel.: +61 3 55633021;

mbuena).

in several fish species has been observed (Buddington et al., 1987), andAtlantic salmon (Salmo salar) have been reported to be able to adapt tochanges in dietary composition of protein sources (Refstie et al., 1998).However, when dietary modifications are specific for dietary lipids,histological modification of enterocytes resulting from differentvegetable oils used in feed have also been observed (Olsen et al.,1999). Furthermore, it was recently shown that lipid digestibility canbemodified over time, suggesting the fish adaptation capacity to dietarysources (Krontveit et al., 2014).

A second consideration is towhat extent environmental temperaturescan impact on nutrient digestibility. Water temperature is known todirectly affect several physiological processes in fish, including digestivephysiology and function (Bowyer et al., 2014; Grans et al., 2013;Neuman et al., in press). Sub-optimal temperatures are increasinglyencountered in aquaculture as the result of climate change relatedphenomena (Cochrane et al., 2009) and/ormarket forces driving farmingof species in areas with environmental characteristics outside theiroptimal physiological range, such as the case of Atlantic salmon farmingin Australia (Neuman et al., in press; Pecl et al., 2011a; Pecl et al.,2011b). Therefore the relationship between water temperature anddietary lipids/fatty acids digestibility in fish has attracted significant

Table 1Formulation (g/kg) of the three experimental diets with different EPA/ARA ratios.

Experimental Diets 1

D-ARA D-ARA/EPA D-EPA

Diet formulation (g/kg)Fish meal2 188 188 188Poultry meal2 168 168 168Soy protein concentrate2 94 94 94Soybean meal2 84 84 84Meat and bones meal3 75 75 75Pregel Starch2 75 75 75Blood meal2 66 66 66Wheat gluten4 47 47 47Whey5 38 38 38Vitamin & mineral mix6 7 7 7Celite7 5 5 5Choline2 4.9 4.9 4.9Monosodium phosphate2 4 4 4Lisine2 1.5 1.5 1.5Methionine2 1 1 1Taurine2 1 1 1Vitamin C2 0.5 0.5 0.5Pigment2 (carophil pink) 0.06 0.06 0.06

OilsARASCO8 33 17 2Incromega EPA TG5009 9 22 34Incromega DHA TG5009 8 4 1Canola Oil10 39 41 45Linseed Oil11 31 31 30High Oleic Sunflower Oil12 13 12 9Palm oil13 7 13 19

1 Experimental diet abbreviations: D-ARA (EPA/ARA ratio = 0.5), D-ARA/EPA (EPA/ARAratio = 1.5) and D-EPA (EPA/ARA ratio = 9).

2 Ridley Agriproducts, Narangba, QLD, Australia.3 The Midfield Group, Warrnambool, VIC, Australia.4 Agrifood Ingredients, Kew East, VIC, Australia.5 Warrnambool Cheese and Butter, VIC, Australia6 DSM Nutritional Products,WaggaWagga, NSW, Australia.7 Merck KGaA, Darmstadt, Germany.8 DSM Nutritional Products, Aquaculture Centre Asia, Bangkok, Thailand.9 CRODA, Snaith, East Yorkshire, UK.10 Black and Gold, Tooronga, VIC. Australia.11 Sceney Chemical Pty., Ltd., Sunshine, VIC, Australia.12 Nuseed Global, Laverton, VIC, Australia.13 ETA Mel-Fry, Goodman Fielder Food Services, Macquarie Park, NSW, Australia.

161C.T. Huguet et al. / Aquaculture 436 (2015) 160–166

research interest (Emery et al., 2014; Guerreiro et al., 2012; Ng et al.,2010).

A third important consideration is that different fatty acids have beenreported to be differently digested (absorbed) not only relative to theirsaturated- (SFA), monounsaturated- (MUFA) and polyunsaturated-(PUFA) fatty acids content (Austreng et al., 1980; Bureau and Meeker,2011) and their chain length (Morais et al., 2005; Ringo, 1991;Sigurgisladottir et al., 1992), but also relative to the position of the firstdouble bond, with omega-3 fatty acids (n-3 PUFA) being reportedto be more easily digested than omega-6 fatty acids (n-6 PUFA)(Eroldogan et al., 2013; Francis et al., 2007). Accordingly, it has alsobeen shown that different dietary n-3/n-6 PUFA ratio can directly affectoverall nutrients (protein, lipid and energy) digestibility (Bandarraet al., 2011).

Last, when fish oil (rich in long chain polyunsaturated fatty acids,LC-PUFA) is replaced by terrestrial oils, which typically do not containany LC-PUFA, the dilution of dietary LC-PUFA is unavoidable. Accordingly,a large body of research has been implemented towards addressing theissue of n-3 LC-PUFA (eicosapentaenoic acid — EPA, 20:5n-3, anddocosahexaenoic acid — DHA, 22:6n-3) in aquafeed over the last twodecades (Turchini et al., 2011), but only recently attention has alsobeen given to the n-6 LC-PUFA, namely arachidonic acid (ARA, 20:4n-6)(Norambuena et al., 2012; Norambuena et al., 2013c).

Bearing in mind all the above mentioned considerations, theobjective of the present studywas to evaluate the effects of altereddietaryn-3/n-6 LC-PUFA ratio (and specifically EPA/ARA ratio), adaptation to dietover time, different water temperatures, and their interactions onnutrients and fatty acids digestibility in juvenile Atlantic salmon.

2. Materials and Methods

The Deakin University Animal Ethic Committee approved allprocedures of this study (Ref. B06-2013), and all possible steps towardsminimizing animal suffering were taken. Juvenile Atlantic salmon(~55 g) were sourced from a private aquaculture farm (Mountain FreshTrout and Salmon Farm, Harrietville, Australia). After transportation tothe Deakin University Aquaculture Research Facility (Warrnambool,Victoria, Australia), fish were acclimatised to the new environmentalconditions for a two week period, during which they were fed acommercial trout diet (Trout-freshwater); 45% protein, 20% lipid;Ridley Aquafeed, Narangba, QLD, Australia). Three experimentaldiets were specifically formulated with the inclusion of a source ofacid insoluble ash (Celite) as an internal marker for digestibility, andwere then manufactured and pelletised using a small scale extruder(DGP-50, Zhengzhou Amisy Machinery Co.ltd, Zhengzhou, Henan,China). Briefly, all dry diet ingredients were combined in a commercialbaker's mixer (MEC, Victoria, Australia) and homogenised for 10 minuntil properly mixed. Dietary oils were added before extrusion andwere slowly poured onto the runningmixer to guarantee an even appli-cation of lipid to dry ingredients. Lipid and dry ingredients were furtherhomogenised for 10min before addition of 6–7 l of water (at 80 °C) per25 kg of diet and were further mixed until a malleable doughy consis-tency was attained. The mash was then loaded on the preconditionedmixer hopper that automatically fed the single-screw extruder, fittedwith a variable speed cutter. Two batches of feed were manufacturedobtaining 25 kg of pellet of 5 mm Ø and 5 mm length and 20 kg with6 mm Ø and 6 mm length. Finished pellets were then dried to 2–3%moisture in a dark, temperature-controlled fan-forced room at 40 °Cover a 48-h period, and stored at−20 ° C in air tight bags until needed.

The formulation of the diets was developed to obtain iso-proteic(~500 mg/g), iso-lipidic (~180 mg/g) and iso-energetic (21.5 KJ/g)diets, varying only in the added dietary lipid sources (Table 1). Threespecifically formulated blends of 7 different oils were used, includingfour vegetable oils (canola, linseed, high oleic acid sunflower and palmoils) and three specialty oils with high content of ARA, EPA and DHA,respectively. The blends of oils were designed specifically to achieve

three experimental diets with the same content of total SFA, MUFA,PUFA, LC-PUFA n-3 C18PUFA, n-6 C18PUFA and DHA, the same sumtotal content of EPA + ARA, but varying specifically in the EPA/ARAratio, and were accordingly named D-ARA (EPA/ARA ratio = 0.5),D-ARA/EPA (EPA/ARA ratio = 1.5) and D-EPA (EPA/ARA ratio = 9).

Two identical multiple-tank (1000 L), thermostatically controlled,recirculating aquaculture systems, equippedwith physical and biologicalfiltration and UV sanitation, were used for the in vivo digestibility trial.For the entire duration of the trial, both systems were maintained on a12:12 h light:dark cycle, a flow rate of 10 L/min per tank. Water qualityparameters (ionised ammonia, nitrite, dissolved oxygen and pH) weremeasured every second day using Aquamerck test kits (Merck,Darmstadt, Germany) and maintained at optimal levels for Atlanticsalmon. Fish (~55 g body weight) were initially stocked in onesystem, with water temperature set at 10 °C, and 540 fish wererandomly distributed into 9 tanks (60 fish per tank) and triplicatetanks were randomly allocated to one of the three experimentaldiets. Fish were fed twice daily to apparent satiation at 0900 and1600 h. After ten weeks, when fish were of ~100 g body weight, faeceswere collected over a period of seven days. At week 14, fish from eachtank were split into two groups, and 20 fish were then moved to thesecond system and 20 fish were returned to their previous tank. Afterfish transfer, thewater temperature of the second systemwas graduallyincreased at a rate of 1.5 °C/day, from 10 °C to 20 °C, whereas the first

Table 3The fatty acid composition (mg/g lipid) of the three experimental dietswith different EPA/ARA ratios.

Experimental diets

D-ARA D-ARA/EPA D-EPA

14:0 7.12 7.41 7.7614:1n-5 0.69 0.71 0.7315:0 1.33 1.33 1.2915:1n-5 0.19 0.21 0.4316:0 96.50 100.62 105.5416:1n-7 13.32 13.61 14.2217:0 1.89 1.71 1.1616:2n-4 0.66 0.95 1.3017:1n-7 1.05 1.04 1.0816:3n-4 1.03 1.76 2.7518:0 44.52 39.73 34.8618:1n-9 t 1.18 2.37 3.5618:1n-7 t 0.62 0.63 0.9618:1n-9 282.07 276.21 268.7818:1n-7 13.28 13.35 13.6218:2n-6 t 0.32 0.33 0.2318:2n-6 86.26 84.31 85.0218:3n-6 3.88 2.69 1.4218:3n-4 0.41 0.65 0.7018:3n-3 74.07 77.46 78.2620:0 2.99 2.58 2.1718:4n-3 2.84 5.67 8.7020:1n-13 0.24 0.46 0.6720:1n-11 0.48 0.36 0.2220:1n-9 3.82 3.50 2.5420:2n-6 1.34 0.90 0.5320:3n-6 3.90 2.30 0.3420:4n-6 49.19 26.92 7.0520:3n-3 0.77 0.42 0.2622:0 3.34 2.23 1.4120:4n-3 0.90 1.18 1.6022:1n-11 0.88 0.56 0.4822:1n-9 0.45 0.35 0.3820:5n-3 20.87 40.93 62.27

162 C.T. Huguet et al. / Aquaculture 436 (2015) 160–166

systemwasmaintained at 10 °C. After an additional fourweeks from thesplitting of the fish into the two systems at different temperatures,when fish were of ~140 g body weight, faeces were collected over aperiod of seven days from all tanks in the two systems. During thefaeces collections periods, all tanks were carefully cleaned after thepm feeding, and the following morning before the am feeding, andfaeces were collected from the solids-collection trays fitted on theoutlets of each individual tank. Collected faeces were immediatelyfrozen and stored at −20 °C until freeze-drying and followinganalyses.

Digestibility of dietary macronutrients and individual fatty acidswere determined by assessing acid insoluble ash (AIA) as described byVankeulen and Young (1977), and successively adapted to rainbowtrout (Atkinson et al., 1984), following standard formulae for apparentdigestibility coefficient determination, as previously described (Franciset al., 2007). Proximate composition analysis was implemented onexperimental diets and faeces samples using standard procedures.Briefly, protein (N × 6.25) was measured using an automated Kjeltec2300 (Foss Tecator, Geneva, Switzerland), moisture by drying samplesin anoven at 80 °C to a constantweight, and ash by incinerating samplesin amuffle furnace (Wit, C & L Tetlow, Australia) at 550 °C for 18 h. Lipidcontent was determined by a modified cold extraction method (Folchet al., 1957) whereby dichloromethane replaced chloroform for healthand safety considerations, and an aliquot of the extracted lipid wassubsequently used for fatty acid analysis. Nitrogen free extract(NFE) was calculated by difference, and energy was computed onthe basis of 23.6, 39.5 and 17.2 kJ/g of protein, fat and carbohydrate,respectively. Fatty acids were separated, identified and quantifiedusing gas chromatography, following procedures previously describedin detail (Norambuena et al., 2013b).

Faeces samples were analysed individually, per tank and persampling point, and all chemical analyses were implemented intriplicate on each individual sample. Analytical replicates were averagedand then the three replicate tanks per treatmentwere used for statisticalanalysis.

All data were reported as mean ± standard error (n = 3). Forstatistical interpretation, the dataset has been split into two separatesub-datasets. The first dataset comprised of data originating fromfish maintained at the same temperature, and comparison wasmade for the two sampling times to assess the effect of the diet, theeffect of time, and their interactions. The second dataset comprisedof data originating at the second sampling time, and comparisonwas made for the two environmental temperatures to assess the effectof the diet, the effect of temperature and their interactions. In bothinstances, comparisons were made amongst treatments using two-way analysis of variance (ANOVA), following confirmation of normalityand homogeneity of variance. The results of the two-way ANOVA testare reported in tables as: n.s. = not significant; * P b 0.05; **P b 0.01;and *** P b 0.001. Data were also subjected to a Student–Newman–Keuls post hoc multiple comparisons test for identifying homogeneoussubsets. All statistical analyses were computed using IBM SPSS statistic20 (IBM Corporation, New York, USA).

Table 2The proximate composition of the three experimental diets with different EPA/ARA ratios.

Experimental diets

D-ARA D-ARA/EPA D-EPA

Moisture (mg/g) 76.9 70.7 69.4Protein (mg/g) 492.6 493.7 491.0Lipid (mg/g) 177.4 173.3 176.3NFE (mg/g) 159.7 170.2 169.0Ash (mg/g) 93.4 92.2 94.4Energy (kJ/g) 21.4 21.4 21.5

3. Results

Theproximate and fatty acid compositions of the three experimentaldiets are reported in Table 2 and Table 3, respectively. The experimentaldiets were iso-proteic, iso-lipidic and iso-energetic, and their fatty acidcomposition was as per objective of the formulation, with almostidentical fatty acid composition, with the only exception of EPA andARA.

For fish held at 10 °C for the entire study no statistically significantdifferences were observed for nutrient digestibility among thethree experimental diets, or between the two sampling time points(Table 4). Individual fatty acid digestibility values showed somestatistically significant effects of the diet and of the sampling timepoint, but not of the interaction between the two (Table 5). Timesignificantly affected the digestibility of 18:1n-7trans, whereas thedietary treatment was responsible for significant differences in15:1n-5, 17:0, 20:1n-9, 20:2n-6, 22:0, 20:4n-6 (ARA) and 20:5n-3

22:4n-6 0.41 0.30 0.2224:0 1.95 1.38 0.8322:5n-6 1.07 0.79 0.6624:1n-9 0.97 0.76 0.5922:5n-3 1.54 1.90 2.3722:6n-3 17.70 16.53 17.33SFA1 159.64 157.00 155.01MUFA2 319.24 314.11 308.25PUFA3 267.16 265.98 271.02n-6 LC-PUFA4 55.91 31.21 8.81n-3 LC-PUFA5 41.77 60.95 83.83Total Fatty acids 746.05 737.09 734.29

1 SFA: saturated fatty acids.2 MUFA: monounsaturated fatty acids.3 PUFA: polyunsaturated fatty acids.4 n-6 LC-PUFA: omega-6 long-chain polyunsaturated fatty acids.5 n-3 LC-PUFA: omega-3 long-chain polyunsaturated fatty acids.

Table 4Apparent digestibility coefficients (%) of macronutrients for the three experimental diets with different EPA/ARA ratios at different times, in juvenile Atlantic salmon.

Time 1 Time 2 P value1

ADC(%) D-ARA D-ARA/EPA D-EPA D-ARA D-ARA/EPA D-EPA Diet Time Diet x Time

Dry Matter 74.85 ± 1.15 75.26 ± 0.38 74.29 ± 0.17 76.38 ± 0.78 76.05 ± 0.62 76.53 ± 0.06 n.s. n.s. n.s.Protein 83.06 ± 1.14ab 83.06 ± 0.74ab 81.26 ± 1.48a 85.47 ± 0.29b 84.65 ± 0.50ab 84.52 ± 0.60ab n.s. n.s. n.s.Lipid 94.36 ± 0.27 94.59 ± 0.24 94.24 ± 0.10 94.48 ± 0.26 94.64 ± 0.08 95.11 ± 0.08 n.s. n.s. n.s.NFE 52.33 ± 3.50 55.32 ± 0.41 57.84 ± 4.96 49.28 ± 3.16 52.74 ± 1.58 55.84 ± 1.94 n.s. n.s. n.s.Energy 82.79 ± 0.94 82.93 ± 0.40 82.22 ± 0.11 83.79 ± 0.65 83.51 ± 0.32 84.16 ± 0.17 n.s. n.s. n.s.

Values in the same rowwith different superscripts are significantly different (P b0.05) as determined by Student-Newman-Keuls post hoc multiple comparisons test. P values relative totwo-way ANOVA statistical test are reported in the last three columns on the right hand side of the table.

1 n.s. = not significant; * P b 0.05; **P b 0.01; and *** P b 0.001.

163C.T. Huguet et al. / Aquaculture 436 (2015) 160–166

(EPA) digestibility. The digestibility of 20:4n-6 (ARA), was lower inthe diets with lower ARA content, whereas the digestibility of20:5n-3 (EPA), was higher in the diets with higher EPA content.

The nutrient digestibility and the fatty acid digestibility for fish at thesecond time point, and as affected bywater temperature, are reported inTable 6 and Table 7, respectively. Statistically significant effects of watertemperature were observed for lipid, NFE and energy digestibility, with

Table 5Apparent digestibility coefficients (%) of fatty acids for the three experimental diets with differ

Time 1 Time 2

ADC (%) D-ARA D-ARA/EPA D-EPA D-ARA

14:0 88.09 ± 0.93 87.44 ± 0.46 88.42 ± 0.32 88.4614:1n-5 94.32 ± 0.54 92.53 ± 1.59 93.97 ± 0.20 93.8815:0 83.21 ± 3.98 76.20 ± 3.92 79.39 ± 0.31 81.7315:1n-5 76.78 ± 2.37 74.88 ± 2.44 55.63 ± 24.23 75.8916:0 87.10 ± 1.08 85.71 ± 0.34 86.00 ± 0.63 87.4816:1n-7 97.54 ± 0.15 98.01 ± 0.16 98.05 ± 0.32 97.5417:0 84.29 ± 1.35b 83.41 ± 3.06b 79.87 ± 0.85b 84.9016:2n-4 97.74 ± 2.26 93.43 ± 2.12 98.81 ± 1.19 90.2917:1n-7 98.77 ± 1.23 98.25 ± 0.90 99.41 ± 0.59 99.4516:3n-4 95.02 ± 0.70 96.27 ± 0.78 98.17 ± 0.20 95.7618:0 83.80 ± 1.39 81.13 ± 0.31 79.98 ± 0.88 84.5218:1n-9 t 1002 100 100 10018:1n-7 t 28.78 ± 15.21a 40.17 ± 14.36a 89.57 ± 3.68b 78.6918:1n-9 97.52 ± 0.06 97.76 ± 0.28 97.46 ± 0.07 97.5518:1n-7 96.73 ± 0.09 97.04 ± 0.30 96.84 ± 0.07 96.6918:2n-6 t 81.19 ± 1.43 81.17 ± 0.43 80.40 ± 0.29 81.6718:2n-6 98.56 ± 0.05 98.69 ± 0.15 98.82 ± 0.22 98.4418:3n-6 98.94 ± 0.03 97.33 ± 1.38 98.61 ± 0.74 98.6418:3n-4 100 99.02 ± 0.98 99.17 ± 0.83 10018:3n-3 99.19 ± 0.03 99.21 ± 0.03 99.28 ± 0.00 99.0320:0 90.79 ± 0.80b 88.92 ± 0.24ab 88.11 ± 0.35a 91.0618:4n-3 100 100 100 10020:1n-13 100 100 100 10020:1n-11 100 100 100 92.5520:1n-9 96.69 ± 0.53b 96.27 ± 0.42b 95.69 ± 0.48a 96.3120:2n-6 93.13 ± 0.38d 91.20 ± 0. 83.60 ± 0.82a 93.4420:3n-6 91.20 ± 0.99 84.79 ± 0.72 - 91.5120:4n-6 99.30 ± 0.02a 99.25 ± 0.09a 98.10 ± 0.14b 99.1320:3n-3 95.09 ± 0.51 95.55 ± 2.68 97.71 ± 2.29 95.6922:0 90.21 ± 0.75b 87.05 ± 0.51ab 84.07 ± 0.79a 90.3520:4n-3 100 85.04 ± 14.96 100 97.4322:1n-11 92.61 ± 0.47b 93.10 ± 1.43b 88.83 ± 0.84a 92.6322:1n-9 86.81 ± 1.42 83.83 ± 1.81 82.72 ± 2.38 84.3620:5n-3 99.18 ± 0.03a 99.48 ± 0.04b 99.52 ± 0.02b 98.9622:4n-6 95.32 ± 2.35 97.89 ± 2.11 100 98.7824:0 88.71 ± 0.90 86.84 ± 0.50 84.73 ± 4.55 89.0822:5n-6 98.70 ± 0.78 100 100 98.6524:1n-9 87.60 ± 0.51 84.50 ± 1.05 82.11 ± 2.14 88.2622:5n-3 99.28 ± 0.36 99.40 ± 0.60 99.33 ± 0.41 99.0622:6n-3 98.52 ± 0.06 98.71 ± 0.11 98.73 ± 0.07 98.59Total Fatty acids 95.49 ± 0.29 95.25 ± 0.20 95.22 ± 0.17 95.55

Values in the same rowwith different superscripts are significantly different (P b0.05) as detertwo-way ANOVA statistical test are reported in the last three columns on the right hand side o

1 n.s. = not significant; * P b 0.05; **P b 0.01; and *** P b 0.001.2 100% = fatty acid not detected in faeces.

higher values recorded infish at the higher temperature;whereas no ef-fects of diet and temperature interactions were recorded. Temperaturewas also responsible for several statistically significant differences inthe digestibility of individual fatty acids, including 16:0, 18:0, 18:1n-9,18:3n-6 and total fatty acids,with, in all instances, higher values recordedin fish held at the higher temperature. The interaction of diet and watertemperature had only minor statistically significant effects.

ent EPA/ARA ratios at different times, in juvenile Atlantic salmon.

P value1

D-ARA/EPA D-EPA Diet Time DietxTime

± 0.75 87.53 ± 0.37 89.12 ± 0.86 n.s. n.s. n.s.± 0.76 93.17 ± 0.15 94.29 ± 0.26 n.s. n.s. n.s.± 1.73 77.63 ± 1.08 81.04 ± 1.58 n.s. n.s. n.s.± 1.49 77.04 ± 0.83 89.45 ± 3.40 ** n.s. n.s.± 0.81 85.81 ± 0.28 86.95 ± 0.86 n.s. n.s. n.s.± 0.33 98.10 ± 0.28 98.30 ± 0.25 n.s. n.s. n.s.± 0.77b 83.34 ± 0.28b 69.60 ± 1.98a *** n.s. n.s.± 4.86 90.18 ± 0.14 94.71 ± 1.00 n.s. n.s. n.s.± 0.55 97.21 ± 2.79 99.39 ± 0.61 n.s. n.s. n.s.± 1.29 97.92 ± 1.04 98.19 ± 0.08 n.s. n.s. n.s.± 0.96 80.73 ± 0.29 80.33 ± 1.40 n.s. n.s. n.s.

100 100 - - -± 4.25b 73.99 ± 0.25b 79.32 ± 4.35b n.s. ** n.s.± 0.32 97.85 ± 0.06 98.03 ± 0.09 n.s. n.s. n.s.± 0.37 96.95 ± 0.08 97.34 ± 0.07 n.s. n.s. n.s.± 0.59 84.90 ± 2.99 77.07 ± 11.54 n.s. n.s. n.s.± 0.29 98.69 ± 0.06 98.89 ± 0.07 n.s. n.s. n.s.± 0.25 97.80 ± 0.34 96.35 ± 0.71 n.s. n.s. n.s.

100 100 n.s. n.s. n.s.± 0.22 99.31 ± 0.06 99.45 ± 0.07 n.s. n.s. n.s.± 0.60b 89.39 ± 0.32ab 88.67 ± 0.67ab n.s. n.s. n.s.

100 99.95 ± 0.05 n.s. n.s. n.s.100 100 - - -

± 7.45 100 100 n.s. n.s. n.s.± 0.94b 96.75 ± 0.07b 94.09 ± 0.28a ***. n.s. n.s.± 0.42d 91.09 ± 0.10c 88.50 ± 0.56b *** n.s. n.s.± 1.45 88.77 ± 0.93 47.58 ± 5.71 n.s. n.s. n.s.± 0.24a 99.19 ± 0.07a 98.53 ± 0.16b *** n.s. n.s.± 1.42 95.73 ± 2.45 98.36 ± 1.64 n.s. n.s. n.s.± 0.55b 86.89 ± 0.68ab 84.72 ± 1.63a ** n.s. n.s.± 2.57 98.21 ± 0.90 99.53 ± 0.47 n.s. n.s. n.s.± 0.41b 89.96 ± 0.57ab 90.31 ± 0.58ab n.s. n.s. n.s.± 0.79 84.61 ± 0.11 88.07 ± 0.75 n.s. n.s. n.s.± 0.16a 99.43 ± 0.04b 99.59 ± 0.04b *** n.s. n.s.± 1.22 100 100 n.s. n.s. n.s.± 0.71 95.31 ± 4.69 92.28 ± 4.92 n.s. n.s. n.s.± 0.75 100 100 n.s. n.s. n.s.± 0.94 84.47 ± 1.08 84.59 ± 2.10 n.s. n.s. n.s.± 0.49 100 99.58 ± 0.21 n.s. n.s. n.s.± 0.20 98.78 ± 0.10 98.97 ± 0.03 n.s. n.s. n.s.± 0.40 95.36 ± 0.01 95.74 ± 0.17 n.s. n.s. n.s.

mined by Student-Newman-Keuls post hoc multiple comparisons test. P values relative tof the table.

Table 6Apparent digestibility coefficients (%) of macronutrients for the three experimental diets with different EPA/ARA ratios when fed to juvenile Atlantic salmon reared at different watertemperatures.

Temperature 10 °C Temperature 20 °C P value1

ADC (%) D-ARA D-ARA/EPA D-EPA D-ARA D-ARA/EPA D-EPA Diet Temp. Diet x Temp.

Dry Matter 76.38 ± 0.78 76.05 ± 0.62 76.53 ± 0.06 78.83 ± 0.92 76.80 ± 1.10 78.47 ± 0.85 n.s. n.s. n.s.Protein 85.47 ± 0.29 84.65 ± 0.50 84.52 ± 0.60 86.30 ± 1.24 83.57 ± 0.59 84.90 ± 1.12 n.s. n.s. n.s.Lipid 94.48 ± 0.26a 94.64 ± 0.08a 95.11 ± 0.08a 96.23 ± 0.19b 96.27 ± 0.03b 96.40 ± 0.29b n.s. *** n.s.NFE 49.28 ± 3.16a 52.74 ± 1.58a 55.84 ± 1.94a 61.20 ± 1.99b 62.40 ± 2.36b 63.97 ± 1.28b n.s. *** n.s.Energy 83.79 ± 0. 83.51 ± 0.32a 84.16 ± 0.17a 86.37 ± 0.63b 84.77 ± 0.64ab 85.87 ± 0.54ab n.s. ** n.s.

Values in the same rowwith different superscripts are significantly different (P b0.05) as determined by Student–Newman–Keuls post hoc multiple comparisons test. P values relative totwo-way ANOVA statistical test are reported in the last three columns on the right hand side of the table.

1 n.s. = not significant; * P b 0.05; **P b 0.01; and *** P b 0.001.

164 C.T. Huguet et al. / Aquaculture 436 (2015) 160–166

4. Discussion

Independently from the dietary treatment, the sampling time point,or thewater temperature, both ARA and EPA recorded high digestibilityvalues. Previous observations described an apparent preferentialdigestibility for n-3 fatty acids versus n-6 fatty acids (Eroldogan et al.,2013; Francis et al., 2007). However, these observations were relativeto C18 PUFA, and not LC-PUFA, such as EPA and ARA, as in the present

Table 7Apparent digestibility coefficients (%) of fatty acids for the three experimental diets with differ

Temperature 10 °C Temper

ADC (%) D-ARA D-ARA/EPA D-EPA D-ARA

14:0 88.46 ± 0.75a 87.53 ± 0.37a 89.12 ± 0.86a 93.67 ±14:1n-5 93.88 ± 0.76 93.17 ± 0.15 94.29 ± 0.26 93.67 ±15:0 81.73 ± 1.73ab 77.63 ± 1.08a 81.04 ± 1.58ab 86.97 ±15:1n-5 75.89 ± 1.49b 77.04 ± 0.83b 89.45 ± 3.40c 6.83 ±16:0 87.48 ± 0.81a 85.81 ± 0.28a 86.95 ± 0.86a 92.73 ±16:1n-7 97.54 ± 0.33a 98.10 ± 0.28ab 98.30 ± 0.25ab 98.50 ±17:0 84.90 ± 0.77b 83.34 ± 0.28b 69.60 ± 1.98a 91.70 ±16:2n-4 90.29 ± 4.86 90.18 ± 0.14 94.71 ± 1.00 89.50 ±17:1n-7 99.45 ± 0.55 97.21 ± 2.79 99.39 ± 0.61 10016:3n-4 95.76 ± 1.29a 97.92 ± 1.04ab 98.19 ± 0.08ab 99.47 ±18:0 84.52 ± 0.96b 80.73 ± 0.29a 80.33 ± 1.40a 90.50 ±18:1n-9 t 1002 100 100 10018:1n-7 t 78.69 ± 4.25ab 73.99 ± 0.25a 79.32 ± 4.35ab 86.53 ±18:1n-9 97.55 ± 0.32a 97.85 ± 0.06ab 98.03 ± 0.09abc 98.23 ±18:1n-7 96.69 ± 0.37a 96.95 ± 0.08ab 97.34 ± 0.07abc 97.60 ±18:2n-6 t 81.67 ± 0.59 84.90 ± 2.99 77.07 ± 11.54 10018:2n-6 98.44 ± 0.29a 98.69 ± 0.06ab 98.89 ± 0.07ab 98.87 ±18:3n-6 98.64 ± 0.25b 97.80 ± 0.34b 96.35 ± 0.71a 98.87 ±18:3n-4 100 100 100 10018:3n-3 99.03 ± 0.22a 99.31 ± 0.06ab 99.45 ± 0.07ab 99.33 ±20:0 91.06 ± 0.60b 89.39 ± 0.32a 88.67 ± 0.67a 94.30 ±18:4n-3 100 100 99.95 ± 0.05 10020:1n-13 100 100 100 95.63 ±20:1n-11 92.55 ± 7.45 100 100 10020:1n-9 96.31 ± 0.94b 96.75 ± 0.07b 94.09 ± 0.28a 97.20 ±20:2n-6 93.44 ± 0.42b 91.09 ± 0.10b 88.50 ± 0.56b 90.83 ±20:3n-6 91.51 ± 1.45b 88.77 ± 0.93b 47.58 ± 5.71a 94.27 ±20:4n-6 99.13 ± 0.24bc 99.19 ± 0.07bc 98.53 ± 0.16a 99.40 ±20:3n-3 95.69 ± 1.42 95.73 ± 2.45 98.36 ± 1.64 99.17 ±22:0 90.35 ± 0.55b 86.89 ± 0.68ab 84.72 ± 1.63a 94.30 ±20:4n-3 97.43 ± 2.57 98.21 ± 0.90 99.53 ± 0.47 98.60 ±22:1n-11 92.63 ± 0.41 89.96 ± 0.57 90.31 ± 0.58 93.30 ±22:1n-9 84.36 ± 0.79a 84.61 ± 0.11a 88.07 ± 0.75ab 90.97 ±20:5n-3 98.96 ± 0.16a 99.43 ± 0.04b 99.59 ± 0.04b 99.37 ±22:4n-6 98.78 ± 1.22 100 100 97.47 ±24:0 89.08 ± 0.71 95.31 ± 4.69 92.28 ± 4.92 10022:5n-6 98.65 ± 0.75 100 100 97.97 ±24:1n-9 88.26 ± 0.94 84.47 ± 1.08 84.59 ± 2.10 89.23 ±22:5n-3 99.06 ± 0.49 100 99.58 ± 0.21 10022:6n-3 98.59 ± 0.20a 98.78 ± 0.10ab 98.97 ± 0.03ab 99.07 ±Total Fatty acids 95.55 ± 0.40a 95.36 ± 0.01a 95.74 ± 0.17a 97.17 ±

Values in the same rowwith different superscripts are significantly different (P b0.05) as determtwo-way ANOVA statistical test are reported in the last three columns on the right hand side o

1 n.s. = not significant; * P b 0.05; **P b 0.01; and *** P b 0.001.2 100% = fatty acid not detected in faeces.

study. Thus, the first important conclusion obtained by the presentstudy was that EPA and ARA are both efficiently and equally digestedand absorbed by Atlantic salmon.

A modification of lipid digestibility over time has been recentlyreported (Krontveit et al., 2014), but in the present study, the possiblealteration of nutrient digestibility, a results of biological adaptation todiet over time, seemed to be minimal. This could likely be due to acombination of two factors. The first one was that the two diets

ent EPA/ARA ratios when fed to juvenile Atlantic salmon at different water temperatures.

ature 20 °C P value1

D-ARA/EPA D-EPA Diet Temp. Diet x Temp.

0.20b 95.40 ± 1.15b 94.37 ± 0.55b n.s. *** n.s.0.12 93.20 ± 0.38 94.70 ± 0.35 n.s. n.s. n.s.0.44b 86.30 ± 2.53b 86.23 ± 1.32b n.s. *** n.s.

1.91a 0.00 ± 0.00 70.50 ± 2.61b *** *** ***0.27b 92.03 ± 0.07b 92.83 ± 0.48b n.s. *** n.s.0.12ab 98.67 ± 0.03b 98.80 ± 0.26b n.s. ** n.s.0.36c 90.40 ± 0.10c 83.13 ± 1.50b *** *** n.s.0.25 91.80 ± 0.32 95.23 ± 0.20 n.s. n.s. n.s.

100 100 n.s. n.s. n.s.0.53b 99.67 ± 0.33b 98.80 ± 0.50ab n.s. n.s. n.s.0.38c 87.90 ± 0.12c 87.97 ± 0.71c ** *** n.s.

100 100 – – –

1.02b 87.30 ± 0.35b 86.93 ± 0.88b n.s. *** n.s.0.19abc 98.73 ± 0.03c 98.50 ± 0.26bc n.s. *** n.s.0.21bc 98.17 ± 0.07c 98.00 ± 0.26c n.s. *** n.s.

97.77 ± 2.23 96.57 ± 3.43 n.s. ** n.s.0.12ab 99.17 ± 0.03b 99.03 ± 0.18ab n.s. n.s. n.s.0.09b 99.47 ± 0.27b 99.07 ± 0.47b n.s. *** n.s.

100 100 – – –

0.09ab 99.57 ± 0.03b 99.47 ± 0.09ab n.s. n.s. n.s.0.42c 92.63 ± 0.24b 92.30 ± 0.26b *** *** n.s.

100 100 n.s. n.s. n.s.4.37 100 99.07 ± 0.93 n.s. n.s. n.s.

100 100 n.s. n.s. n.s.0.32b 97.70 ± 0.06b 95.43 ± 0.73ab ** n.s. n.s.1.75b 86.30 ± 1.27b 77.23 ± 5.79a n.s. n.s. n.s.0.81b 86.27 ± 0.84b 54.03 ± 1.47a *** n.s. n.s.0.06c06a 99.40 ± 0.06c 98.70 ± 0.15ab *** n.s. n.s.0.83 95.13 ± 2.74 100 n.s. n.s. n.s.0.40c 90.47 ± 0.84b 88.17 ± 0.39b *** *** n.s.0.72 98.67 ± 0.69 100 n.s. n.s. n.s.1.41 89.83 ± 2.22 84.97 ± 5.56 n.s. n.s. n.s.1.84bc 85.77 ± 0.44a 93.00 ± 1.01c *** *** n.s.0.09b 99.57 ± 0.03b 99.63 ± 0.07b *** n.s. n.s.2.53 100 100 n.s. n.s. n.s.

98.80 ± 1.20 100 n.s. n.s. n.s.2.03 100 100 n.s. n.s. n.s.3.08 91.90 ± 0.62 89.13 ± 1.03 n.s. n.s. n.s.

100 100 n.s. n.s. n.s.0.07b 99.17 ± 0.03b 99.20 ± 0.12b n.s. ** n.s.0.12b 97.20 ± 0.00b 97.27 ± 0.23b n.s. *** n.s.

ined by Student–Newman–Keuls post hoc multiple comparisons test. P values relative tof the table.

165C.T. Huguet et al. / Aquaculture 436 (2015) 160–166

were relatively similar, including, 18:2n-6, 18:3n-3 and 22:6n-3 (DHA),and varying only in the ARA/EPA ratio. Therewere no extremedifferencesin their fatty acid composition such as a reduced LC-PUFA content andgreatly increased C18 PUFA, as is the case where abundant vegetableoils are used to replace fish oil, andwhich have been shown to potentiallyimpart histological modification of enterocytes (Olsen et al., 1999). Thesecond factor could have been that the length of time between thecommencement of the trial and the first sampling point was long enoughto allow fish to adapt to the different diets, and thus no further adaptationandmodificationwas recorded at the subsequent sampling point. Accord-ingly, quite efficient capabilities of phenotypic adaptations to diet havebeen shown for different fish species (Buddington et al., 1987; Refstieet al., 1998). Thus, in normal feeding practices, where the same feed isused for relatively long periods of time, the possible effects on nutrientsdigestibility resulting from the use of different lipid sources are of littleconcern. However, if more rapid and frequent feed alternations areused, as recently investigated and proposed to potentially maximise fishperformance and lipid metabolism by exploiting physiological dailyrhythms (Betancor et al., 2014; Brown et al., 2010), then clearly furtherstudies specifically focusing on these aspects are warranted.

Sub-optimal environmental temperatures are increasinglyencountered in aquaculture, and interesting new insights are beinggenerated, including the resulting effects on metabolome and endo-crine responses (Kullgren et al., 2013), and digestive physiology andfunction (Bowyer et al., 2014; Grans et al., 2013; Neuman et al., inpress). When it comes to nutrient digestibility, and specifically lipiddigestibility, several studies have already highlighted the existence ofa direct correlation. In general, most trials reported increased nutrientdigestibility with increasing temperature (Bogevik et al., 2010).Accordingly, in the present study, it was clearly shown that lowerwater temperature was responsible for lower lipid digestibility, lowertotal fatty acids digestibility, but also lower total nitrogen free extracts,and consequently also lower total energy digestibility. These findingswere consistent with previous studies on salmonids (Bogevik et al.,2010; Ng et al., 2004; Ng et al., 2010; Olsen and Ringo, 1998), and otherspecies as well (Guerreiro et al., 2012; Peres and Oliva-Teles, 1999).

Digestibility of individual fatty acids was also significantly affectedby environmental temperature, and it was particularly evident for themain SFA (14:0, 16:0 and 18:0), the main MUFA (namely 18:1n-9)and, amongst the PUFA, interestingly DHA (22:6n-3) was significantlyaffected by the temperature as well. As mentioned in the introduction,it has been well documented that in fish, fatty acids are digested andabsorbed differently relative to their chemical characteristics (chainlength and degree of unsaturation), which fundamentally determinetheir melting point (Austreng et al., 1980; Bureau and Meeker, 2011;Morais et al., 2005; Ringo, 1991; Sigurgisladottir et al., 1992). Thus,considering that themelting point of a lipid source is a key characteristicfor its digestibility, it appears to be evident that any variations inenvironmental temperature, and thus amodification of the lipid fluidity(solid tofluid status) could directly impact its digestibility. This has beenclearly demonstrated predominantly for SFA andMUFA (Ng et al., 2003;Ng et al., 2004; Ng et al., 2010; Olsen and Ringo, 1998), whereas PUFAhave been reported to be susceptible to suchmodifications but to a lesserextent, most likely because of their much lower melting points. Interest-ingly, in the present study, an increased DHA apparent digestibility forfish at the higher temperature was recorded but, admittedly, this was arelatively minor (~0.5%) increase from a quantitative point of view, andDHA was also digested at a very high rate at low temperature.

In this study we also showed that varying EPA/ARA ratio in the diethad only minor direct effects on digestibility, with no direct effecton overall nutrients digestibility, and fundamentally only statisticallysignificant effects in the fatty acid digestibility of EPA and ARAthemselves. Thus, in light of the recently increased interest in theroles of dietary ARA, as an important and somewhat overlooked LC-PUFA (Alves Martins et al., 2012; Bell and Sargent, 2003; Norambuenaet al., 2013a), this study can be considered instrumental also for providing

evidence that any inclusion of, or even fortification with ARA, should notcause concern about possible negative effects on nutrients and fatty acidsdigestibility.

Acknowledgements

This researchwas supported under theAustralian ResearchCouncil'sDiscovery Projects funding scheme (Project DP1093570). The viewsexpressed herein are those of the authors and are not necessarilythose of the Australian Research Council. The funding agency had norole in study design, data collection and analysis, decision to publish,or preparation of the manuscript. The authors wish to thank also Dr.Jacques Gabaudan (DSM Nutritional Products, Aquaculture Centre AsiaPacific) and DSM Nutritional Products for kindly donating theARASCO oil, Dr. Giorgio Palmeri (Warrnambool Cheese and ButterFactory) for kindly donating whey protein and Dr. Richard Smullen(Ridley Agriproducts/ Aquafeed) for kindly donating several of theraw materials used for experimental feed manufacturing.

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