Growth performance, proximate and histological analysis of rainbow trout fed diets containing Camelina sativa seeds, meal (high-oil and solvent-extracted) and oil

    Growth performance, proximate and histological analysis of rainbow trout fed diets containing camelina sativa seeds, meal (high-oil and solvent-extracted) and oil Christina N. Bullerwell, Stephanie A. Collins, Santosh P. Lall, Derek M. Anderson PII: DOI: Reference:

S0044-8486(15)30236-2 doi: 10.1016/j.aquaculture.2015.11.008 AQUA 631906

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

10 August 2015 5 November 2015 6 November 2015

Please cite this article as: Bullerwell, Christina N., Collins, Stephanie A., Lall, Santosh P., Anderson, Derek M., Growth performance, proximate and histological analysis of rainbow trout fed diets containing camelina sativa seeds, meal (high-oil and solvent-extracted) and oil, Aquaculture (2015), doi: 10.1016/j.aquaculture.2015.11.008

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ACCEPTED MANUSCRIPT Growth performance, proximate and histological analysis of rainbow trout fed diets

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containing Camelina sativa seeds, meal (high-oil and solvent-extracted) and oil

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Christina N. Bullerwella, Stephanie A. Collinsa, Santosh P. Lallb, Derek M. Andersona*

Dalhousie University, Faculty of Agriculture, Department of Plant and Animal Sciences, Truro,

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NS, Canada B2N 5E3

National Research Council of Canada, Institute for Marine Biosciences, Halifax, NS, Canada

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B3H 3Z1

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Corresponding author. Tel.: +1 902 893 6651; fax: +1 902 895 6734.

E-mail address: [email protected] (D. M. Anderson).

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ACCEPTED MANUSCRIPT Abstract Two 112-day feeding trials were conducted to examine the effects of including camelina

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(Camelina sativa) products in rainbow trout diets. In the first experiment, 20 tanks (40L) of

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rainbow trout (Oncorhynchus mykiss; 2.36±0.18g, 30 fish/tank) were fed one of five diets containing solvent-extracted camelina meal (SECM) at dietary inclusion levels of 0, 50, 100, 150

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or 200g/kg. Weight gain and specific growth rate (SGR) of fish fed 200g/kg SECM were

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significantly lower than those fed 0 and 50g/kg SECM. The protein efficiency ratio (PER) of fish fed 50g/kg SECM (2.4) was significantly higher than fish fed 0, 150 and 200g/kg SECM (all

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2.1). There were no significant differences among treatments for total feed consumption or FCR, although during earlier periods of the trial, fish fed 150 and 200g/kg SECM had higher FCR than

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fish fed 0 and 50g/kg SECM (P<0.05). In the second experiment, 24 tanks (40L) of rainbow trout (1.0±0.1 g, 30 fish/tank) were fed one of eight diets: a control diet; six diets containing full-

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fat camelina seed or high oil residue camelina meal (HORM), at dietary inclusion levels of 100, 200 and 300g/kg; or a diet that contained 100g/kg SECM and 151g/kg camelina oil (SECM+CO), with no added fish oil. After 112 days on feed, fish fed diet containing 300g/kg camelina seed and 300g/kg HORM gained less weight than fish fed the control diet (P<0.05). Fish that consumed 300g/kg camelina seed also gained significantly less weight than fish fed 100g/kg camelina seed and 100g/kg HORM. Feed consumption was not affected by diet. FCR was higher in fish fed 300g/kg HORM than in fish fed 100g/kg camelina seed and the PER of the fish fed 300g/kg HORM was lower than that of fish fed 100g/kg camelina seed and HORM (P<0.05). There was no treatment effect on the intestinal histology of fish fed these diets (P<0.05). Fish fed up to 100g/kg SECM, camelina seed and HORM, as well as the SECM+CO diet did not perform any differently than fish fed the control diet (P>0.05). Although there were a

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ACCEPTED MANUSCRIPT few negative effects on performance in fish fed 200g/kg camelina seed and HORM early in the trial, this did not remain consistent for the entire trial. The maximum dietary inclusion threshold

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of these camelina products in juvenile rainbow trout diets is likely between 100 and 200g/kg,

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although further research will be required to determine more definite values.

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Keywords: rainbow trout; camelina; seed; meal; oil; growth; histology

Abbreviations: HORM, High oil residue camelina meal; FCR, Feed conversion ratio; PER,

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Protein efficiency ratio; SECM, pre-press solvent-extracted camelina meal; SGR, Specific

1. Introduction

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growth rate

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Camelina (Camelina sativa) is an ancient oilseed crop in the Brassicaceae family that includes mustard, cabbage, broccoli and rapeseed.

In recent years, renewed interest has

developed in camelina as a crop for biofuel, human consumption and animal feedstuffs. Products derived from camelina, including camelina seeds, oil, pressed meal and solventextracted camelina meal (SECM) may have the potential to be included in aquaculture feeds. Camelina is attractive as a potential aquafeed ingredient due to its oil content (40.447.7% of camelina seed) and fatty acid composition (as high as 90% of the total fatty acid content). The essential fatty acids α-linolenic acid and linoleic acid comprise approximately 35 and 15% of the total fatty acids, respectively (Zubr, 1997). Camelina seeds are also 23-30% protein (Marquard and Kuhlmann, 1986). Cold pressing camelina oil from camelina seeds yields a presscake that can be ground to form a meal. This meal contains 10% residual oil and is 45%

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ACCEPTED MANUSCRIPT crude protein and 12.8% crude fibre (Zubr, 1997). It may be referred to as high oil residue meal (HORM). The pressed meal can be further processed by solvent extraction (SECM), to remove

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oil from the meal and increase its overall protein content (Korsrud et al., 1978).

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Early work has already been made to determine the effects of camelina meal and oil as a feed ingredient for rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and

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Atlantic cod (Gadus morhua) (Betancor et al., 2015; Ye, 2016; Hixson et al., 2014a; Hixson et

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al., 2014b; Morais et al., 2012). Hixson et al. (2014b) were able to completely replace fish oil with camelina oil in Atlantic salmon diets without impacting weight gain, although concurrently

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including camelina oil and HORM (dietary inclusion of 195g/kg and 100g/kg, respectively) in the same diet did significantly reduce growth as compared with control-fed fish. Morais et al.

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(2012) also found completely replacing fish oil with camelina oil had no impact on growth performance, although it did significantly increase intestinal lipid levels in addition to altering

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the structural properties of the intestinal muscle of Atlantic cod. Pan et al. (2011) found dietary inclusion levels of high-oil residue camelina meal (HORM) up to 160g/kg did not negatively affect growth performance or carcass composition of rainbow trout. Ye et al. (2016) fed SECM to Atlantic salmon and found 50g/kg SECM in the diet did not affect fish performance, but inclusion levels of 100g/kg and higher did. The negative impact of HORM-containing diets on fish growth observed by Hixson et al. (2014b), may have been due to antinutrients present in the meal. Antinutrients, or antinutritional factors (ANF) are compounds that serve a metabolic or protective function in the plant, but impair the nutritive value of animal feeds (Francis et al., 2001; Bennett and Wallsgrove, 1994). Antinutritional factors found in camelina meal include glucosinolates, sinapine, phytic acid and

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ACCEPTED MANUSCRIPT condensed tannins (Matthäus and Zubr; 2000, Schuster and Friedt, 1998), as well as indigestible carbohydrates (Zubr, 2010; Zubr, 1997).

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The objectives of this study were to assess the effects of camelina products on the

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performance of rainbow trout to establish the maximum inclusion rates of camelina products for use in practical diets. This was accomplished by examining the effects of camelina seeds,

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HORM, SECM and camelina oil on rainbow trout growth and feed intake-related parameters, as

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well their intestinal histology and carcass composition.

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2. Materials and Methods 2.1. Experimental ingredients

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Four camelina products were tested in the two experiments detailed in this study: ground camelina seed, camelina oil, pre-press solvent-extracted camelina meal (SECM) and high-oil

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residue camelina meal (HORM).

Camelina (Calena cultivar) was grown and harvested in Canning, Nova Scotia (Canada) under the supervision of staff of Dalhousie University, Faculty of Agriculture (Truro, NS, Canada). Camelina oil was extracted from the seeds by Atlantic Oilseed Processing, Ltd. (Kinkora, Prince Edward Island, Canada), using a KEK P-0500 expeller-press (EGON KELLER GMBH and CO. KG, Remscheid, Germany). The remaining presscake was hammer-milled (screen size 8 mm) to yield a camelina meal with a high oil content, or HORM. To prevent oxidation during storage, ethoxyquin (60% ethoxyquin, 40% silica) was added to the camelina oil and HORM at 0.2% of the predicted oil content. Petroleum ether was used to solvent-extracted HORM at a ratio of 3:1 (1 hour, stirring once every 15 min) at the Dalhousie University Faculty of Agriculture, then air-dried for two

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ACCEPTED MANUSCRIPT hours in a fume hood to produce SECM. To prepare the full-fat camelina seed as a feed ingredient, it was ground for 20 seconds using a coffee grinder.

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The chemical composition of the four camelina products tested in this experiment are

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presented in Table 1.

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2.2. Diet preparation

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All diets were formulated to be isocaloric, isonitrogenous and to meet the nutrient requirements of rainbow trout (NRC, 2011). They were formulated to contain 45% protein (as

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fed) and 4156 kcal/kg estimated digestible energy.

All diets were mixed and pelleted at the Dalhousie University, Faculty of Agriculture

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Campus. A Hobart mixer (Hobart Corporation; Model L-800; Troy, OH, USA) was used to mix

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diets, which were then steam-pelleted in a laboratory-scale pellet mill (California Pellet Mill Co.,

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Crawfordsville, Indiana, USA). The feed used in Experiments 1 was pelleted using a 2.5mm die. The feed used in Experiment 2 was pelleted through a 3mm die. The pelleted feed was dried for 3h at 55ºC in a forced air oven, sifted to remove any fines, and stored at -20ºC. During the early stages of Experiments 1, feed pellets were ground to form a crumble and sifted through a 1mm screen. As the size of the fish increased, the particle size of the feed was gradually increased to 2.5mm. In Experiment 2, the particle size of the feed was initially 1.5mm and gradually increased to 2.5mm.

2.3. Fish husbandry The data presented in this manuscript was collected in two experiments. The trials were conducted at the Dalhousie University, Faculty of Agriculture Campus. In both experiments, fish were housed in experimental tanks in flow-through systems that were provided with continuous 6

ACCEPTED MANUSCRIPT aeration and a flow of nitrogen degassed fresh water at a rate of approximately 2L / min. Water temperature was maintained at 14±2ºC and recorded daily. Fish were hand-fed three times per

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day, seven days per week, to apparent satiation. Tanks were purged two times daily to remove

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any excreta. Mortalities were removed immediately from the tanks and their weights in addition to the feed weights for that tank were recorded. Prior to beginning each feeding trial, the control

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diet to be used for that experiment was fed to all experimental fish in the housing system for a

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minimum of one week as an acclimation period. Fish were cared for according to the guidelines

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of the Canadian Council on Animal Care (2005).

2.4. Growth trials

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2.4.1. Experiment 1 - Performance of rainbow trout fed graded levels of pre-press solvent extracted Camelina sativa meal

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One control diet was formulated, as well as four test diets that contained pre-press solvent extracted camelina meal (SECM) at inclusion levels of 50, 100, 150 and 200g/kg (Table 2). Diets were randomly assigned to 20 tanks, with four replicates per treatment. Rainbow trout (initial weight: 2.3±0.1g) were randomly assigned to 40L tanks (30 fish /tank). The fish were fed experimental diets for 16 weeks, from January to May 2011, under a natural photoperiod (45.3647°N, 63.2800°W). Feed intake was recorded for the duration of the trial. Batch weights of the fish were taken on days 0, 28, 56, 84 and 112.

2.4.2. Experiment 2 - Performance of rainbow trout fed camelina seeds and high oil residue camelina meal

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ACCEPTED MANUSCRIPT One control diet and seven test diets were used in this experiment. Three contained ground camelina seeds at inclusion levels of 10, 20 or 30%. Three contained high oil residue

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camelina meal (HORM) at inclusion levels of 10, 30 or 30%. A double substitution diet was also

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formulated, which contained 10% HORM and a total replacement of added fish oil with camelina oil (Table 3). This experiment was conducted using 24 tanks (40L) from December,

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2011 to April, 2012. The lighting regime for these fish followed a natural photoperiod

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(45.3647°N, 63.2800°W). Diets were fed to rainbow trout (initial weight: 1.0±0.1g). There were 30 fish / tank with three replicates / treatment, and diets were assigned randomly to tanks. The

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fish were fed the experimental diets for 16 weeks and batch weights of each tank were measured

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on days 0, 28, 56, 84 and 112. Feed intake was recorded for the duration of the trial.

2.5. Growth response and related factors

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The batch weights and feed intake data taken in these growth trials was used to calculate growth response and related parameters for each period within a trial, as well as for the entirety of each experiment. Mean body weight, weight gain, specific growth rate (SGR), thermal growth coefficient (TGC), average feed consumption, feed conversion ratios (FCR) and protein efficiency ratio (PER) were calculated with the following equations: mean body weight = body weight / number of fish, weight gain = final body weight – initial body weight, SGR = (ln (final body weight) – ln (initial body weight)) / number of days in period *100, feed consumption = (final weight of feed – initial weight of feed) / number of fish, FCR = (final weight of feed – initial weight of feed) / final body weight – initial body weight, PER = (final body weight – initial body weight) / feed consumed * percentage of protein in feed.

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ACCEPTED MANUSCRIPT 2.6. Tissue sampling At the end of Experiment 2, six fish from each tank were euthanized by an overdose of

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tricaine methanesulfonate (TMS; Syndel Laboratories Ltd., Nanaimo, BC, Canada). The body

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cavities of the fish were opened along the ventral midline. The digestive tract (esophagus to anus) and all the attached organs, except heart and kidney, were carefully removed. Hindgut

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samples were taken by removing a section of approximately 2cm from the most distal point of

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the intestinal tract. Midgut samples were taken by removing an approximately 2cm section of intestine from the center of the intestine, which was defined as the smooth-surfaced region of the

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intestine occurring caudal to the point of the most distal pyloric ceca and rostral to the hindgut, which was identified based on its increase in diameter and visibility of circular folds and

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sacculations.

Intestinal samples were immediately placed in scintillation vials filled with 10% neutral

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phosphate-buffered formalin (3.7% formaldehyde) for future histological analysis. Fish carcasses (intestinal tracts and attached organs removed) were frozen at -20oC.

2.7. Histology analysis

Histological slides were prepared by the Animal Health Laboratory, Agriculture and Food Operations Branch (Nova Scotia Department of Agriculture, Truro, NS, Canada). Formalin-fixed tissues were subjected to the wax tissue processing method (Drury and Wallington, 1980). They were dehydrated using a graded series of alcohol baths, then cleared of alcohol using xylene in a Tissue-Tek VIP (Sakura Finetek USA Inc., Torrance, CA, USA). Tissues were then infiltrated with paraffin wax using a Tissue-Tek TEC (Sakura Finetek USA

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ACCEPTED MANUSCRIPT Inc., Torrance, CA, USA). A 5µm cross-sectional section was cut from the embedded samples, placed in a 35.5oC water bath, mounted on a slide and stained with haematoxylin and eosin.

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Histological slides and one 1mm calibration slide were scanned using a Nikon Coolscan

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4000ED (Nikon Inc., Japan). Images were captured using Nikon Scan 4.0.2 (Nikon Inc., Japan)

using a 2-point re-scaling on the 1.0mm calibration.

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and viewed using SigmaScan Pro 5 (SPSS Inc., Chicago, IL). Distance and area were calibrated

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Villus height, villus width, villus area, crypt depth and mucosa width were measured. Points of measurement are shown in Figure 1. Villi were determined measurable by being fully

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visible from tip to the submucosa, with no shattered edges. Villus width was measured at the midpoint of each villus. Crypt depth was measured from the top of the crypt to the inner edge of

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the muscularis mucosae. The intestinal wall thickness was measured from the inner edge of the muscularis mucosae to the outer edge of the serosa. As many villi as possible were measured, up

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to 10 villi per slide and no less than five. If more than 10 were able to be measured, the villi were chosen to be as evenly spaced around the intestine sample as possible. Slides with fewer than six suitable villi per slide were excluded.

2.8. Chemical analysis

Fish carcasses were pooled by tank and ground in a commercial hand-operated meat grinder (4mm die). Ground tissue from each tank was split between three aluminum pie plates. Each pie plate was frozen at -20oC and freeze-dried overnight. Dry matter was determined by weighing the pie plates prior to adding samples and after the sample was added. Weights of samples were taken before and after freeze-drying and percent moisture was calculated (moisture = ((wet weight-dry weight) / wet weight)*100). Feeds, ingredients and freeze-dried fish tissues

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ACCEPTED MANUSCRIPT were ground using a coffee grinder to a particle size of 1mm. Feeds and ingredients were analyzed for moisture (AOAC, 2005, method no. 934.01). The ash content of the fish tissues was

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determined (AOAC 2005; method no. 942.05). Test feed ingredients, experimental diets and fish

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tissues were analyzed for dry matter (100-moisture), crude fat (AOCS, 2005; method Am 5-04) and crude protein (AOAC, 2005; method no. 990.03). In determining crude fat, an ANKOM

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XT15 extraction system (ANKOM Technology, Macedon, NY, USA) was used to extract fat

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from samples. Crude protein was determined using a Leco protein / N analyzer (Model FP-528, Leco Corp., St. Joseph, MI, USA). The test ingredients were also analyzed to determine their

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mineral composition (AOAC, 2003; method no. 968.08).

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2.9. Statistical analysis

Each experiment was conducted in a completely randomized design. The General Linear

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Model procedure of IBM SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used to analyze growth parameters and histological data. For all growth and feed intake parameters measured, tank was the experimental unit. For histological parameters, fish was the experimental unit. Differences between means were calculated using the Ryan-Einot-Gabriel-Welsch F-test. Means were considered significantly different when P<0.05. Linear and quadratic regression analysis was conducted using IBM SPSS 20.0 to determine the effect of ingredient (camelina seed, HORM and SECM) inclusion rate on all measured growth and feed intake parameters. Regressions were considered significant when P<0.05.

3. Results

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ACCEPTED MANUSCRIPT 3.1. Experiment 1 - Performance of rainbow trout fed graded levels of pre-press solvent extracted Camelina sativa meal

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The growth of fish (Table 4) showed that on Day 28, fish fed 15 and 20% SECM diets

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had lower weight gains and SGR and higher FCR than fish fed the other treatments (P<0.05). Fish fed 10% SECM gained less than fish fed 0 and 5% SECM. Fish fed 15 and 20% SECM had

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a significantly lower PER than fish fed 10, 5 or 0% SECM. The PER of fish fed 10% SECM was

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also lower than those of fish fed 5 and 0% SECM (P<0.05).

On day 56, the FCR of fish fed 20% SECM was higher than fish fed 0 and 5% SECM and

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that of fish fed 15% SECM was higher than fish fed the control diet (P<0.05). Both of these diets also had a significantly lower PER than fish fed the control diet.

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Over the 56-84 day period, fish fed 15 and 20% SECM gained less than fish fed the other three treatments. Fish fed 10% SECM also gained less than those fed 0 and 5% SECM (P<0.05).

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A drop in feed consumption was observed in fish fed 15 and 20% SECM. Fish that consumed 15% SECM had significantly lower feed intake than those fed diet containing 5 and 10% SECM and fish fed 20% SECM ate less than those fed 0, 5 and 10% SECM (P<0.05). The PER of fish fed 20% SECM was significantly lower than those fed 0 and 5% SECM and that of fish fed 10% SECM was significantly lower than fish who consumed the control diet. On day 112, the SGR of fish fed 20% SECM was significantly higher than that of control-fed fish and the FCR was significantly higher than fish fed 0, 5 and 10% SECM. The FCR of fish fed 15% SECM was higher than fish fed 0 and 5% SECM (P<0.05). In this experiment, fish fed 20% SECM gained less weight and had a lower SGR than fish fed 0 and 5% SECM (P<0.05) and fish fed 15 and 20% SECM had a lower PER than fish fed 5% SECM (P<0.05). There were no other differences among treatments for weight gain or

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ACCEPTED MANUSCRIPT PER, nor were there any differences for the other parameters (P>0.05). Significant, negative linear relationships determined an increase in the dietary inclusion rate of camelina seed led to

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reduced feed consumption and SGR. No significant regressions were determined the other

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growth or feed intake parameters measured (Figure 2).

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3.2. Experiment 2 - Performance of rainbow trout fed camelina seeds and high oil residue

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camelina meal

The growth of fish in this experiment (Table 5) showed that on day 28, fish fed 30%

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camelina seed and 30% HORM gained significantly less weight than fish fed all other experimental diets, except for the 20% HORM diet. Fish fed diets containing 20% camelina seed

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and 20% HORM gained less weight than fish fed the control diet (P<0.05). The SGR of fish fed diets containing 30% camelina seed and HORM were significantly lower than those of fish fed

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all treatments except for the 20% camelina seed and HORM diets. Fish fed the 20% camelina seed diet had a significantly lower SGR than fish fed the control. Fish receiving the diet containing 30% camelina seed had significantly lower feed consumption than fish fed the control and SECM+CO diets. Fish fed the 10% camelina seed diet also ate less than fish fed the DR diet (P<0.05). None of the FCR or PER of the experimental treatments differed significantly from fish fed the control diet. However, fish fed 10% camelina seed had a lower FCR and a higher PER than fish fed the 30% HORM and SECM+CO diets (P<0.05). Fish fed 10% HORM diet had a lower FCR than fish fed 30% HORM and a higher PER than fish fed diets containing 20 and 30% camelina seed, 20 and 30% HORM, as well as the SECM+CO diet (P<0.05). The weight gains of fish fed the 300g/kg camelina seed, 300g/kg HORM and SECM+CO diets were significantly less than fish fed the control and 10% HORM diets on day 56. Fish fed

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ACCEPTED MANUSCRIPT 300g/kg camelina seed also gained less weight than fish who ate diets containing 100g/kg camelina seed and 200g/kg HORM. Fish fed with diets containing 300g/kg HORM gained less

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weight than fish fed 100g/kg camelina seed in their feed (P<0.05).

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On day 84, the weight gain of all fish fed the experimental treatments was similar to that of fish fed the control diet (P>0.05). Fish fed diets containing 300g/kg camelina seed gained less

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weight than fish fed 100g/kg HORM in their feed (P<0.05).

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The overall results showed that fish fed diet containing 300g/kg camelina seed and 300g/kg HORM gained significantly less weight than fish fed the control diet. Fish fed 30%

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camelina seed gained significantly less weight than fish fed the other experimental diets. The PER of fish fed all experimental diets were similar to the PER of fish fed the control diet

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(P>0.05). Fish fed 100g/kg camelina seed and fish fed 10% HORM had a PER that was significantly higher than that of fish fed 300g/kg HORM.

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There was a negative linear effect of increasing the dietary inclusion rate of SECM on feed intake (P<0.05). Significant, negative quadratic relationships were determined for the SGR of both the HORM-fed and SECM-fed fish. Increased dietary inclusion rates of these feed ingredients were associated with reduced SGR. There was also a significant, negative quadratic effect of increasing the dietary inclusion rate of HORM on PER. No other significant regressions were determined (Figure 2). All histological measurements including villus height, villus mid-width, villus area, crypt depth and mucosa width were similar for all diets for the midgut and hindgut samples. The carcass composition analysis showed no difference in dry matter, ash, fat or protein among fish fed the experimental diets (Table 6).

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ACCEPTED MANUSCRIPT 4. Discussion Performance of rainbow trout was not affected when 50g/kg SECM was included in the

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diet, however rainbow trout fed SECM at dietary inclusion levels of 150 and 200g/kg had

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decreased performance compared to those fed the control diet. This correlates with the reduced feed intake and PER also seen in fish fed diets containing higher levels of SECM. In the first

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experiment, the fish fed diets containing 100g/kg SECM exhibited reduced growth performance,

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which was seen in a PER that was significantly lower than that of the control-fed and 50g/kg SECM-fed fish, but this reduced PER did not affect their weight gain. In the second experiment,

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fish fed the SECM+CO diet, which contained 100g/kg SECM and 151g/kg camelina oil (with no added fish oil) exhibited similar growth performance and feed intake as fish fed the control diet

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(P>0.05). This differs from the results of Hixson et al. (2014a), those diets were fed to Atlantic salmon, rather than rainbow trout. Evidence indicates these two salmonid species respond

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differently to similar feeds (Krogdahl et al., 2004). Additionally, the total levels of camelina oil included in this diet was lower (44g/kg) and SECM was used in the current trial, rather than HORM.

Growth data for the fish fed diets containing 150g/kg and 200g/kg SECM in their feed indicate that they were beginning to acclimatize to these higher inclusion levels. On day 112, the SGR of fish fed 200g/kg SECM in their diets was significantly higher than that of control-fed fish, as was their feed intake, whereas the feed intake of the 200g/kg SECM-fed fish was significantly lower than that of the control-fed fish the previous month. These fish adjusted to their feed later in the trial and went through a period of compensatory gain (Sevgili, et al., 2012). Although the total weight gain of the fish fed 200g/kg SECM in their feed was significantly lower than that of the control-fed fish, in the final month of this trial, there were no significant

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ACCEPTED MANUSCRIPT differences between treatments with respect to weight gain. Extending the length of future trials may provide further information on the ability of rainbow trout to recover from the initial decline

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in growth observed in the first few months of the fish fed higher levels of SECM.

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The significantly reduced feed intake of fish fed 300g/kg camelina seed in the first month of feeding may have been a result of the glucosinolate levels of this diet. The dietary

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glucosinolate level that rainbow trout can tolerate has been determined to be in the range of

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1,400-19,300 µmol/g (Burel et al., 2001; Tripathi and Mishra, 2007) and the glucosinolate content of the 300g/kg camelina seed diet was within this range. Schuster and Friedt (1998)

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determined the total glucosinolate content of ten genotypes of camelina ranged between 13.236.2 µmol/g, which would place the glucosinolate content of this diet in the range of 3,960-

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10,860 µmol/g. At levels beyond this tolerance range, rainbow trout growth performance is negatively influenced (Burel et al., 2001; Tripathi and Mishra, 2007) and even when fed at low,

2013).

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non-toxic levels, glucosinolates cause a decrease in feed intake of rainbow trout (Collins et al.,

Growth and feed intake of rainbow trout fed 100g/kg camelina seed or HORM in their diets did not differ from that of control-fed fish (P>0.05). After the first 28 days of this trial, fish fed HORM at dietary inclusion levels greater than 100g/kg had lower body weights than controlfed fish (P<0.05). Differences between treatments became less marked as time progressed, indicating again, that an adaptation period had passed. With the exception of the first month, feed consumption and SGR were similar among all diets for the duration of the trial (P>0.05). In terms of total performance, after 112 days on feed, the 100g/kg camelina seed, 100g/kg HORM and the SECM+CO treatments were tolerated well by rainbow trout. Anderson et al. (2008) found that utilization of full-fat camelina seeds in rainbow trout diets at an inclusion rate of

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ACCEPTED MANUSCRIPT 150g/kg had similar SGR and PER as fish feed herring meal control diet and Pan et al. (2011) reported rainbow trout fed diets containing 160g/kg HORM would have a similar SGR and FCR

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as control-fed fish. This agrees with the findings of the current study as levels of 100g/kg were

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accepted early in the trial, but not a level of 200g/kg, indicating that the acceptable inclusion level for these feed ingredients fits within this range.

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Further investigation will be required to determine the maximum inclusion level of these

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feed ingredients for rainbow trout. Additional evidence of the suitability of these camelina products is that although high inclusion levels may cause reductions in feed intake and growth at

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certain points during the feeding period, at dietary inclusion levels up to 200g/kg, camelina meal and HORM does not affect the intestinal morphology or tissue composition of rainbow trout

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(also with the inclusion of a combination of 100g/kg SECM and 151g/kg camelina oil ). The histological analysis data indicate there were no alterations in intestinal morphology

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due to the inclusion of these camelina products in rainbow trout diets. Other studies investigating the inclusion of plant proteins such as soybean meal (Venold et al., 2011) and potato protein concentrate (Tusche et al., 2015) have observed morphological differences in fish fed these feed ingredients as compared with control-fed fish. This effect may be attributed to the antinutrients found in these plant proteins, such as saponins and alkaloids (Francis et al., 2001; Tusche et al., 2015), neither of which have been reported present in camelina. However, to ensure that these camelina products do not induce inflammation in the digestive tract of rainbow trout, futher studies would benefit from investigating the presence or absence of inflammatory gene expression markers and other immune responses that may also occur (Mansfield et al., 2010; Venold et al., 2011; Ramos et al., 2015).

17

ACCEPTED MANUSCRIPT The use of more advanced processing technology, such as research into the removal of ANF from camelina products or the development of a protein concentrate, may improve the

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quality of camelina products to be included at a higher level in aquafeeds. Similarities in the

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equations for the relationship between the dietary inclusion levels of camelina seed and SECM on rainbow trout feed consumption and SGR indicate that it would be beneficial to investigate

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the ANF in these feed ingredients that affect feed intake, such as glucosinolates. Removal of

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these ANF may reduce feed aversion, allowing for greater dietary inclusion levels. Increasing the nutrient density of these products, through the production of camelina protein concentrates, may

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also be a method of increasing the dietary inclusion level of camelina products in the diets of rainbow trout. Future research should focus on developing processing methods that would

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prevent reductions in feed consumption and growth performance related to increasing levels of these camelina seed, HORM and SECM, as illustrated in the performance curves provided in

diets.

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Figure 2. If effective, this may allow for higher inclusion levels of camelina products in salmonid

5. Conclusion

The information gained from this trial will be useful in the formulation of practical diets using these feed ingredients. Performance of rainbow trout was not affected by the inclusion of 100g/kg camelina seed, HORM or SECM in the diets, or by feeding a diet containing 100g/kg SECM and 151 g/kg camelina oil with no additional fish oil. Results demonstrate that at these inclusion levels, camelina products are suitable ingredients for use in rainbow trout diets. Further studies on protein concentration and the effects of the ANF in these products may increase the maximum inclusion level of camelina protein products in salmonid feeds.

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Acknowledgements

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Genome Atlantic, The Atlantic Canada Opportunities Agency - Atlantic Innovation Fund

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and the Province of Nova Scotia Aquaculture Development Fund provided funding for this project. Thank you to Cara Kirkpatrick, Michael McConkey, Scott Jeffrey, Paul MacIsaac, Jamie

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Fraser, Zhiyu Chen, Minhao Fu, Jennifer Cousineau, Terra MacDonald, Graham Ripley and

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Dalhousie University, Faculty of Agriculture’s NUTR 3002 students (2011) for their assistance

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with this research.

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ACCEPTED MANUSCRIPT References Anderson, D.M., MacPherson, M.J., MacIsaac, P.F. 2008. Feeding full-fat oilseeds as partial

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replacement for fish meal and fish oil in practical diets for rainbow trout fingerlings. Can. J. Anim. Sci. 88, 166.

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ANKOM Technology. 11 April 2014. Method 5 – Acid Detergent Fiber in Feeds – Filter Bag

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Technique for A200. ANKOM Technology, Macedon, NY, USA. Accessed July 24, 2015

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from www.ankom.com.

ANKOM Technology. 11 April 2014. Method 6 – Neutral Detergent Fiber in Feeds – Filter Bag

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Technique for A200. ANKOM Technology, Macedon, NY, USA. Accessed July 24, 2015 from www.ankom.com.

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AOAC (Association of Official Analytical Chemists), 2005. Official Methods of Analysis. 18th Edition. AOAC, Gaithersburg, MD, USA.

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AOAC (Association of Official Analytical Chemists), 2003. Official Methods of Analysis. 17th Edition. AOAC, Gaithersburg, MD, USA. AOCS (American Oil Chemists Society), 2005. AOCS Official Procedure. Approved Procedure Am 5-04. Rapid determination of oil/fat utilizing high temperature solvent extraction. AOCS, Urbana, IL, USA.

Bennett, R.N., Wallsgrove, R.M. 1994. Tanley Review No. 72. Secondary metabolites in plant defense mechanisms. New Phytol. 127, 617-633. Betancor, M.B., Sprague, M., Usher, S., Sayanova, O., Campbell, P.J., Napier, J.A., Tocher, D.R. 2015. A nutritionally-enhanced oil from transgenic Camelina sativa effectively replaces fish oil as a source of eicosapentaenoic acid for fish. Sci. Rep. 5, 8104.

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ACCEPTED MANUSCRIPT Burel, C., Boujard, T., Kaushik, S.J., Boeuf, G., Mol, K.A., Van der Geyten, S., Darra, V.M., Kühn, E.R., Pradet-Balade, B., Quérat, B., Quinsac, A., Krouti, M., Ribaillier, D. 2001.

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rainbow trout. Gen. Comp. Endocrinol. 124, 343-358.

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Effects of rapeseed meal-glucosinolates on thyroid metabolism and feed utilization in

Canadian Council on Animal Care. 2005. Guide to the care and use of fish in research

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teaching and testing, Canadian Council on Animal Care, Ottawa, ON, Canada.

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Collins, S.A., Mansfield, G.S., Desai, A.R., Van Kessel, A.G., Hill, J.E., Drew, M.D. 2013. Structural equation medeling of antinutrients in rainbow trout diets and their impact on

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feed intake and growth. Aquaculture 416-417, 219-227. Drury, A.A., Wallington, E.A. 1980. Carleton’s histological technique. 5th Edition.,

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Oxford University Press, New York, USA. Francis, G., Makkar, H.P.S., Becker, K. 2001. Antinutritional factors present in plant-derived

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alternate fish feed ingredients and their effects in fish. Aquaculture 199, 197-227. Hixson, S.M., Parrish, C.C., Anderson, D.M. 2014a. Full substitution of fish oil with camelina (Camelina sativa) oil, with partial substitution of fish meal with camelina meal, in diets for farmed Atlantic salmon (Salmo salar) and its effect on tissue lipids and sensory quality. Food Chem. 157, 51-61. Hixson, S.M., Parrish, C.C., Anderson, D.M. 2014b. Use of camelina oil to replace fish oil in diets for farmed salmonids and Atlantic cod. Aquaculture 431, 44-52. Korsrud, G.O., Keith, M.O., Bell, J.M. 1978. A comparison of the nutritional value of crambe and camelina seed meals with egg and casein. Can. J. Anim. Sci. 58, 493-499.

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ACCEPTED MANUSCRIPT Krogdahl, Å., Sundby, A., Olli, J.J. 2004. Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) digest and metabolize nutrients differently. Effects of water

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salinity and dietary starch level. Aquaculture 229, 335-360.

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Mansfield, G.S., Desai, A.R., Nilson, S.A., Van Kessel, A.G., Drew, M.D., Hill, J.E. 2010. Characterization of rainbow trout (Oncorhynchus mykiss) intestinal microbiota and

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inflammatory marker gene expression in a recirculating aquaculture system. Aquaculture

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307, 95-104.

Matthäus, B., Zubr, J. 2000. Variability of specific components in Camelina sativa oilseed cakes.

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Ind. Crop Prod. 12, 9-18.

Morais, S., Edvardsen, R.B., Tocher, D.R., Bell, J.G. 2012. Transcriptomic analysis of intestinal

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gene expression of juvenile Atlantic cod (Gadus morhua) fed diets with Camelina oil as replacement for fish oil.

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National Research Council (NRC). 2011. Nutrient requirements of fish and shrimp. National Academy Press. Washington, DC, USA. Pan, W., Xie, W., Caldwell, C. D. and Anderson, D. M., 2011. Growth performance and carcass composition of rainbow trout (Oncorhynchus mykiss) fed practical diets containing graded levels of high fat residue Camelina meal. Can. J. Anim. Sci. 91, 484. Ramos, M.A., Gonçalves, J.F.M., Batista, S., Costas, B., Pires, M.A., Rema, P., Ozório, R.O.A. 2015. Growth, immune responses and intestinal morphology of rainbow trout (Oncorhynchus mykiss) supplemented with commercial probiotics. Fish Shellfish Immunol. 45, 19-26. Schuster, A., Friedt, W. 1998. Glucosinolate content and composition as parameters of quality of Camelina seed. Ind. Crop Prod. 7, 297-302.

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ACCEPTED MANUSCRIPT Sevgili, H., Belgin, H., Emre, Y., Kanɩlmaz, M. 2012. Compensatory growth after various levels of dietary protein restriction in rainbow trout, Oncorhynchus mykiss. Aquaculture 344-

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349, 126-134.

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Tripathi, M.K., Mishra, A.S. 2007. Glucosinolates in animal nutrition: a review. Anim. Feed Sci. Technol. 132. 1-27.

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Tusche, K., Wuertz, S., Susenbeth, A., Schultz, C. 2011. Feeding fish according to organic

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aquaculture guidelines EC 710/2009: Influence of potato protein concentrates containing various glycoalkaloid levels on health status and growth performance of rainbow trout

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(Oncorhynchus mykiss). Aquaculture 319, 122-131. Venold, F.F., Penn, M.H., Krogdahl, Å., Overturf, K. 2012. Severity of soybean meal induced

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distal intestinal inflammation, enterocyte proliferation rate, and fatty acid binding protein (Fabp2) level differ between strains of rainbow trout (Oncorhynchus mykiss).

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Aquaculture 364-365, 281-292.

Ye, C.L., Anderson, D.M., Lall, S.P. 2016. The effects of camelina oil and solvent extracted camelina meal on the growth, carcass composition and hindgut histology of Atlantic salmon (Salmo salar) parr in freshwater. Aquaculture 450, 397-404. Zubr, J. 1997. Oil-seed crop: Camelina sativa. Ind. Crop Prod. 6, 113-199. Zubr, J. 2010. Carbohydrates, vitamins and minerals of Camelina sativa seed. Nutr. Food Sci. 40, 523-531.

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ACCEPTED MANUSCRIPT

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Figure 1. Points of measurements for histological analysis of intestinal cross-sections: (A) villus length; (B) villus width; (C) crypt depth; (D) intestinal wall thickness; (E) villus area.

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Figure 2. Significant (P<0.05) regressions (mean ± SD) for the feed consumption, protein efficiency ratio (PER) and specific growth rate (SGR) of rainbow trout fed graded levels of camelina seed, highoil residue camelina meal (HORM) and pre-press solvent-extracted camelina meal (SECM): (A) feed consumption when fed camelina seed; (B) SGR when fed camelina seed; (C) PER when fed HORM; (D) SGR when fed HORM; (E) feed consumption when fed SECM; (F) SGR when fed SECM.

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ACCEPTED MANUSCRIPT Table 1 Nutrient composition (as is basis) of experimental ingredients Camelina oil

Calena seed

HORM

SECM

89.5 35.7 9.9 4653 5.89 0.50 0.98 1.35 0.39 36.37 11.22 71.12 18.3 40.0

90.8 39.0 2.8 4270 6.29 0.51 1.06 1.52 0.44 37.46 13.54 77.51 18.0 33.9

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HORM=High oil residue camelina meal. SECM=Solvent-extracted camelina meal.

91.9 25.2 34.6 6123 3.78 0.25 0.69 0.95 0.28 22.39 7.67 48.67 23.6 37.1

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. . 100 9382 . . . . . . . . . .

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Dry matter Crude protein Crude fat Gross energy Ash Calcium Phosphorus Potassium Magnesium Manganese (ppm) Copper (ppm Zinc (ppm) Acid detergent fibre Neutral detergent fibre

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Nutrient (%, unless otherwise stated)

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ACCEPTED MANUSCRIPT Table 2 Ingredient and analyzed nutrient composition (as fed basis) of experimental diets used to evaluate performance of rainbow trout fed pre-press solvent extracted camelina meal (SECM) as a feed ingredient in practical diets.

a

91.72 46.46 21.57 1.42 1.06 0.45 0.59 0.15 140 8 205

100 g/kg

50.0 315.0 100.0 80.0 179.0 133.5 50.0 50.0 25.0 3.0 2.0 2.0 2.0 2.5 3.0 1000.0

100.0 297.0 100.0 80.0 180.0 100.5 50.0 50.0 25.0 3.0 2.0 2.0 2.0 2.5 3.0 1000.0

150.0 277.0 100.0 80.0 184.0 66.5 50.0 50.0 25.0 3.0 2.0 2.0 2.0 2.5 3.0 1000.0

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MA 91.78 44.09 22.37 1.49 1.09 0.32 0.68 0.17 135 17 147

150 g/kg

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50 g/kg

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0.0 335.0 100.0 80.0 175.0 167.5 50.0 50.0 25.0 3.0 2.0 2.0 2.0 2.5 3.0 1000.0

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Nutrient (as fed) Dry matter (%) Crude protein (%) Crude fat (%) Calcium (%) Phosphorus (%) Sodium (%) Potassium (%) Magnesium (%) Manganese (ppm) Copper (ppm) Zinc (ppm)

Control

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Composition Ingredient (g/kg, as fed) SECM Herring meal Soybean meal 48% Corn protein concentratea Herring oil Wheat middlings Feather meal Poultry by-product meal Lignisolb Iodized saltc Choline chlorided DL-Methioninee Vitamin / mineral premixf Pigment premixg L-Lysineh Total

90.29 45.26 22.19 1.18 0.96 0.37 0.63 0.16 149 10 133

91.34 45.24 22.08 1.53 1.11 0.32 0.66 0.17 145 11 142

200 g/kg 200.0 268.0 100.0 80.0 180.0 32.5 50.0 50.0 25.0 3.0 2.0 2.0 2.0 2.5 3.0 1000.0

88.47 44.44 21.25 1.20 0.96 0.38 0.58 0.15 156 14 142

Empyreal 75; Cargill Corn Milling; Blair, NE, USA. Arbo Temstik; Tembec; Temiscaming, QC, Canada. c Iodized stock salt (Canadian Stockman); Sifto Canada Corp., A Compass Minerals Company, Mississauga, ON, Canada. d Choline chloride 60%; Jefo Nutrition Inc.; Saint-Hyacinte, QC, Canada. e DL-Methionine, Feed Grade, 99%; Evonik Corporation; Theodore, AL, USA. f Vitamin / mineral premix contains (IU or mg/kg): zinc, 77.5 mg; manganese, 125 mg; iron, 84mg; copper, 2.5 mg; iodine, 7.5 mg; vitamin A, 5000 IU; vitamin D, 4000 IU; vitamin K, 2 mg; vitamin B12, 0.004 mg; thiamine, 8 mg; riboflavin, 18 mg; pantothenic acid, 40 mg; niacin, 100 mg; folic acid, 4 mg; biotin, 0.6 mg; pyridoxine, 15 mg; inositol, 100mg; ethoxyquin, 42 mg; wheat shorts, 1372 mg. g Pigment premix contains (/mgkg): selenium, 0.220 mg; vitamin E, 250IU; vitamin C, 200mg; astaxanthin, 60 mg; wheat shorts, 1988 mg. h L-Lysine HCl 78.8%; Jefo Nutrition Inc.; Saint-Hyacinte, QC, Canada. b

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ACCEPTED MANUSCRIPT Table 3 Ingredient and analyzed nutrient composition of diets used to evaluate performance of rainbow trout fed ground camelina seed and high oil residue camelina meal (HORM) at increasing dietary inclusion levels, as well as a a diet containing 100g/kg solvent-extracted camelina meal and 151g/kg camelina oil (SECM+CO).

a

92.36 46.29 18.24 1.55 1.17 0.69 0.66 0.16 127 8 116

D

90.73 45.07 20.25 1.40 1.10 0.62 0.66 0.16 130 13 113

93.41 45.33 21.91 1.42 1.16 0.69 0.76 0.19 146 11 115

92.69 45.76 23.82 1.35 1.10 0.65 0.74 0.19 146 8 130

HORM (%) 20

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0.0 0.0 300.0 0.0 267.0 102.0 80.0 84.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

0.0 0.0 0.0 100.0 308.0 190.0 80.0 155.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

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0.0 0.0 200.0 0.0 294.0 159.0 80.0 100.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

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0.0 0.0 100.0 0.0 318.0 218.0 80.0 117.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

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Nutrient (as fed) Dry matter (%) Crude protein (%) Crude fat (%) Calcium (%) Phosphorus (%) Sodium (%) Potassium (%) Magnesium (%) Manganese (ppm) Copper (ppm) Zinc (ppm)

0.0 0.0 0.0 0.0 330.0 286.0 80.0 137.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

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Ingredient (g/kg, as fed) SECM Camelina oil Ground camelina seed HORM Herring meal Ground wheat Corn protein concentratea Herring oil Wheat glutenb Feather meal Poultry by-product meal Salt (iodized)c Choline chlorided DL-Methioninee Vitamin / mineral premixf Pigment premixg L-Lysineh Total

Camelina seed (%) 20 30

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10

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Control

89.11 44.50 20.54 1.42 1.10 0.63 0.61 0.17 127 8 109

0.0 0.0 0.0 200.0 274.0 103.0 80.0 176.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

90.08 44.32 23.05 1.32 1.06 0.59 0.68 0.18 130 8 115

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SECM +CO

0.0 0.0 0.0 300.0 241.0 14.0 80.0 198.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

100.0 151.0 0.0 0.0 306.0 196.0 80.0 0.0 50.0 50.0 50.0 3.0 5.0 1.7 2.0 2.5 3.0 1000.0

92.77 45.24 24.65 1.33 1.09 0.55 0.71 0.20 133 10 125

90.32 45.02 19.33 1.43 1.13 0.67 0.65 0.17 139 10 123

Empyreal 75; Cargill Corn Milling; Blair, NE, USA. Vital Wheat Gluten; Jäckering Muhlen –und Nahrmittelwerke GmbH; Hamm, Germany. c Iodized stock salt (Canadian Stockman); Sifto Canada Corp., A Compass Minerals Company, Mississauga, ON, Canada. d Choline chloride 60%; Jefo Nutrition Inc.; Saint-Hyacinte, QC, Canada. e DL-Methionine, Feed Grade, 99%; Evonik Corporation; Theodore, AL, USA. f Vitamin / mineral premix contains (IU or mg/kg): zinc, 77.5 mg; manganese, 125 mg; iron, 84mg; copper, 2.5 mg; iodine, 7.5 mg; vitamin A, 5000 IU; vitamin D, 4000 IU; vitamin K, 2 mg; vitamin B12, 0.004 mg; thiamine, 8 mg; riboflavin, 18 mg; pantothenic acid, 40 mg; niacin, 100 mg; folic acid, 4 mg; biotin, 0.6 mg; pyridoxine, 15 mg; inositol, 100mg; ethoxyquin, 42 mg; wheat shorts, 1372 mg. g Pigment premix contains (/mgkg): selenium, 0.220 mg; vitamin E, 250IU; vitamin C, 200mg; astaxanthin, 60 mg; wheat shorts, 1988 mg. h L-Lysine HCl 78.8%; Jefo Nutrition Inc.; Saint-Hyacinte, QC, Canada. b

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ACCEPTED MANUSCRIPT Table 4 Growth performance of rainbow trout fed graded levels of pre-press solvent extracted camelina meal (SECM, g/kg) (n=4). Control

50

100

2.2±0.1

2.3±0.1

2.3±0.0

Day 0-28

6.5±0.4a

6.2±0.3a

5.4±0.5b

Day 28-56

19.6±1.3

19.5±2.3

17.6±3.4

Day 56-84

48.5±3.4a

46.3±3.6a

41.3±5.3ab

Day 84-112

55.1±12.0

66.6±7.1

61.5±6.0

129.2±12.5

138.5±8.4

a

125.8±12.4

Day 0-28

4.9±0.2a

4.7±0.2a

Day 28-56

3.2±1.9

4.3±0.2

Day 56-84

3.6±0.1

Total (Day 0-112) Feed consumption (g/fish)

3.6±0.1a

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1.9±0.3

3.4±0.4c 12.9±2.4

36.5±3.2bc

32.4±4.7c

55.6±4.5

56.7±9.3

abc

111.1±5.9

bc

105.3±13.9c

4.3±0.3a

3.4±0.3b

3.2±0.3b

4.2±0.4

4.5±0.1

4.2±0.4

3.5±0.2

3.6±0.3

2.7±0.3a

3.5±0.1ab

3.5±0.1ab

3.4±0.1b

5.7±0.7

5.9±0.6

5.4±0.6

5.5±0.3

3.7±0.1a

2.4±0.2

ab

3.6±0.3 ab

2.3±0.2

ab

D

Day 84-112

3.5±0.1 b

3.7±0.4c

15.3±1.0

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SGR

2.4±0.1

2.4±0.1

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Total (Day 0-112)

ab

200

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Weight gain (g/fish)

150

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Parameter Initial body weight (g/fish)

2.4±0.2

5.6±0.5

Day 28-56

12.6±8.0

17.0±1.8

17.9±1.7

16.3±0.8

15.9±1.4

Day 56-84

42.7±1.8ab

43.8±3.2a

44.5±1.3a

38.0±2.2bc

36.6±3.7c

Day 84-112

61.3±7.0

63.8±4.9

62.9±6.8

58.6±4.4

58.0±7.3

130.7±8.3

129.3±7.1

118.3±5.1

113.1±18.2

0.9±0.0b

0.9±0.1b

1.1±0.1b

1.5±0.2a

1.6±0.1a

0.8±0.2c

0.9±0.1bc

1.0±0.2abc

1.1±0.1ab

1.2±0.2a

0.9±0.0b

1.0±0.0ab

1.1±0.2a

1.1±0.1ab

1.1±0.1a

1.1±0.2

1.0±0.1

1.0±0.1

1.1±0.0

1.0±0.0

1.1±0.2

0.9±0.1

1.0±0.1

1.1±0.1

1.1±0.1

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Day 0-28

Total (Day 0-112) FCR Day 0-28 Day 28-56 Day 56-84 Day 84-112

Total (Day 0-112)

136.3±25.7

PER Day 0-28

2.5±0.1a a

2.5±0.2a

2.1±0.2b

1.5±0.2c

1.4±0.1c

b

1.8±0.2b

2.9±0.8

Day 56-84

2.5±0.1a

2.4±0.1ab

2.1±0.3bc

2.1±0.1abc

2.0±0.2c

Day 84-112

1.9±0.4

2.4±0.1

2.2±0.1

2.1±0.1

2.2±0.1

Total (Day 0-112)

2.1±0.2

2.4±0.1

a

2.1±0.3

ab

Day 28-56

b

2.6±0.3

ab

2.2±0.2

ab

2.1±0.1

2.1±0.1

b

2.1±0.1b

abc

Means within rows with different superscripts are significantly different (P < 0.05). SGR=Specific growth rate. FCR=Feed conversion ratio. PER=Protein efficiency ratio.

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ACCEPTED MANUSCRIPT Table 5 Growth performance of rainbow trout fed ground camelina seed and high oil residue camelina meal (HORM) at increasing dietary inclusion levels, as well as a diet containing 100g/kg solvent-extracted camelina meal and 151g/kg camelina oil (SECM+CO) (n=3).

Day 0-28

100

200

1.0±0.0

3.4±0.4a a

1.0±0.1

2.9±0.2abc

2.3±0.2cde

100

1.0±0.1

1.1±0.1

1.9±0.2e

1.0±0.0

3.2±0.2ab

1.1±0.2

0.9±0.1

2.6±0.3bcd

2.1±0.2de

2.8±0.3abc

abc

cd

7.1±0.4bcd

20.6±2.0ab

18.7±2.3ab

16.1±2.1b

22.0±1.3a

20.6±2.5ab

16.4±2.2ab

16.9±1.8ab

Day 84-112

45.5±7.2

41.4±4.3

35.9±4.7

31.0±0.5

39.5±0.6

36.2±6.0

32.5±5.0

34.1±6.3

73.5±7.2

c

abc

6.6±0.8

bc

60.9±8.2abc

73.9±2.6

5.0±0.2ab

4.5±0.4abcd

3.9±0.7cd

5.2±0.3ab

3.9±0.3

4.1±0.1

4.3±0.1

4.0±0.3

3.8±0.1

3.6±0.1

3.8±0.3

3.4±0.2

3.6±0.1

3.5±0.2

3.4±0.2

2.9±0.2

2.9±0.2

2.7±0.1

2.6±0.2

2.9±0.1

2.8±0.2

3.7±0.1

3.6±0.1

3.8±0.1

3.7±0.2

3.6±0.3

3.8±0.1

3.6±0.5abc

3.0±0.4c

3.9±0.4abc

4.2±0.5abc

4.2±0.7abc

5.4±1.2a

64.2±7.3

SGR 5.4±0.3a

4.8±0.1abc

4.3±0.3bcd

Day 28-56

4.0±0.4

4.2±0.2

4.1±0.0

Day 56-84

3.3±0.6

3.5±0.1

Day 84-112

3.1±0.4

2.9±0.2

Day 0-112

3.9±0.0

3.9±0.1

54.5±2.3

3.9±0.3d

D

MA

Day 0-28

Feed consumption (g/fish)

ab

8.4±0.4

NU

76.3±4.4

SC

20.4±3.8ab

Day 0-112

9.2±0.7

a

SECM +CO

300

Day 56-84

abc

5.5±0.4

d

200

9.0±1.0

ab

7.3±0.8

abcd

300

Day 28-56

a

8.6±1.1

ab

HORM (g/kg)

PT

1.0±0.0

Camelina seed (g/kg)

RI

Parameter Initial body weight (g/fish) Weight gain (g/fish)

Control

67.7±9.0

57.6±7.6

5.1±0.3ab

3.5±0.5bc

Day 28-56

7.4±0.4

7.7±1.5

5.8±1.1

6.2±0.1

9.0±1.1

7.1±1.4

8.4±4.4

6.0±0.5

Day 56-84

18.0±1.9

17.8±1.0

17.2±0.6

16.0±2.5

18.3±3.5

19.2±3.3

18.3±3.5

16.8±1.3

Day 84-112

36.0±1.8

32.5±5.2

28.0±3.9

26.0±0.9

31.1±2.5

30.1±4.9

31.4±6.7

29.9±3.5

Day 0-112

70.0±6.5

61.4±8.2

54.6±6.1

51.1±3.3

62.3±3.0

60.6±9.8

62.4±14.3

58.1±5.4

AC CE P

FCR

TE

Day 0-28

Day 0-28

1.5±0.1abc

1.2±0.1c

1.6±0.1abc

1.6±0.3abc

1.2±0.1bc

1.6±0.2abc

2.0±0.4a

1.9±0.3ab

Day 28-56

0.8±0.1

0.9±0.1

0.8±0.1

1.1±0.1

1.0±0.1

0.9±0.2

1.3±0.6

0.8±0.0

Day 56-84

0.9±0.1

0.9±0.0

0.9±0.1

1.0±0.1

0.8±0.1

0.9±0.1

1.1±0.1

1.0±0.1

Day 84-112

0.8±0.1

0.8±0.1

0.8±0.0

0.8±0.0

0.8±0.1

0.8±0.1

1.0±0.1

Day 0-112

0.9±0.1

ab

0.8±0.0

b

0.9±0.0

ab

0.9±0.0

ab

0.8±0.0

ab

0.9±0.1

ab

1.1±0.1

0.9±0.1 a

1.0±0.0ab

PER Day 0-28

1.5±0.1abc

1.8±0.2ab

1.4±0.1bc

1.4±0.3bc

1.9±0.1a

1.4±0.2bc

1.1±0.2c

1.2±0.2c

Day 28-56

2.7±0.4

2.5±0.2

2.8±0.4

2.0±0.1

2.3±0.2

2.7±0.5

2.0±0.8

2.7±0.1

Day 56-84

2.5±0.2

2.6±0.1

2.4±0.2

2.2±0.2

2.8±0.5

2.4±0.3

2.0±0.2

2.2±0.1

Day 84-112

2.7±0.2

Day 0-112

2.4±0.3

2.9±0.3 ab

2.7±0.1

2.9±0.2 a

2.6±0.0

2.6±0.1 ab

2.3±0.1

2.9±0.3 ab

2.7±0.1

2.7±0.4 a

2.6±0.3

2.3±0.1 ab

2.1±0.2

2.5±0.2 b

2.3±0.1ab

abcdMeans

within rows with different superscripts are significantly different (P < 0.05). SGR=Specific growth rate. FCR=Feed conversion ratio. PER=Protein efficiency ratio.

30

ACCEPTED MANUSCRIPT

Control

Camelina seed (g/kg) 100

RI

Parameter

200

300

0.35±0.08

0.13±0.03

0.15±0.04

0.15±0.01

0.14±0.02

0.14±0.03

0.05±0.01

0.07±0.03

0.07±0.01

0.06±0.01

0.06±0.02

0.08±0.01

0.08±0.01

0.08±0.01

0.07±0.01

0.07±0.01

0.12±0.03

0.12±0.01

0.12±0.01

0.11±0.02

0.10±0.02

0.40±0.09

0.40±0.10

0.47±0.12

0.39±0.06

0.39±0.05

0.40±0.09

0.19±0.03

0.19±0.03

0.18±0.02

0.18±0.02

0.18±0.02

0.18±0.02

0.08±0.02

0.08±0.03

0.10±0.03

0.08±0.02

0.08±0.01

0.08±0.02

0.06±0.01

0.06±0.01

0.06±0.01

0.06±0.01

0.06±0.01

0.06±0.00

0.11±0.03

0.09±0.03

0.09±0.02

0.10±0.02

0.09±0.02

0.08±0.03

Villus width (µm)

0.15±0.05

0.15±0.03

0.13±0.01

Villus area (µm )

0.06±0.03

0.07±0.01

0.06±0.01

Crypt depth (µm)

0.08±0.01

0.07±0.01

0.08±0.02

Intestinal wall thickness (µm)

0.12±0.02

0.11±0.01

0.12±0.02

Villus height (µm)

0.35±0.08

0.40±0.09

Villus width (µm)

0.18±0.04

0.18±0.04

Villus area (µm )

0.07±0.02

0.08±0.02

Crypt depth (µm)

0.07±0.02

0.06±0.02

Intestinal wall thickness (µm)

0.11±0.02

0.11±0.03

PT ED

CE

AC

27.53±1.00

27.88±0.41

27.33±0.67

27.45±0.61

28.03±0.84

27.71±0.65

27.62±0.12

7.11±1.02

6.59±0.28

6.58±0.18

7.22±0.52

6.89±0.56

7.04±1.04

6.55±0.81

6.91±0.54

Protein

60.45±0.93

61.68±2.89

60.93±1.58

60.66±0.74

59.86±1.79

59.14±2.61

57.95±1.49

61.21±0.59

Fat

30.00±1.88

30.36±1.87

28.54±0.51

27.81±0.40

30.26±1.29

30.57±0.82

31.26±1.45

29.50±0.90

Ash

27.61±0.50

0.33±0.04

NU

0.38±0.07

MA

0.40±0.05

Dry matter

300

0.34±0.06

0.38±0.10

Carcass composition (%, as is)

200

0.40±0.04

Villus height (µm)

2

100

SECM +CO

0.37±0.10

Midgut

Hindgut

HORM (g/kg)

SC

Histological measurements

2

PT

Table 6 Mean histological measurements of midgut and hindgut samples and carcass composition of rainbow trout fed ground camelina seed and high oil residue camelina meal (HORM) at increasing dietary inclusion levels, as well as a diet containing 100g/kg solvent-extracted camelina meal and 151g/kg camelina oil (SECM+CO).

ab

Means within rows with different superscripts are significantly different (P < 0.05).

31

ACCEPTED MANUSCRIPT

Highlights We determine rainbow trout fed up 100g/kg camelina seed, high oil residue camelina

PT



meal (HORM) and solvent-extracted camelina meal (SECM) performed similarly to fish

We determine rainbow trout fed a combination of 100g/kg SECM and 151g/kg camelina

SC



RI

fed a control diet.

oil performed similarly to fish fed a control diet.

We develop feed intake and growth response curves for rainbow trout fed camelina seed,

NU



We determine feeding up to 300g/kg camelina seed and HORM, as well as a combination

D

of 100g/kg SECM and 151g/kg camelina oil will not alter the intestinal morphology or

TE

carcass proximate composition of rainbow trout, as compared with fish fed a control diet.

AC CE P



MA

HORM and SECM.

32