Apparent digestibility of proximate nutrients, energy and fatty acids in nutritionally-balanced diets with partial or complete replacement of dietary fish oil with microbial oil from a novel Schizochytrium sp. (T18) by juvenile Atlantic salmon (Salmo salar L.)

Aquaculture 520 (2020) 735003

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Apparent digestibility of proximate nutrients, energy and fatty acids in nutritionally-balanced diets with partial or complete replacement of dietary fish oil with microbial oil from a novel Schizochytrium sp. (T18) by juvenile Atlantic salmon (Salmo salar L.)

T

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Sean M. Tibbettsa, , Mark A. Scaifeb, Roberto E. Armentab,c a b c

National Research Council of Canada, Aquatic and Crop Resource Development Research Centre, 1411 Oxford Street, Halifax, Nova Scotia B3H 3Z1, Canada Mara Renewables Corporation, 101A Research Drive, Dartmouth, Nova Scotia B2Y 4T6, Canada Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Digestibility Docosahexaenoic acid Fish oil replacement Salmon Schizochytrium Thraustochytrid

Oils derived from thraustochytrid microorganisms, particularly Schizochytrium sp., are rapidly gaining attention as sustainable feedstocks to replace conventional edible oils for many industrial applications. A novel strain of Schizochytrium sp. (T18) has shown rapid growth under optimized heterotrophic cultivation, efficiently using inexpensive carbon and nitrogen feedstocks and capable of producing a high docosahexaenoic acid (DHA)-rich oil. This is the first study directed towards its nutritional evaluation as a potential feed resource for salmonid aquaculture. The study examined the effects of partial or complete replacement of dietary marine fish oil with Schizochytrium sp. (T18) microbial oil on apparent digestibility (AD) of dietary proximate nutrients, energy and fatty acids (FAs) when fed to juvenile (32 g) Atlantic salmon (Salmo salar L.). Four isonitrogenous (50% crude protein), isolipidic (20% lipid) and isocaloric (19 MJ/kg digestible energy) practical-ingredient experimental diets were formulated to replace 0, 33, 66 and 100% of dietary fish oil with extracted Schizochytrium sp. (T18) oil. The oil used contained 50% polyunsaturated FAs (PUFA); of which 82% (e.g., 42% of total FAs) was comprised of the dietary n-3 long-chain PUFA (n-3 LC-PUFA) DHA and contained extremely low levels of harmful contaminants (e.g., heavy metals, lipid oxidation products, pathogens). Partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil had no significant (P ≥ .064) effects on AD of dietary dry matter (76–77%), protein (94%), lipid (89–90%) or energy (83–84%). Similarly, AD was unaffected (P ≥ .647) for dietary PUFA (97%) and n-3 PUFA (98%) with only minor differences (P ≤ .046) detected for monounsaturated FAs (MUFA; 91–92%) and n-6 PUFA (95–96%). Alternatively, AD of dietary saturated FAs (SFA) and DHA were significantly (P < .001) improved (70–76% and 95–98%, respectively) with increasing replacement of fish oil with Schizochytrium sp. (T18) oil. Results suggest that conventional fish oil can be completely replaced in juvenile farmed salmon feeds by Schizochytrium sp. (T18) oil with no negative effects on digestibility of dietary proximate nutrients, energy or fatty acids and, in fact, promoting a significant linear dose-response increase in AD of dietary SFA and a statistically significant increase in dietary DHA digestibility.

1. Introduction Aquaculture is the world's fastest growing food production sector. Currently supplying nearly 60% of all dietary aquatic protein, essential fatty acids and other dietary nutrients to the global seafood market; this industry has an annual value of $232 billion USD (FAO, 2018). Salmon farming, in particular, is presently worth approximately $15 billion USD annually but has a disproportionately heavy reliance on the finite

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and costly supplies of traditional marine resources (e.g., fish meals and fish oils) and less-expensive terrestrial plant-based protein and lipid sources (e.g., soy, corn, canola, etc.) (FAO, 2018; Foroutani et al., 2018). While the replacement of dietary fish meal protein (to meet essential amino acid requirements) with nutritious and lower-cost plant-protein feedstocks has been widely successful, the replacement of dietary fish oil (to maintain n-3 LC-PUFA content of the final consumer product) has been more challenging (Lenihan-Geels et al., 2013;

Corresponding author. E-mail address: [email protected] (S.M. Tibbetts).

https://doi.org/10.1016/j.aquaculture.2020.735003 Received 10 December 2019; Received in revised form 20 January 2020; Accepted 21 January 2020 Available online 22 January 2020 0044-8486/ Crown Copyright © 2020 Published by Elsevier B.V. All rights reserved.

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production of DHA at very high levels, they have become a particularly interesting group of organisms for mass fermentation, oil extraction and use in aquaculture feeds (Adarme-Vega et al., 2012; Tocher, 2015; Tocher et al., 2019). These products may have tremendous potential to reduce pressures on wild fish stocks harvested for fish meal and fish oil reduction. In addition, because of their highly-controlled fermentation processes, the DHA-rich edible oils produced are more likely to be free of the environmental contaminants often problematic with conventional fish oils produced from harvested pelagic marine fish (Hites et al., 2004; Sprague et al., 2015; Bélanger-Lamonde et al., 2018; Harwood, 2019). Several studies have been conducted to evaluate the use of dry whole-cell thraustochytrid meals in farmed salmonid feeds. It seems that dietary inclusion for Atlantic salmon and rainbow trout feeds must be limited to low levels (generally < 10% of the diet) before growth performance and digestibility is negatively impacted as a result of the highly recalcitrant cell walls and indigestible non-starch polysaccharides (NSPs) components of these thraustochytrid meals (Carter et al., 2003; Zhang, 2013; Kousoulaki et al., 2015; Sprague et al., 2015; Betiku et al., 2016; Lyons et al., 2017). As such, the use of an extracted DHA-rich thraustochytrid oil at higher dietary levels is likely to be better utilized by salmonid fish as a source of essential LC-PUFA and, to this point, just one study has evaluated extracted oil of a related Schizochytrium strain in the feeds of farmed Atlantic salmon (Miller et al., 2007). A handful of companies are now involved in this emerging industrial sector, commercial food-grade DHA products may soon be added to the feedstock portfolios of large aquafeed producers (Tibbetts, 2018) and new thraustochytrid strains with good potential continue to be isolated and explored for industrial development (Shene et al., 2019). In order to evaluate the extracted oil from a novel strain, it must first be demonstrated that the fatty acid profile of the extracted oil is adequate for use in aquafeeds, that it is free of harmful contaminants and that a high level can be included in the diet without negatively impacting diet palatability and feed consumption. Secondly, the effect of inclusion of the novel oil on the digestibility of major dietary nutrients, energy and fatty acids must be established with the specific target fish species and growth phase. Lastly, nutritional evaluations must be conducted that demonstrate the impacts of dietary inclusion of the novel oil on various production parameters such as growth rate, feed efficiency, animal health, fillet quality and the economics of feed production. The present study evaluated the extracted oil of a novel proprietary thraustochytrid known as Schizochytrium sp. strain T18 which was isolated from Advocate Harbour, Nova Scotia, Canada (Burja et al., 2006). A handful of studies have been published on Schizochytrium sp. (T18) with a foci on cultivation optimization (Burja et al., 2006), harvesting methods and fatty acid extraction (Burja et al., 2007), food safety and toxicity (Schmitt et al., 2012) and low-cost nutrient feedstocks (Lowrey et al., 2016a, 2016b, 2016c); while the present study is the first to evaluate its potential application in farmed salmon aquafeeds. The first objective of this study was to characterize Schizochytrium sp. (T18) oil for its general composition, selected lipid quality parameters, concentrations of harmful contaminants, key hygienic properties and fatty acid profile. The second objective was to determine the effects on apparent digestibility of proximate nutrients, energy and fatty acids by low (33%), medium (66%) or complete (100%) replacement of dietary fish oil with Schizochytrium sp. (T18) oil in nutritionally-balanced juvenile Atlantic salmon (Salmo salar L.) feeds.

Tocher, 2015). Presently, the lipid fraction of many farmed salmon feeds contain double the amount of terrestrial-derived oils (e.g., vegetable oils and rendered animals fats) as conventional marine fish oils (Aas et al., 2019). While these edible oils provide the fish with excellent sources of digestible energy; fish health and the consumer products produced from those fish have become compromised in recent years. This troubling and well documented outcome is the result of a systematic reduction of the dietary n-3/n-6 ratio of their feeds; typified by a reduced content of C20 and C22 n-3 LC-PUFA; particularly docosahexaenoic acid (22:6n-3; DHA) in favour of elevated dietary levels of C18 n-6 series fatty acids such as linoleic acid (18:2n-6; LA) (Sprague et al., 2016; Bou et al., 2017a, 2017b; de Roos et al., 2017; Cheng et al., 2018; Sissener, 2018). In both farmed fish and the consumers of their products, dietary n-3 LC-PUFA play important and physiologically-complex roles in several key metabolic functions such as cell membrane structure, cholesterol metabolism, prostaglandin synthesis, plasma triglyceride transport and efficient intestinal absorption of fat-soluble vitamins (Khazrai et al., 2004; Tocher, 2015). On the other hand, in situations where these n-3 LC-PUFA are lacking in the diet resulting in low n-3/n-6 ratio, such is the present case with modern salmon feeds, compromised fish and human health can occur as a result of disease pathogenesis related to cardiovascular and nervous system issues, decreased cell membrane fluidity, endogenous over-production of arachidonic acid and impaired anti-inflammatory response and immune function while higher ratios promote an anti-inflammatory response in humans and animals resulting in improved cardiac and nervous system health and increased cell membrane fluidity (Simopoulos, 2002; Bodnar and Wisner, 2005; Hixson, 2014; Harwood, 2019). The reality is that the n-3 LC-PUFA content of most farmed Atlantic salmon has declined by ~66% over the past decade; requiring consumers to double their number of portions to obtain the weekly recommended intake of these critical n-3 LC-PUFA (Sprague et al., 2016). Nutrition and health effects aside, aquaculture feed resources derived from terrestrial oilseeds and livestock are rising in cost and the direct competition as human food pose growing concerns with regard to their judicious use of land, freshwater and production resources (Pahlow et al., 2015; Fry et al., 2016). A search for new edible oils that can increase dietary n-3 LC-PUFA levels, shift n-3/ n-6 feed ratios back to desirable ranges and restore muscle DHA levels in farmed salmon products is of urgency; and this urgency is further confounded by anticipated shortfalls in oceanic primary production of n-3 LC-PUFA as a result of climate change (Tocher, 2015; Colombo et al., 2019). To emphasize this need, members of the Global Salmon Initiative (GSI) have stated that they could immediately use 200,000 t annually of alternative novel DHA-rich oils; if they were readily available to salmon aquafeed manufacturers (Sprague et al., 2017). The group of microorganisms known as thraustochytrids are often referred to in the published literature and for marketing purposes as ‘microalgae’ and, indeed, they can be mass-cultivated very much like many types of algae. However, they are not microalgae (Armenta and Valentine, 2013; Leyland et al., 2017) and are actually classified taxonomically as marine or brackish fungal protists; composed of the 5 genera Thraustochytrium, Aplanochytrium, Japonochytrium, Ulkenia and Schizochytrium (Barclay et al., 1994; Burja et al., 2006). More specifically within the genus of Schizochytrium sensu lato, three different genera have been proposed; Schizochytrium sensu stricto, Aurantiochytrium and Oblongichytrium gen. Nov., a proposed taxonomic rearrangement that has been gaining increased acceptance (Yokoyama and Honda, 2007). Under optimized heterotrophic cultivation conditions, many thraustochytrids have demonstrated good capability to produce very high lipid concentrations (> 70% of their mass); rich in essential long-chain polyunsaturated fatty acids (LC-PUFA) by using a vast array of carbon and nitrogen feedstocks (Lewis et al., 1999). This is possible as a result of a single multi-subunit key enzyme (e.g., type I PUFA fatty acid synthase) that is highly active in these microorganisms (Hauvermale et al., 2006). Since these organisms are cable of industrial

2. Materials and methods 2.1. Schizochytrium sp. (T18) cultivation The extracted oil used in this study was produced from a proprietary thraustochytrid strain known as Schizochytrium sp. (T18) isolated from the Bay of Fundy in Nova Scotia, Canada (Burja et al., 2006). 2

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Thraustochytrid biomass was produced using a fermentation medium containing (on a per litre basis): 100 g glucose, 2 g soy peptone, 4 g magnesium sulfate heptahydrate, 2.2 g potassium phosphate monobasic, 2.4 g potassium phosphate dibasic, 20 g ammonium sulfate, 0.1 g calcium chloride dihydrate, 0.003 g iron chloride, 0.003 g copper sulfate pentahydrate, 0.0015 g sodium molybdate dihydrate, 0.003 g zinc sulfate heptahydrate, 0.0015 g cobalt chloride hexahydrate, 0.0015 g manganese chloride tetrahydrate, 0.0015 g nickel sulfate hexahydrate, 0.00003 g vitamin B12, 0.00003 g biotin and 0.006 g thiamin hydrochloride. Batch-fed fermentation was performed in a 30 L reactor at 20 °C, controlled at pH 6 (with NaOH), air was introduced into the culture via sparge bar at a rate of 0.33 vvm (based on initial volume), glucose concentration was maintained at 30 g/L and mixing was maintained at a range of 500–600 rpm.

Table 2 Fatty acid profile of the Schizochytrium sp. (T18) oil and the other dietary oils used to balance the lipid profile of the experimental diets (as-is basis).

Fatty acid (% of total FA) 12:0 14:0 14:1n-7 15:0 16:0 16:1n-7 16:2n-4 16:2n-6 16:3n-4 16:4n-1 17:0 18:0 18:1n-7 18:1n-9 18:2n-6 18:3n-3 (ALA) 18:4n-3 20:0 20:1n-9 20:2n-6 20:3n-6 20:3n-3 20:4n-3 20:5n-3 (EPA) 21:5n-3 22:1n-9 22:5n-3 (DPAn-3) 22:5n-6 (DPAn-6) 22:6n-3 (DHA) Σ SFA Σ MUFA Σ PUFA Σ n-3 PUFA Σ n-6 PUFA n-3/n-6 ratio

2.2. Oil extraction Cell walls were disrupted using the enzymatic method described by Dennis and Armenta (2017). Specifically, the fermentation broth was subjected to enzymatic hydrolysis using 0.1% (v/v) of a commercial bacterial protease produced from Bacillus licheniformis (Alcalase®) at pH 8 and 55 °C for 16 h under nitrogen blanketing. After hydrolysis, the enzyme was deactivated by heating (100 °C for 20 min) and the broth was centrifuged (4600 rpm, 20 min, 40 °C) to separate the media from the concentrated disrupted biomass (approximately 80% media removal). The media was then passed through a 0.25 μm filter and the concentrated broth containing disrupted cell biomass was treated with 5% (w/v) sodium sulfate and heated with shaking to 70 °C for 60 min. The sample was then centrifuged (4600 rpm, 20 min, 40 °C) to separate the extracted oil from the spent biomass and the recovered oil passed through a 5 μm filter. 2.3. Experimental diets To evaluate the effects of partial or complete replacement of fish oil with Schizochytrium sp. (T18) oil on the digestibility of dietary proximate nutrients, energy and fatty acids, four nutritionally-balanced experimental diets were formulated (Table 3) to be isonitrogenous (50% crude protein), isolipidic (20% fat) and isocaloric (19 MJ/kg

Schizochytrium sp. (T18) oil

1.1 0.3 9353.1 39.1 3.8 11.5 19.1 0.5

Contaminating heavy metal concentrations (ppm) Arsenic Cadmium Lead Mercury

< 0.1 < 0.001 < 0.001 < 0.005

Microbial hygienic parameters Bacteria (cfu/g) Coliforms (MPN/g) Escherichia coli (MPN/g) Staphylococcus aureus (cfu/g) Yeasts (cfu/g) Molds (cfu/g)

< 10 <3 <3 <5 < 10 < 10

a b

Fish oil

Poultry fat

Canola oil

0.8 12.0 – 1.5 26.3 4.5 – – – – 0.3 0.9 2.9 0.7 0.3 – 0.2 – – – – – 0.4 0.8 – – – 7.5 40.9 41.9 8.1 50.0 42.3 7.8 5.4

0.1 8.2 – 0.6 19.9 8.4 1.1 – 1.4 2.0 0.6 4.3 2.9 9.7 2.1 1.1 2.6 0.5 1.1 – – 1.1 0.8 15.5 0.7 0.7 2.0 0.4 11.9 34.3 22.8 42.8 35.8 2.5 14.2

0.1 0.6 0.2 – 23.4 6.3 – 0.2 – – 0.1 5.7 1.9 37.4 21.2 1.9 – – 0.4 0.1 0.2 0.4 – – – – – – – 29.9 46.2 23.9 2.3 21.7 0.1

– – – – 4.3 – – – – – – 1.8 3.1 60.4 21.2 9.1 – – – – – – – – – – – – – 6.1 63.5 30.3 9.1 21.2 0.4

digestible energy). Additionally, these diets were balanced for their contents of saturated fats (SFA, 5%), monounsaturated fats (MUFA, 7%), polyunsaturated fats (PUFA, 6%), n-3 PUFA (3%) and n-6 PUFA (3%) using varying ratios of poultry fat and canola oil (2–7% of the diet). The experimental diets were formulated to replace 0, 33, 66 and 100% of dietary fish oil; representing a range of dietary inclusion levels of 0, 3, 6 and 9% Schizochytrium sp. (T18) oil. All experimental diets were supplemented with chromic oxide (Cr2O3, 0.5% w/w basis) as the inert digestion indicator. Dry ingredients were finely ground to a powder (≤500 μm) at 10,000 rpm using a laboratory ultra-centrifugal mill (model ZM200, Retsch GmbH., Haan, Germany) equipped with a Retsch pneumatic auto-feeder (model DR100). Micronutrients (e.g., vitamins, minerals, amino acids) were pre-mixed with wheat flour using a Globe® benchtop planetary mixer (model SP-20, Globe Food Equipment Company, Dayton, OH) prior to addition to the main ingredient mixture. All ingredients were thoroughly blended in a Hobart® floor planetary mixer (model H600T, Rapids Machinery Corporation, Troy, OH) and compression steam pelleted into 2.5 mm pellets (model CL-2, California Pellet Mill Co., San Francisco, CA). The pellets were dried in a forced-air drier at 80 °C for 90 min to form dry, sinking pellets and stored in air-tight containers at −20 °C until use. Diets were screened to remove fines prior to feeding.

Table 1 Lipid quality criteria, contaminating heavy metal concentrations and microbial hygienic parameters of the Schizochytrium sp. (T18) oil (as-is basis).

Lipid quality criteria Moisture (%) Ash (%) Energy (cal/g) Energy (MJ/kg) Peroxide value (meq/kg) p-Anisidine valuea Totox scorea,b Free fatty acids (%)

Schizochytrium sp. (T18) oil

2.4. Digestibility trial In vivo apparent digestibility coefficients (ADCs) for dry matter, protein, lipid, energy and fatty acids of the experimental diets were measured using the indirect digestibility determination method (NRC,

Unit-less. Totox score = (p-Anisidine value + [2 × Peroxide value]). 3

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with guidelines set out by the Canadian Council on Animal Care (CCAC, 2005).

Table 3 Formulation of the nutritionally-balanced experimental diets used to measure the effects of partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil on in vivo apparent digestibility by Atlantic salmon (Salmo salar) diets (as-is basis).

2.5. Analytical techniques

Replacement (%) of dietary fish oil

Ingredient Fish meal (71% CPa) Soy protein concentrate (63% CP) Corn protein concentrate (78% CP) Wheat gluten meal (81% CP) Poultry by-product meal (71% CP) Blood meal (91% CP) Wheat flour Fish oilb Schizochytrium sp. (T18) oilc Poultry fatd Canola oile Trace ingredientsf Total

0

33

66

100

20.00 20.00 9.50 8.60 3.80 4.00 9.57 9.00 – 6.75 2.25 5.35 100.00

20.00 20.00 9.50 8.60 3.80 4.00 9.66 5.94 2.97 6.30 2.70 5.35 100.00

20.00 20.00 9.50 8.60 3.80 4.00 9.75 2.97 5.94 4.41 4.50 5.35 100.00

20.00 20.00 9.50 8.60 3.80 4.00 9.77 – 9.00 2.20 6.60 5.35 100.00

Experimental diets and lyophilized faecal samples were analyzed using similar procedures. Moisture and ash contents were determined gravimetrically by drying in an oven at 105 °C and by incineration in a muffle furnace at 550 °C for 18 h. Nitrogen (N) contents were determined by elemental analysis (950 °C furnace) using a Leco N analyzer (model FP-528, Leco Corporation, St. Joseph, MI) with ultra-high purity oxygen as the combustion gas and ultra-high purity helium as the carrier gas and crude protein content calculated as N × 6.25. Lipids were extracted by methanolic HCl in-situ transesterification (McGinn et al., 2012) and the corresponding fatty acid methyl esters (FAMEs) were separated and quantified by GC-FID (Omegawax 250 column, Agilent 7890). Individual FAs, along with an internal standard (C19:0; methyl nonadecanoate, Fluka), were identified by comparing retention times to two FA reference mixtures (Supelco 37 and PUFA No. 3, SigmaAldrich). Lipid quality criteria, contaminating heavy metal concentrations and microbial hygienic parameters of the Schizochytrium sp. (T18) oil were evaluated as peroxide value (AOAC method 965.33), p-anisidine value (AOCS method Cd 18–90), free fatty acid content (AOCS Ca 5a-40), ICP-AES (methods 6010C and 7471B) and standard lab bacteriology plate counts. Gross energy (MJ/kg) contents were measured using an isoperibol oxygen bomb calorimeter (model 6200, Parr Instrument Company, Moline, IL) equipped with a Parr 6510 water handling system for closed-loop operation. Chromic oxide concentrations were determined by flame atomic absorption spectrophotometry (model iCE 3000 Series AA, Thermo Fisher Scientific, Waltham, MA) following phosphoric acid and potassium bromide digestion (Williams et al., 1962). All analytical work was conducted in triplicate.

a

Crude protein (N × 6.25). Fish oil (12% DHA, 16% EPA, 34% SFA). c Schizochytrium sp. (T18) oil (41% DHA, < 1% EPA, 42% SFA). d Poultry fat (0% DHA, 0% EPA, 30% SFA). e Canola oil (0% DHA, 0% EPA, 6% SFA). f Corn starch, dextrinized (1.18%); Calcium phosphate, monobasic (2.89%); Vitamin/Mineral mixtureg (0.8%); Chromic oxide (0.5%); Choline chloride (0.4%); L-Lysine (0.39%); Salt, NaCl (0.25%); L-Methionine (0.06%); Vitamin C, ascorbic acid ‘Stay-C 35’ (0.03%); Vitamin E, α-tocopherol (0.03%). g Freshwater salmonid mixture (Corey Nutrition, Fredericton, NB, Canada). b

2011). Specially-designed tanks as described in Tibbetts et al. (2006) were used for passive collection of naturally egested faecal material from fish voluntarily consuming the various experimental diets. Digestibility measurements were made using 413 juvenile Atlantic salmon (average weight; 31.9 ± 1.2 g/fish) obtained from a commercial hatchery (Marine Harvest Fish Hatchery, Cardigan, PE, Canada). The fish were gradually weaned from a commercial feed onto their respective experimental diets over a 7-day period, at which time they were hand-fed to apparent voluntary satiety four times daily (08:00, 11:00, 13:00 and 15:00 h). The commercial feed (3.0 mm extruded salmonid feed, EWOS/Cargill Canada, Surrey, BC, Canada) contained 6% moisture, 50% crude protein, 19% lipid, 11% ash and 23 MJ/kg gross energy (as-fed basis). Once fully acclimated to their respective experimental diets, the fish were fed for an additional 4-days prior to sampling. Faecal samples were then collected daily from each tank for 7 consecutive days until a minimum of 40 g of wet faecal material was collected from every tank. Each of the 4 experimental diets was fed to triplicate tanks (initial stocking density, 11.0 ± 0.4 kg/m3). De-gassed and oxygenated freshwater from a well was supplied to each tank (120L) at a flow rate of 0.4 L/min in a flow-through system and water temperatures (13.9 ± 0.1 °C) and dissolved oxygen levels (> 100% saturation) were recorded daily. During the experimental period, fish were hand-fed to apparent voluntary satiety four times daily (08:00, 11:00, 13:00 and 15:00 h). The tanks were checked daily for dead or moribund fish and none were found throughout the trial. Each day, after the final feeding, the tanks and faecal collection columns were thoroughly cleaned with a brush to remove residual particulate matter (faeces and uneaten feed) and rinsed thoroughly with freshwater. Faecal samples were collected each morning at 08:00 h (~16 h after the final feeding) into 50 mL plastic conical bottom tubes, centrifuged (4000 rpm [2560 ×g] for 20 min at 4 °C) and the supernatant carefully decanted and discarded and each sample stored in a sealed container at −20 °C for the duration of the collection period. Faecal samples were lyophilized for 72 h at a low shelf temperature (≤5 °C) to a final moisture content of < 4%. The study was conducted in compliance

2.6. Calculations and statistical methods In vivo ADCs of dry matter (DM), protein (P), lipid (L), energy (E) and fatty acids (FA) of the experimental diets were calculated on a dryweight basis according to the following equations (NRC, 2011):

ADC of DM (%) = 100 − 100 ×

Chromic oxide in diet (%) Chromic oxide in faeces (%)

ADC of P, L, E or FA (%) Chromic oxide in diet (%) = 100 − 100 × Chromic oxide in faeces (%) P, L, E or FA in faeces (%or cal per g) × P, L, E or FA in diet (%or cal per g) Data are reported as mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance, ANOVA (SigmaStat® v.3.5) with a 5% level of probability (P < .05). Where significant differences were observed, treatment means were differentiated using pairwise comparisons using the Tukey test. Correlations between response variables were calculated by Pearson correlation analysis (r) using SigmaStat® v.3.5. Raw data was checked for normality using the Kolmogorov-Smirnov test (SigmaStat® v.3.5). 3. Results 3.1. Composition of the Schizochytrium sp. (T18) oil Lipid quality criteria, contaminating heavy metal concentrations and microbial hygienic parameters of the Schizochytrium sp. (T18) oil are reported in Table 1. Fatty acid profiles of the Schizochytrium sp. (T18) oil and the other dietary oils used to balance the lipid profile of the experimental diets are shown in Table 2. The tested oil was 4

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(T18) oil (from 0 to 9% of the diet).

Table 4 Composition of the nutritionally-balanced experimental diets used to measure the effect of partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil on in vivo apparent digestibility by Atlantic salmon (Salmo salar) diets (as-is basis).

3.3. Fish performance Throughout the trial, survival was 100% for fish consuming all experimental diets. The experimental diets were fed to each tank of fish four times daily to apparent satiety and they were consumed at a statistically equal rate (0.55 ± 0.02 g feed/fish/day; P = .083); amounting to ~2% of their BW per day. This suggests that partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil at the inclusion levels studied (3–9% of the diet) caused no positive or negative chemosensory effects in the test feeds; relative to the practical-ingredient control diet (free of T18 oil) which was representative of industrial juvenile farmed Atlantic salmon feeds used in Canada. Although this digestibility trial was too short to be considered a comprehensive growth performance study, juvenile salmon fed diets with increasing replacement of dietary fish oil with the tested oil displayed an upward and nearly significant trend (P = .052–0.057) in their growth performance with regard to final body weights (39–41 g/ fish) and thermal growth coefficients (0.15–0.23 g⅓/degree day). Overall, fish in this trial fed experimental diets with partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil performed equally as well as the control diet with no significant differences observed for final body weight (40.3 ± 1.3 g/fish; P = .052), weight gain (8.4 ± 0.7 g/fish; P = .211), thermal growth coefficient (0.19 ± 0.04 g⅓/degree day; P = .057) or feed conversion ratio (0.71 ± 0.04 g feed/g gain; P = .621). A longer-term growth performance and fish health study is required to evaluate these encouraging results.

Replacement (%) of dietary fish oil 0

33

66

100

Proximate nutrients Moisture (%) Ash (%) Crude protein (%) Lipid (%) Carbohydratea (%) Gross energy (MJ/kg) Chromic oxide (%)

4.9 8.0 49.1 20.4 17.6 22.8 0.52

5.0 8.2 48.9 20.4 17.5 22.9 0.54

5.3 8.4 49.3 20.5 16.5 22.8 0.54

5.4 8.2 49.2 20.4 16.8 22.7 0.51

Fatty acid (% of diet) 14:0 15:0 16:0 18:0 16:1n-7 16:2n-4 16:3n-4 16:4n-1 18:1n-7 18:1n-9 18:2n-6 18:3n-3 (ALA) 18:4n-3 20:0 20:1n-9 20:3n-3 20:4n-3 20:5n-3 (EPA) 22:1n-9 22:5n-3 (DPAn-3) 22:5n-6 (DPAn-6) 22:6n-3 (DHA) Σ SFA Σ MUFA Σ PUFA Σ n-3 PUFA Σ n-6 PUFA n-3/n-6 ratio

0.6 0.1 3.6 0.8 1.0 0.1 0.1 0.1 0.5 4.8 2.5 0.4 0.2 0.1 0.4 0.1 0.1 1.0 0.4 0.1 0.0 0.9 5.1 7.1 5.5 2.8 2.5 1.1

0.7 0.1 3.7 0.7 1.0 0.1 0.1 0.1 0.5 4.8 2.5 0.4 0.1 0.0 0.4 0.1 0.1 0.8 0.4 0.1 0.2 1.6 5.3 7.0 6.2 3.2 2.8 1.2

1.0 0.1 3.9 0.6 0.9 0.0 0.0 0.1 0.5 5.1 2.6 0.5 0.1 0.0 0.4 0.0 0.1 0.6 0.5 0.1 0.4 2.3 5.6 7.3 6.7 3.7 3.0 1.2

1.1 0.1 3.7 0.4 0.6 0.0 0.0 0.0 0.6 5.3 2.5 0.6 0.0 0.0 0.3 0.0 0.0 0.2 0.4 0.0 0.5 2.6 5.5 7.2 6.4 3.4 3.0 1.1

a

3.4. Apparent digestibility coefficients (ADCs) In vivo ADCs of dry matter, protein, lipid, energy and fatty acids in the experimental diets are shown in Tables 5 and 6. Diets with partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil were highly digested and at statistically the same level as the highquality control diet for all proximate nutrients; including dry matter (76–77%; P = .410), protein (93–94%; P = .182), lipid (89–90%; P = .064) and energy (83–84%; P = .116). Increasing dietary inclusion of Schizochytrium sp. (T18) oil significantly increased (70–76%; P < .001) the digestibility of saturated fatty acids (Σ SFA) in a significant linear dose-response manner (r = 0.924, R2 = 0.855, P < .001). This scenario was reflective of significant increases in ADCs for all dietary SFAs including 14:0 (75–81%; P = .011), 15:0 (73–77%; P = .008), 16:0 (71–76%; P = .001) and 18:0 (65–71%; P < .001). Digestibility of dietary monounsatured fatty acids (Σ MUFA) was virtually unaffected (91–92%; P = .046) by partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil. This was reflected by the lack of ADC differences observed for the MUFAs 16:1n-7 (93–94%; P = .132), 18:1n-7 (90–91%; P = .081) and 18:1n-9

Calculated as: (100 – [moisture + ash + crude protein + lipid]).

composed primarily (~90%) of docosohexaenoic acid (DHA, 22:6n-3; 41%), palmitic acid (16:0; 26%), myristic acid (14:0; 12%) and docosapentaenoic acid (DPAn-6, 22:5n-6; 8%). The oil was high in total saturated fatty acids (Σ SFA; 42%) and polyunsaturated fatty acids (Σ PUFA; 50%) and low in monounsaturated fatty acids (Σ MUFA; 8%). The Σ PUFA lipid fraction was composed primarily (~85%) of n-3 PUFA; which was almost completely comprised of DHA at 97% of n-3 PUFA, 82% of total PUFA and 41% of total FA. (See Table 2.) 3.2. Composition of experimental diets

Table 5 Apparent digestibility coefficientsa (in vivo ADCs, %) of proximate nutrients and energy in the nutritionally-balanced experimental diets with partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil fed to juvenile Atlantic salmon (Salmo salar).

Composition of the experimental diets is shown in Table 4. As formulated, the nutritionally-balanced experimental diets had highly similar levels of moisture (5%), ash (8%), crude protein (49%), lipid (20%), carbohydrate (17–18%), gross energy (23 MJ/kg) and chromic oxide (0.5%). Additionally, the intended (formulated as % of diet) balance of fatty acid groupings was generally achieved for Σ SFA (5–6%), Σ MUFA (7%), Σ PUFA (6–7%), Σ n-3 PUFA (3–4%) and Σ n-6 PUFA (3%). Due to fatty acid balancing achieved by inclusion of graded levels of poultry fat and canola oil, the composition of most fatty acids in the experimental diets are highly similar with the exception of decreasing levels of EPA (from 1.0 to 0.2%) and increasing levels of DHA (from 0.9 to 2.6%). This scenario is reflective of the graded replacement of dietary fish oil (from 9 to 0% of the diet) with Schizochytrium sp.

Replacement (%) of dietary fish oil 0 Dry matter Protein Lipid Energy

77.5 94.0 89.0 84.2

33 ± ± ± ±

0.2ns 0.1ns 0.1ns 0.3ns

76.4 93.8 88.7 83.4

66 ± ± ± ±

0.3 0.2 0.6 0.2

76.6 93.5 89.6 83.5

100 ± ± ± ±

0.6 0.3 0.8 0.6

77.3 93.7 90.0 83.9

± ± ± ±

0.5 0.3 0.4 0.4

a Values within the same row having different superscript letters are significantly different (P < .05).

5

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consideration of its freshness, heavy metal concentrations and microbial hygienic quality. Many aquaculture feeds (particularly farmed salmon feeds) contain very high lipid levels (generally 20–40% depending upon the growth phase) so freshness and hygienic quality of the added oils is highly desirable to maintain oxidative stability and to reduce off-flavours in the finished product. It is worthy to note that the media nutrients added to the small-scale 30 L fermentation batch to produce the Schizochytrium sp. (T18) oil for this study are the same as the ones presently used in large-scale fermenters for commercial oil production. Under this industrial level production, both the media nutrient feedstocks and the final products are rigorously monitored under a strict certified quality control system, which also extends to product transport and storage. Thus, a similar level of product quality and safety can be expected under the present industrial conditions. The Schizochytrium sp. (T18) oil used in this study was comprised of 50% total PUFA, while the remaining half was predominantly SFA (42%) with a small amount of MUFA (8%). Of the PUFA content of this Schizochytrium sp. (T18) oil sample, the vast majority (82%) was the essential n-3 LC-PUFA docosahexaenoic acid (DHA); representing 41% of the product. This result demonstrates that continual optimization of the culture conditions of Schizochytrium sp. (T18) over the past two decades have led to increased DHA yields as initial production trials, conducted soon after its domestication, produced lower levels (16–31% DHA) (Burja et al., 2006, 2007). The FA profile of Schizochytrium sp. (T18) oil was composed almost entirely (> 85% of total FAs) of the four FAs, myristic acid (14:0; 12%), palmitic acid (16:0; 26%), docosapentaenoic acid (22:5n-6; 8%) and docosahexaenoic acid (22:6n-3; 41%). This relatively simple profile is reflective of other thraustochytrid strains, where these four FAs are documented in the ranges of 1–12% (myristic acid), 14–46% (palmitic acid), 1–21% (docosapentaenoic acid) and 3–68% (docosahexaenoic acid) (Yaguchi et al., 1997; Lewis et al., 1999, 2002; Perveen et al., 2006; Jakobsen et al., 2008; Jasuja et al., 2010; Sarker et al., 2016; Sprague et al., 2016). For farmed salmon in the freshwater phase, a source of essential n-3 LC-PUFA must be provided in the feed to meet the fishes' daily metabolic demands; which are approximately 0.5–1% of the diet of combined DHA + EPA (NRC, 2011; Qian et al., 2020). As previously discussed, the essential n3 LC-PUFA content of commercial salmon feeds has greatly declined over the past several decades; which has been concomitant with increasing dietary replacement of n-3 LC-PUFA-rich marine fish oils with alternative resources rich in n-6 FAs of terrestrial origin. A foreseeable result of this scenario has been a reduction in the amount of DHA + EPA that the consumer obtains from consuming farmed salmon products. In fact, the EPA + DHA content of some farmed Atlantic salmon has declined by two-thirds over the past decade; requiring consumers to double their number of weekly farmed salmon portions to obtain the recommended intake of these critical n-3 LC-PUFA (Sprague et al., 2016). The n-3/n-6 ratio of Schizochytrium sp. (T18) oil is > 5; which is far higher than most terrestrial animal fats and vegetable oils (at < 2) and in the range of most conventional, albeit unsustainable, marine fish oils (3–24) (NRC, 2011). Of critical note is that n-3 PUFA is a broad grouping that generally includes α-linolenic acid (ALA, 18:3n3), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3); the latter is which is of greatest physiological importance for animal, fish and human health (Harwood, 2019). In this case, at 41% DHA, Schizochytrium sp. (T18) oil represents a superiorlyrich source of DHA than conventional marine fish and krill oils (at 2–23% DHA) and heavily-used terrestrial animal fats and vegetable oils; which are fully devoid of DHA (NRC, 2011; Sissener, 2018; Harwood, 2019). On the other hand, it is important to note that the general lack of EPA in Schizochytrium sp. (T18) oil would likely preclude the full replacement of marine fish oil due to its well-established roles in fish health. Farmed salmonids have a dietary need for essential n-3 LC-PUFA and this requirement is typically expressed as either total ‘n-3 LC-PUFA’ or as ‘EPA + DHA’ (NRC, 2011). However, while EPA and DHA are somewhat interchangeable in their physiological roles, ingested and

Table 6 Apparent digestibility coefficientsa (in vivo ADCs, %) of fatty acids in the nutritionally-balanced experimental diets with partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil fed to juvenile Atlantic salmon (Salmo salar). Replacement (%) of dietary fish oil 0 14:0 15:0 16:0 18:0 20:0 16:1n-7 16:2n-4 16:3n-4 16:4n-1 18:1n-7 18:1n-9 18:2n-6 18:3n-3 (ALA) 18:4n-3 20:1n-9 20:3n-3 20:4n-3 20:5n-3 (EPA) 22:1n-9 22:5n-3 (DPAn3) 22:5n-6 (DPAn6) 22:6n-3 (DHA) Σ SFA Σ MUFA Σ PUFA Σ n-3 PUFA Σ n-6 PUFA

33

75.1 ± 72.8 ± 71.2 ± 64.8 ± 57.7 ± 94.2 ± 100.0 100.0 100.0 90.5 ± 92.8 ± 95.5 ± 98.8 ± 100.0 88.3 ± 100.0 100.0 100.0 84.8 ± 100.0

0.0a 0.2a 0.1a 0.1a 0.2 0.1ns

0.2ns 0.1ns 0.3ns 2.0ns 0.3ab

0.3ab

– 95.2 70.0 92.1 97.2 98.3 95.5

66

79.1 ± 75.5 ± 72.3 ± 65.7 ± – 93.4 ± 100.0 100.0 100.0 89.5 ± 91.9 ± 94.9 ± 97.5 ± 100.0 86.8 ± 100.0 100.0 100.0 83.1 ± 100.0

2.7ab 1.6ab 1.2a 1.0a 0.3

0.4 0.2 0.2 2.2 0.5a

1.0a

100.0 ± ± ± ± ± ±

0.6a 0.1a 0.1ab 0.4ns 0.5ns 0.3ab

96.7 71.7 91.1 96.9 98.1 95.2

± ± ± ± ± ±

100

81.3 ± 76.8 ± 74.8 ± 69.0 ± – 94.2 ± – – 100.0 90.8 ± 93.1 ± 95.5 ± 97.0 ± 100.0 89.7 ± – 100.0 100.0 87.2 ± 100.0

2.5b 1.5b 1.3b 1.2b 0.8

0.9 0.8 0.4 0.4 1.1b

1.3b

100.0 0.3b 1.4a 0.3a 0.3 0.4 0.2a

97.8 74.9 92.5 97.2 98.2 96.1

± ± ± ± ± ±

81.2 ± 76.8 ± 75.5 ± 70.6 ± – 93.5 ± – – – 90.5 ± 93.2 ± 95.6 ± 97.0 ± – 88.2 ± – – 100.0 84.8 ± –

0.0b 0.2b 0.3b 0.3b 0.3

0.4 0.4 0.5 0.7 0.7ab

1.3ab

100.0 0.3bc 1.5b 0.9b 0.3 0.2 0.4ab

98.0 76.0 92.3 97.2 98.0 96.3

± ± ± ± ± ±

0.7c 0.3b 0.4ab 0.5 0.6 0.4b

a Values within the same row having different superscript letters are significantly different (P < .05).

(92–93%; P = .057) and minimal ADC differences observed for 20:1n-9 (87–90%; P = .011) and 22:1n-9 (83–87%; P = .011). In a similar manner, digestibility of dietary polyunsaturated fatty acids (Σ PUFA) was unaffected (97%; P = .647) by partial or complete replacement of dietary fish oil with Schizochytrium sp. (T18) oil. Correspondingly, ADC values for dietary Σ n-6 PUFA and Σ n-3 PUFA were virtually identical at 95–96% (P = .016) and 98% (P = .804), respectively. Digestibility of specific n-6 PUFAs, including 18:2n-6 and 22:5n-6 (DPAn-6), were statistically the same (P ≥ .126) across all experimental diets at 95–96% and 100%, respectively. Digestibility of minor dietary n-3 PUFAs (e.g., 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3, 20:5n-3 and 22:5n-3) were very high (97–100%; P ≥ .465) regardless of dietary treatment. As would be expected, the digestibility of DHA was very high (95%) for the fish oil-only control diet, yet increasing replacement of dietary fish oil with Schizochytrium sp. (T18) oil significantly (P < .001) increased DHA digestibility by a further 2–3% (97–98%). 4. Discussion Edible oils extracted from other Schizochytrium strains have been shown to be safe for human and animal consumption (Hammond et al., 2001a, 2001b, 2001c; Hammond et al., 2002; Abril et al., 2003; Federova-Dahms et al., 2011a, 2011b; Dahms et al., 2019). Published results have shown this to be the case for Schizochytrium sp. strain T18 oil as well; having a food-grade level of safety, as demonstrated by a lack of genotoxicity, acute toxicity and sub-chronic toxicity in studies conducted with bacteria (Salmonella typhimurium, Escherichia coli), human cell lines (peripheral blood lymphocytes) and laboratory rodents (Sprague-Dawley rats) (Schmitt et al., 2012). Our results also suggest the safety of this particular batch of Schizochytrium sp. (T18) oil with 6

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length, degree of unsaturation, melting point and n-3/n-6 ratio (Hua and Bureau, 2009; Huguet et al., 2015) and these factors are also influenced by the well-established effect of fish culture water temperature (Cho et al., 1982). It is well recognized that lipolytic enzymes in the fish intestine have a high affinity for PUFA, followed by MUFA and, lastly, SFA (Colombo-Hixson et al., 2011). This scenario was fully reflected in the present study where AD of PUFA, MUFA and SFA of the experimental diets were 97% > 91–93% > 70–76%, respectively. In addition, the notion that within the SFA group, carbon chain length is inversely proportional to AD in fish, was also evident in the present study where the average AD for 14:0, 15:0, 16:0, 18:0 and 20:0 were 79% > 75% > 73% > 67% > 58%, respectively. However, the ‘magnitude’ of improvement in AD amongst these SFAs from the fish oil-only diet to the Schizochytrium sp. oil-only diet remained relatively unchanged at 4–6%; so the improvement in AD of dietary SFA cannot be attributed to any specific SFA in this study. With particular regard to the n-3/n-6 ratio, in fact a study examining the effects of dietary inclusion of an n-6 FA-rich freshwater chlorophytic microalgae on AD in Atlantic salmon suggested a correlated dose-response of a 3-fold decrease in dietary n-3/n-6 ratios with concomitant decreases in dietary lipid AD; despite balanced dietary SFA content (Tibbetts et al., 2017). As mentioned, all experimental diets used in the present study had balanced dietary n-3/n-6 ratios and, indeed, the lipid AD was statistically the same; regardless of the level of replacement of dietary fish oil with Schizochytrium sp. (T18) oil. This result is in accordance with previous work demonstrating that, while all dietary LC-PUFA is highly digestible to farmed coldwater fish, n-3 LC-PUFA is generally more highly digestible than n-6 PUFA (Francis et al., 2007; Bandarra et al., 2011; Eroldogan et al., 2013). This is the first study to report the AD of dietary proximate nutrients, energy and fatty acids of salmonid feeds with partial (33 and 66%) or complete (100%) replacement of dietary fish oil with any extracted Schizochytrium oil, while a previous study reported the AD of strictly 4 fatty acid groupings and 4 individual fatty acid and only at complete (100%) fish oil replacement (Miller et al., 2007). In that study, the authors effectively showed that extracted oil from a related Schizochytrium strain could fully replace dietary fish oil without significantly affecting production performance of juvenile Atlantic salmon; however the AD of total MUFA and the resulting fillet EPA concentrations were significantly compromised. As noted, the aforementioned study did not examine the effects of partial replacement of fish oil with Schizochytrium oil (only complete replacement) and also neglected to determine the effects of this dietary substitution on AD of non-lipid proximate nutrients and energy. Subsequently, comparisons are limited to the AD of certain fatty acid groupings and specific fatty acids and only at the 100% replacement level and the results do not entirely agree. In both studies, AD values were similar and high for dietary total n-3 PUFA (96–98%), total n-6 PUFA (93–96%), DHA (97–98%), linoleic acid (96–100%) and DPA (97–100%). However, the former study showed consistently lower dietary AD than the present study for total SFA (71 vs. 76%, respectively), total MUFA (75 vs. 92%, respectively) and EPA (91 vs. 100, respectively). Since the initial fish size and water temperature in the studies were highly similar (32–40 g and 14–15 °C, respectively) and faecal collections were made by sedimentation in both studies, it is possible that the aforementioned imbalances of dietary MUFA, PUFA, n-3 PUFA, n-6 PUFA and n-3/n-6 ratio between the 2 test feeds of Miller et al. (2007) may account for these differences. Differences in experimental diet formulation likely also contribute to the results as the experimental diets used in each study contained different ingredients and were rather ranging in their contents of dietary protein (40–50%), fat (15–20%) and gross energy (20–23 MJ/kg); not to mention the use of different sources of fish meal and Schizochytrium oil. Regardless, both studies have demonstrated the exceptional potential of extracted Schizochytrium oils for sustainable reduction and/or substitution of conventional dietary marine fish oils. All other reports found in the literature have used dry whole-cell meals; which has

metabolized DHA and EPA do, in fact, play several different roles in the body and are each associated with certain unique metabolic processes (Emery et al., 2016; Horn et al., 2019). Of largest importance is that DHA plays a far more critical role in the structural maintenance of cell membrane fluidity and cell signaling (Stillwell and Wassall, 2003). In addition, it is believed that certain classes of anti-inflammatory, antiapoptotic and pro-neuroprotective compounds known as protectins and D-series resolvins are derived solely from DHA; and not EPA (Schwab et al., 2007; Serhan and Chiang, 2008). By contrast, unlike DHA, EPA plays a more pivotal role in the cellular production of anti-inflammatory eicosanoids such as prostaglandins, thromboxanes, leukotrienes, corticosteroids and E-series resolvins; which in turn enhance the anti-inflammatory response when under conditions of stress or illhealth (Swanson et al., 2012; Emery et al., 2016; Harwood, 2019). Conventional marine fish oils used in aquafeeds provide both EPA (at 7–17% of total FAs) and DHA (at 3–17% of total FAs) (NRC 2011) while most DHA-rich thraustochytrid products on the market (or in development) contain very low EPA levels and the Schizochytrium sp. (T18) oil used in the present study shares this characteristic (< 1% of total FAs). Given the unique physiological roles of this essential n-3 LCPUFA, this must be an important consideration in terms of feed formulation, growth and fish health in further nutritional studies and for commercialization of this novel product. Given that the experimental diets used in this study were formulated with highly similar total levels and mixing ratios of protein-rich ingredients (66% of diet), added oils (18% of diet), carbohydrate-rich ingredients (11% of diet) and micronutrients (5% of diet), it is not unexpected that the experimental diets had the same proximate composition (e.g., 5% moisture, 8% ash, 49% crude protein, 20% lipid, 17% carbohydrate, 23 MJ/kg gross energy). The only distinct differences were directly related to the source of the edible oil added to the experimental diets. Stearic acid (SA; 18:0) was reduced by half (e.g., from 0.8% in the control diet to 0.4% in the complete replacement diet) and, in a similar manner, eicosapentaenoic acid (EPA; 20:5n-3) was reduced by 80% (e.g., from 1.0% in the control diet to 0.2% in the complete replacement diet). In the opposite fashion, docosapentaenoic acid (DPAn-6; 22:5n-6) and docosahexaenoic acid (DHA; 22:6n-3) levels were increased from 0.0 and 0.9% in the control diet, respectively to 0.5 and 2.6% in the complete replacement diet, respectively. These dietary changes are consistent with the only other published study to evaluate an extracted Schizochytrium sp. oil at full replacement of dietary fish oil in Atlantic salmon feeds (Miller et al., 2007). They showed similar reductions in dietary SA (0.6 to 0.2%) and EPA (1.8 to 0.3%); concomitant with increases in dietary DPA (0.0 to 2.1%) and DHA (1.1 to 5.3%). It is well documented that the SFA content of feeds can influence nutrient digestibility in poikilothermic animals such as salmonid fish (Austreng et al., 1979; Menoyo et al., 2003; Ng et al., 2004; NRC, 2011). As such, experimental diets in the present study were formulated with graded levels of poultry fat and canola oil (at 2–7% of diet) so that total dietary SFA content was balanced at 5% across all diets. This was also the case for diets used by Miller et al. (2007) who conducted a digestibility trial using oil from a related Schizochytrium sp. strain. In fact, the lipid fraction of the experimental diets used in the present study were also highly balanced in regards to total MUFA (7%), total PUFA (6%), total n-3 PUFA (3%) and total n-6 PUFA (3%), resulting in balanced dietary n-3:n-6 ratios of 1. In contrast, this was not the case for Miller et al. (2007) where levels ranged between Schizochytrium sp. oil-only and fish oil-only diets, respectively, for MUFA (< 1 to 4%), total PUFA (9 to 5%), total n-3 PUFA (6 to 4%), total n-6 PUFA (3 to 1%) and n-3/n-6 ratio (2–3). As a result of the high degree of dietary proximate constituent and fatty acid balancing in the present study, differences in AD of dietary proximate nutrients, energy or fatty acids are unlikely to be attributed to dietary formulation imbalances; but rather as a direct result of the tested variable (e.g., added oil blend). There are several edible oil characteristics that can affect dietary lipid AD in farmed salmonids; most notably carbon chain 7

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Table 7 Summary of the main effects of dietary inclusion of dry whole-cell Schizochytrium meals for salmonid fish. Fish species

Product

Inclusion levels

Main finding

Atlantic salmon

Thraustochytrid strain ACEM 6063

0–10%

Atlantic salmon

A-DHA (Aurantiochytrium sp.)

0–20%

Atlantic salmon

Schizochytrium sp./Yeast extract blend AquaGrow Gold® (Schizochytrium sp.)

0–15%

Rainbow trout

A-DHA (Aurantiochytrium sp.)

0–20%

Rainbow trout

DHA-Gold® (Schizochytrium sp.)a

Rainbow trout

DHA-Gold® (Schizochytrium sp.)

a

Rainbow trout

ALL-G-Rich™ (S. limacinum)

0–5%

Rainbow trout

S-Type Gold Fat (Schizochytrium sp.)

0–9%

Can be included up to 10% without negative effects on growth performance, feed utilization, body composition and blood chemistry. However, fish were less robust to respond to stressors (e.g., seawater transfer and disease challenge) than those fed a fish oil-based control diet; which may be related to low EPA levels in thraustochytrids (Carter et al., 2003). Can be included at 13% without reducing digestibility of dry matter and protein but levels above 7% reduced digestibility of lipid and fatty acids (Zhang, 2013). Can be included up to 6% before negative effects were observed on growth performance, feed utilization, nutrient digestibility, product quality and intestinal health (Kousoulaki et al., 2015). Growth performance and feed utilization were compromised above 5.5%; while 11% reduced harmful persistent organic pollutants in the feed and fillets and restored fillet DHA levels (but not EPA) to those of the fish oil-based control diet (Sprague et al., 2015). Can be included at 20% without reducing digestibility of protein and essential amino acids but levels above 13% reduced digestibility of dry matter, energy, lipid and fatty acids (Zhang, 2013). Can be included up to 9% without negative effects on growth performance, feed utilization, blood chemistry and fillet DHA levels, but fillet EPA concentrations were reduced (Betiku et al., 2016). At 30% inclusion, lipid digestibility was similar to conventional fish oils, but very poor for dry matter, protein, energy and amino acids (Betiku et al., 2016). At 5% inclusion, growth performance was unaffected and the diversity of the intestinal microbiome was enhanced; particularly the lactic acid bacteria (LAB) group (generally considered as beneficial to healthy intestinal epithelium and may protect against harmful pathogens). Unclear whether this enhancement was related to intracellular DHA of S. limacinum meal or structural cell wall polysaccharides (Nayak, 2010; Lyons et al., 2017). Inclusion at 9% (in combination with 15% canola oil) reduced flesh contaminants (e.g., toxaphenes, organochlorine pesticides and PCB's) without affecting digestibility or production performance of the fish (Bélanger-Lamonde et al., 2018).

Atlantic salmon

a

0–11%

0–9% 0–30%

Erroneously reported as derived from Crypthecodinium cohnii.

Acknowledgements

unfortunately limited their possible inclusion tolerance to low levels (generally < 10% of the diet) for salmonid fish (see summary in Table 7). This is consistent with our preliminary studies where we observed significant reductions (by up to 26%) in the AD of dietary dry matter, lipid and energy when dry whole-cell Schizochytrium sp. (T18) meal was included in juvenile Atlantic salmon feeds at 10–15%; while protein AD was relatively unaffected (unpublished data). Despite these generally discouraging results, dry whole-cell Schizochytrium meals included at a low dietary level (≤11%) in salmonid feeds have been shown to promote some beneficial effects with regard to intestinal microbiome, gut health, feed contaminant levels and product quality (Table 7). In the case of intestinal health, however; it is unclear whether the enhancement is related to the intracellular DHA of Schizochytrium meals or their structural cell wall polysaccharides. In conclusion, this was the first study directed towards the nutritional evaluation of Schizochytrium sp. (T18) microbial oil as a novel and sustainable ‘low trophic’ resource for farmed salmonid feeds. This novel product can be produced at commercial scale using low-cost nutrient feedstocks, is safe for human, animal and fish consumption and contains a very high level of the most physiological-important n-3 LCPUFA docosahexaenoic acid (DHA; 22:6n-3). The results demonstrate that conventional, unsustainable marine fish oils can be completely replaced in juvenile farmed Atlantic salmon feeds by Schizochytrium sp. (T18) oil without any negative effects on the digestibility of key essential dietary proximate nutrients, energy or fatty acids. Further evaluations are warranted to determine the optimum dietary inclusion level and/or blend of Schizochytrium sp. (T18) oil based on growth performance and fish health, nutrient utilization efficiency and final product quality for the consumer.

This work was supported in part by the National Research Council of Canada, Industrial Research Assistance Program (IRAP Certificate Program #A0028962) and contributions from the National Research Council of Canada, Aquatic and Crop Resource Development (NRCACRD) Research Centre. The authors acknowledge the valuable expertise of Drs. Dominic Nanton, Santosh Lall and André Dumas. We are grateful for the highly skilled technical assistance provided by Paula Mercer, Shane Patelakis, Amanda Smith and Crystal Lalonde. We thank Yves Gohier (DSM Nutritional Products Canada Inc.), Alan Donkin (Northeast Nutrition Ltd.) and Michael Klapperich (Cargill Inc.) for kindly donating some of the dietary ingredients used in the experimental test feeds. Valuable consultations, logistical support and critical review of Drs. Patrick McGinn and Debbie Plouffe are greatly appreciated. This is NRCC publication no. 56460. References Aas, T.S., Ytrestøyl, T., Åsgård, T., 2019. Utilization of feed resources in the production of Atlantic salmon (Salmo salar) in Norway: an update for 2016. Aquac. Rep. 15. https://doi.org/10.1016/j.aqrep.2019.100216. Abril, R., Garrett, J., Zeller, S.G., Sander, W.J., Mast, R.W., 2003. Safety assessment of DHA-rich microalgae from Schizochytrium sp. part V: target animal safety/toxicity study in growing swine. Regul. Toxicol. Pharmacol. 37, 73–82. Adarme-Vega, T.C., Lim, D.K., Timmins, M., Vernen, F., Li, Y., Schenk, P.M., 2012. Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production. Microb. Cell Factories 11. https://doi.org/10.1186/1475-2859-11-96. Armenta, R.E., Valentine, M.C., 2013. Single-cell oils as a source of omega-3 fatty acids: an overview of recent advances. J. Am. Oil Chem. Soc. 90, 167–182. Austreng, E., Skrede, A., Eldegard, A., 1979. Effect of dietary fat source on the digestibility of fat and fatty acids in rainbow trout and mink. Acta Agr. Scand. 29, 119–126. Bandarra, N.M., Rema, P., Batista, I., Pousao-Ferreira, P., Valente, L.M.P., Batista, S.M.G., Ozorio, R.O.A., 2011. Effects of dietary n-3/n-6 ratio on lipid metabolism of gilthead seabream (Sparus aurata). Eur. J. Lipid Sci. Technol. 113, 1332–1341. Barclay, W.R., Meager, K.M., Abril, J.R., 1994. Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J. Appl. Phycol. 6, 123–129. Bélanger-Lamonde, A., Sarker, P.K., Ayotte, P., Bailey, J.L., Bureau, D.P., Chouinard, P.Y., Dewailly, É., Leblanc, A., Weber, J.P., Vandenberg, G.W., 2018. Algal and vegetable oils as sustainable fish oil substitutes in rainbow trout diets: an approach to reduce contaminant exposure. J. Food Qual. https://doi.org/10.1155/2018/7949782. Betiku, O.C., Barrows, F.T., Ross, C., Sealey, W.M., 2016. The effect of total replacement of fish oil with DHA-gold® and plant oils on growth and fillet quality of rainbow trout

Declaration of Competing Interest We declare that MAS and REA are employees of Mara Renewables Corporation; however, any reference to commercial companies, product names and/or organism identifiers is for accuracy and does not represent endorsement by the authors.

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