Apparent digestibility of protein, amino acids and gross energy in rainbow trout fed various feed ingredients with or without protease

Aquaculture 524 (2020) 735270

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Apparent digestibility of protein, amino acids and gross energy in rainbow trout fed various feed ingredients with or without protease Seunghan Leea, M.A. Kabir Chowdhuryb, Ronald W. Hardya, Brian C. Smalla, a b

T

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University of Idaho, Aquaculture Research Institute, Hagerman Fish Culture Experiment Station, 3059-F National Fish Hatchery Road, Hagerman, ID 83332, USA Jefo Nutrition Inc., Saint-Hyacinthe, Quebec J2S 7B6, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Digestibility Ingredients Protease Rainbow trout

Apparent digestibility coefficients (ADCs) of dry matter, crude protein, amino acids, and gross energy in various feed ingredients with or without protease supplementation (Jefo Nutrition Inc., Quebec, Canada) were determined for rainbow trout in a two-year study. The ingredients consisted of two feather meals, two poultry byproduct meals, two meat and bone meals, sardine meal, menhaden meal, black soldier fly larvae meal, Methanococcus maripaludis single cell protein, soybean meal, canola meal, distiller's dried grains with solubles (DDGS), cottonseed meal, peanut meal, sunflower meal, and algae (Spirulina sp.) meal. A reference diet with 0.1% yttrium oxide as inert marker was mixed with test ingredients in a 70:30 ratio to produce a series of test diets. Fish were fed their respective diets and fecal samples were collected by stripping. ADC of dry matter for rainbow trout ranged 51.0–86.6% for animal products and single cell protein and 33.1–70.1% for plant products without protease supplementation. ADC (without protease supplementation) of protein and energy ranged from 55.4–84.5% and 58.1–90.2%, respectively, for animal products, and 70.0–83.8% and 32.9–76.0%, respectively, for plant products. Supplementation with the commercial protease (175 mg protease complex/kg of diet) resulted in ingredient-specific ADC increases for dry matter, energy, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, alanine, aspartic acid and glutamic acid, with most ingredients having improved digestibility of at least one amino acid. Protease supplementation had the most profound improvement on ADCs for soybean meal, including dry matter and the majority of individual amino acids. Overall, this research demonstrates the benefit of protease supplementation on the digestibility of feed ingredients commonly used in rainbow trout and other commercially cultured fish feeds, although the degree of improvement in digestibility varied among ingredients. Further studies with this protease are recommended to evaluate its effects at higher inclusion levels and in other species of cultured fishes.

1. Introduction Protein is the primary and most expensive major ingredient in fish and crustacean feeds. It is also considered to be growth limiting due to the dietary requirements for essential amino acids. Fishmeal is a protein-rich ingredient in feeds for carnivorous finfish and marine shrimp (Naylor et al., 2009). Although fishmeal inclusion levels in aquaculture feeds have shown a significant downward trend for the last two decades, it is still being considered a primary protein source used in aquafeed due to its well-balanced amino acid profile, high digestibility and palatability (Hardy, 2010). The rapid growth of aquaculture has boosted demand for feeds, which primarily depend on fishmeal as the major protein source due to its excellent nutritional profile. Many alternative feed ingredients of animal and plant origin have been

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investigated and used in aquaculture feeds to replace fishmeal and reduce the cost of fish production (Gatlin et al., 2007). However, imbalanced amino acid profiles, poor digestibility and palatability, and presence of anti-nutritional factors (ANFs) associated with these protein sources, such as phytate, trypsin inhibitor, and non-starch polysaccharides, limit their use in aquafeeds (NRC, 2011; Sookying and Davis, 2011). Several strategies have been employed to improve digestibility of these alternative protein sources, including fermentation to reduce ANFs (Yamamoto et al., 2010; Seong et al., 2018), amino acid supplementation to balance amino acid profiles (Gaylord and Barrows, 2009; Mokrani et al., 2019), and enzyme treatment to improve the nutrient digestibility and availability (Lin et al., 2007; Yao et al., 2019). Among enzymes, proteases have potential use in reducing ANFs, such as

Corresponding author at: University of Idaho, Aquaculture Research Institute, Hagerman Fish Culture Experiment Station, USA. E-mail address: [email protected] (B.C. Small).

https://doi.org/10.1016/j.aquaculture.2020.735270 Received 19 February 2020; Received in revised form 19 March 2020; Accepted 19 March 2020 Available online 20 March 2020 0044-8486/ © 2020 Elsevier B.V. All rights reserved.

Aquaculture 524 (2020) 735270

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protease inhibitors, and breaking down macromolecular proteins (Li et al., 2016). Furthermore, proteases added to the feed may compensate for deficiencies in endogenous enzymes, especially in young animals (Shi et al., 2016). The use of exogenous protease has been extensively studied to improve the quality of alternative protein sources in poultry (Angel et al., 2011; Mahmood et al., 2017; Walk et al., 2018). Some evaluations have been published on the supplementation of protease in fish feeds (Shi et al., 2016; Yigit et al., 2018; Li et al., 2019), but the efficacy of supplemental protease across a wide range of protein ingredients has not been previously investigated. Therefore, this study was conducted to evaluate the effects on apparent digestibility coefficients (ADCs) of dry matter (DM), crude protein (CP), amino acids (AAs), and gross energy (GE) of a dietary protease added to 17 different protein ingredients using rainbow trout as a model species.

Table 2 Proximate composition and gross energy of ingredients (% dry-matter basis).

2. Materials and methods 2.1. Experimental design and diets In vivo digestibility was determined for 17 ingredients with and without protease supplementation (175 g kg −1) fed to rainbow trout. The protease complex (Jefo Nutrition Inc., Quebec, Canada) is a mixture of dehydrated yeast culture and dehydrated soluble extracted from bacterial fermentation. A reference diet (Table 1) containing practical ingredients and 0.1% yttrium oxide was prepared. Proximate and amino acid compositions of the test ingredients are shown in Tables 2 and 3, respectively. A batch of test diet containing 30% test ingredient and 70% reference diet mash (combined on a dry-matter basis) was prepared and analyzed (Tables 4 and 5). Dry ingredients used in the experimental feeds were ground to a particle size of < 500 μm using a pulverizer. All ingredients were mixed together and cold pelleted at the University of Idaho's Hagerman Fish Culture Experiment Station (HFCES) using a laboratory-scale California pellet mill fitted with a 4mm die. After 36 h drying in a hot-air dryer at 37 °C, the feeds were stored at ambient temperature (20–22 °C).

Inclusion level

Fishmeal, sardinea Soy protein concentrateb Corn protein concentratec Wheat floura Wheat gluten meala Dicalcium phosphatea Choline chloride (60%)a Vitamin C (Stay C, 35%)a Vitamin premix, ARS 702d Trace mineral mix, Trouw Nutritione Fish oil, Alaska pollockf Yttrium oxideg Total

33.0 13.9 10.0 18.0 7.10 1.20 0.60 0.20 0.80 0.10 15.0 0.10 100

Dry matter

Crude protein

Lipid

Ash

Gross energy (kcal/kg)

Feather meal-1a Feather meal-2b Poultry by-product-1a Poultry by-product-2b Meat and bone meal-1c Meat and bone meal-2b Sardine meala Menhaden meala Black soldier fly larvaed Single cell proteine Soybean meala Canola meala Distiller's dried grains with solublesa Cottonseed mealb Peanut mealf Sunflower mealb Algae meala

90.8 91.8 96.9 97.1 96.7 97.3 92.3 92.7 98.0 91.4 91.0 89.5 90.4

94.4 90.5 67.1 66.9 54.7 53.7 72.2 69.3 44.7 64.3 52.7 41.5 30.1

5.63 6.85 11.3 15.6 12.5 17.7 11.7 10.4 11.7 17.8 1.36 2.49 4.58

1.48 1.02 20.9 12.2 27.1 24.7 17.3 22.1 9.60 16.2 6.71 7.36 5.83

5838 5925 4816 5502 4350 4894 5038 4685 5076 5225 4668 4736 4944

88.8 97.3 89.6 93.4

49.5 57.6 38.2 73.3

2.77 11.2 0.30 0.32

7.86 4.99 7.34 8.25

4741 5329 4640 5411

a b c d e f

Rangen Inc., Buhl, ID, USA. Cargill, Inc., Blair, NE, USA. Darling ingredient Inc., Irving, TX, USA. Enviroflight, LLC., Maysville, KY. Methanococcus maripaludis, Sasya LLC., Plymouth, MN, USA. Golden Peanut Company, LLC., Alpharetta, GA, USA.

2.2. Fish and feeding trial A domesticated strain (TroutLodge, Sumner, WA) of rainbow trout (average weight, 250 g) was used in the digestibility trials. Rainbow trout were selected from a larger population, weighed, and systematically interspersed into twenty-two 145-L fiberglass tanks to provide 30 fish per tank, each supplied with 13 L/min of untreated spring water with a constant temperature (15 °C) at the Hagerman Fish Culture Experiment Station. The trial was conducted over a two-year period in four rounds, with reference and 10 test diets fed in rounds 1–3 and reference plus 4 test diets fed in round four. Each of the experimental diets (reference and 34 test diets) was fed to two replicate tanks of fish in a completely randomized design to apparent satiation twice daily, 6 days per week for 3 weeks. After a week of acclimation, 18–20 h postprandial, fish were anesthetized with tricaine methanosulfonate (MS222, 100 mg L−1). Feces were expelled from each fish using gentle pressure on the lower one third of the abdomen and collected in aluminum cups. Fish were stripped for feces twice per week for 2 weeks. Feces samples were pooled by tank, frozen at −20 °C during the fecal collection period and dried at 70 °C in an oven for 24 h for proximate, energy, amino acids and minerals including yttrium analyses. All fish handling and sampling, plus the experimental protocols used in the digestibility trials were approved in advance by the University of Idaho's Institutional Animal Care and Use Committee.

Table 1 Ingredient composition of reference mash (%, as-fed basis). Ingredient

Test ingredients

2.3. Sample collection and analysis

a

Rangen Inc., Buhl, ID, USA. b Profine VF, The Solae Company, St. Louis, MO, USA. c Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. d Vitamin premix supplied the following per kg diet: vitamin A, 2.4 mg; vitamin D, 0.15 mg; vitamin E, 267 mg; vitamin K as menadione sodium bisulfite, 20 μg; thiamin as thiamin mononitrate, 32 mg; riboflavin, 64 mg; pyridoxine as pyridoxine-HCl, 64 mg; pantothenic acid as Ca-d-pantothenate, 192 mg; niacin as nicotinic acid, 240 mg; biotin, 0.56 mg; folic acid, 12 mg; vitamin B12, 50 μg; and inositol as meso-inositol, 400 mg. e Trace miner mix supplied the following per kg diet: Zn (as ZnSO4.7H2O), 75 mg; Mn (as MnSO4), 20 mg; Cu (as CuSO4.5H2O), 1.54 mg; I (as KIO3), 10 mg. f Skretting USA, Tooele, UT, USA. g Sigma Aldrich, St. Louis MQ, USA.

Feed and fecal samples were finely ground by mortar and pestle. Proximate composition of ingredients, feed and fecal samples were determined using AOAC (2000) procedures. Briefly, samples were dried in a convection oven at 105 °C for 12 h to determine moisture level. Samples were analyzed for crude protein (total nitrogen × 6.25) using the combustion method with a nitrogen determinator (TruSpec N, LECO Corporation, St. Joseph, MI). Crude lipid was analyzed using an ANKOM XT15 extractor (ANKOM Technology, Macedon, NY) with petroleum ether as the extracting solvent, and ash by incineration at 550 °C in a muffle furnace for 5 h. Energy content of samples were determined using an isoperibol bomb calorimeter (Parr 6300, Parr Instrument Company Inc., Moline, IL). Analyses of amino acids in samples 2

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Table 3 Amino acid composition of ingredients (% dry-matter basis). Test ingredients

Essential and semi-essential amino acids

Feather meal-1 Feather meal-2 Poultry by-product-1 Poultry by-product-2 Meat and bone meal-1 Meat and bone meal-2 Sardine meal Menhaden meal Black soldier fly larvae Single cell protein Soybean meal Canola meal Distiller dried grains with soluble Cottonseed meal Peanut meal Sunflower meal Algae meal

Non-essential amino acids

Arg

Cys

His

Iso

Leu

Lys

Met

Phe

Thr

Trp

Tyr

Val

Ala

Asp

Glu

Gly

Pro

Ser

Tau

4.67 6.33 4.51 4.48 3.83 3.58 3.52 4.23 1.81 3.47 3.67 2.27 1.19 4.62 5.65 2.29 3.32

1.62 2.07 0.66 0.74 0.60 1.23 0.57 0.52 0.33 0.36 0.76 1.03 0.58 0.75 0.41 0.31 0.38

1.57 1.17 1.32 1.59 1.22 0.77 1.88 1.39 1.17 1.23 1.35 1.05 0.75 1.18 1.52 0.90 1.54

2.98 4.46 2.32 2.69 1.63 1.95 2.56 3.19 1.75 1.97 2.47 1.62 1.13 1.44 1.73 1.40 3.07

5.10 7.69 4.14 4.59 3.30 3.46 4.46 5.07 3.14 3.41 4.04 2.73 3.08 2.52 3.73 2.09 4.91

3.96 2.57 4.06 4.20 3.02 2.12 4.76 4.16 1.83 3.65 3.36 2.30 0.89 1.93 1.18 1.18 2.76

1.24 0.72 1.25 1.34 0.80 0.66 1.97 1.59 0.80 1.44 0.71 0.78 0.51 0.67 0.31 0.25 1.08

2.85 4.58 2.34 2.62 1.80 2.04 2.41 2.70 1.75 1.92 2.72 1.57 1.34 2.34 2.93 1.48 2.67

2.81 4.33 2.34 2.52 1.78 1.86 2.71 3.05 1.57 2.11 2.09 1.67 1.10 1.39 1.53 1.25 2.93

0.53 0.47 0.54 0.57 0.34 0.22 0.24 0.53 0.15 0.43 0.71 0.44 0.18 0.47 0.30 0.68 0.48

2.37 2.64 1.18 2.32 1.39 1.32 2.25 1.60 0.89 1.80 1.78 1.07 1.04 1.24 0.96 0.34 1.74

3.86 4.06 2.75 3.25 2.34 2.80 3.02 3.58 2.22 2.38 2.51 2.03 1.43 1.95 2.18 1.63 3.70

4.25 4.41 4.42 4.34 3.88 3.60 4.00 4.66 2.67 3.73 2.25 1.70 1.87 1.66 2.49 1.50 4.74

5.48 6.23 5.17 5.26 4.05 3.65 5.86 6.40 3.47 4.73 5.93 2.74 1.80 3.86 7.26 3.20 6.00

8.75 9.62 8.40 8.36 6.78 6.04 8.25 9.43 6.71 6.85 9.30 6.51 3.78 8.20 9.81 6.77 8.99

6.12 6.84 7.13 6.27 7.03 6.22 4.40 4.42 2.18 5.55 2.18 1.95 1.08 1.78 3.48 2.00 3.36

4.65 8.35 4.62 4.00 4.23 4.43 2.21 3.29 2.91 4.73 2.75 2.42 2.33 1.70 3.04 1.97 3.44

3.97 9.40 2.57 2.52 2.13 2.90 2.57 2.90 1.78 2.27 2.26 1.37 1.18 1.53 2.85 1.47 3.03

0.33 0.30 0.41 0.41 0.11 0.08 0.75 0.55 0.12 0.67 0.07 0.12 0.10 0.10 0.02 0.02 0.03

2.4. Calculations

Table 4 Proximate composition and gross energy of reference and test diets (% drymatter basis) without and with the protease complex (+P). Diets

Dry matter

Crude protein

Lipid

Ash

Gross energy (kcal/kg)

Reference diet Feather meal-1 Feather meal-1 + P Feather meal-2 Feather meal-2 + P Poultry by-product-1 Poultry by-product-1 + P Poultry by-product-2 Poultry by-product-2 + P Meat and bone meal-1 Meat and bone meal-1 + P Meat and bone meal-2 Meat and bone meal-2 + P Sardine meal Sardine meal +P Menhaden meal Menhaden meal +P Black soldier fly larvae Black soldier fly larvae +P Single cell protein Single cell protein +P Soybean meal Soybean meal +P Canola meal Canola meal +P Distiller dried grains with soluble Distiller dried grains with soluble +P Cottonseed meal Cottonseed meal +P Peanut meal Peanut meal +P Sunflower meal Sunflower meal +P Algae meal Algae meal +P

92.6 92.1 91.2 92.0 91.2 92.7 91.8 92.5 92.0 92.1 92.0 93.0 92.8 93.2 92.9 92.7 92.5 93.3 91.8 93.2 92.6 92.8 92.7 92.5 92.6 93.1

48.4 61.1 61.2 60.2 60.9 53.8 53.5 54.4 54.0 50.6 49.9 52.6 52.0 56.8 56.0 55.9 56.5 49.8 50.9 53.5 54.6 50.2 50.2 46.9 47.3 43.6

21.2 15.1 16.4 15.5 16.0 18.3 18.5 19.0 19.1 17.2 17.9 20.2 20.1 17.9 18.2 18.0 18.2 18.1 18.3 19.7 19.1 15.0 15.0 15.3 15.4 16.5

9.76 7.11 7.54 7.21 7.41 11.2 11.2 9.76 10.5 14.3 14.1 13.2 12.8 10.9 11.2 12.2 12.7 9.50 9.40 11.0 10.7 8.54 8.36 8.45 8.56 8.18

5489 5484 5508 5560 5591 5337 5356 5431 5422 5162 5119 5410 5401 5442 5449 5376 5365 5446 5494 5481 5481 5289 5282 5302 5315 5360

93.9

44.7

16.5

7.92

5346

92.4 93.2 93.8 92.9 93.3 92.2 92.8 91.6

48.6 48.5 52.5 52.9 45.8 46.8 56.4 57.2

15.2 15.3 19.0 18.5 15.3 14.4 16.4 15.8

8.64 8.77 6.50 6.70 7.30 7.10 7.60 7.90

5299 5299 5495 5519 5296 5341 5481 5492

ADC of diets and ingredients, for dry matter, protein, amino acids and energy were calculated using the following formula described by Bureau et al. (1999):

ADCdiet = 1 − [(F/D) × (Di/Fi)] where D = % nutrient of diet, F = % nutrient of feces, Di = % digestion indicator of diet, Fi = % digestion indicator of feces.

ADCingredient = ADCT + [((1 − s) DR)/s DI] × (ADCT − ADCR) where ADCT = ADC of test diet, ADCR = ADC of reference diet, DR = % nutrient of reference diet, DI = % nutrient of test ingredient, s = proportion of test ingredient in test diet (0.3). 2.5. Statistical analysis Apparent digestibility was calculated using fecal material pooled from 30 fish/tank (n = 2 tanks), and all data are expressed as the mean ± standard error of the mean (SE). Data were subjected to a Student's t-test to test for protease effect using SPSS Version 20.0 (SPSS Inc., Chicago, IL, USA). Protease effects were considered significant a P < .1. 3. Results The ADC for dry matter, crude protein, and energy of the test ingredients fed to rainbow trout without and with the protease complex (+P) are summarized in Table 6. ADC for dry matter ranged from 32.4 to 86.8%, for crude protein from 55.4 to 91.3%, and for energy from 32.9 to 86.7%. Digestible energy was higher (P = .001) for protease supplemented ingredients compared to ingredients without protease addition (69.2 vs. 66.6%), whereas ADC of dry matter was numerically but not significantly higher (P = .069, 60.8 vs. 62.5%) with the addition of protease. ADC of crude protein was unaffected by protease (P = .198). In particular, the ADC for dry matter of feather meal-2 and soybean meal was significantly higher with protease supplementation (P = .003) and a modest gain improvement in dry matter ADC was observed when feeding soybean meal (P = .082). Specific improvements in digestible energy were also noted for feather meal-2 (P = .095), as well as poultry by-product-2 (P = .041) and single cell

were conducted using a BioChrom 30+ amino acid analyzer. Analyses of minerals including yttrium were conducted by the Department of Agricultural Chemistry, Louisiana State University Agricultural Center, Baton Rouge, LA, using inductively-coupled plasma (ICP). All analyses were done in duplicate.

3

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Table 5 Amino acid composition of reference and test diets (% dry-matter basis), without and with the protease complex (+P). Diet

Reference diet Feather meal-1 Feather meal-1 + P Feather meal-2 Feather meal-2 + P Poultry by-product-1 Poultry by-product-1 + P Poultry by-product-2 Poultry by-product-2 + P Meat and bone meal-1 Meat and bone meal-1 + P Meat and bone meal-2 Meat and bone meal-2 + P Sardine meal Sardine meal +P Menhaden meal Menhaden meal +P Black soldier fly larvae Black soldier fly larvae +P Single cell protein Single cell protein +P Soybean meal Soybean meal +P Canola meal Canola meal +P Distiller dried grains with soluble Distiller dried grains with soluble +P Cottonseed meal Cottonseed meal +P Peanut meal Peanut meal +P Sunflower meal Sunflower meal +P Algae meal Algae meal +P

Essential and semi-essential amino acids

Non-essential amino acids

Arg

Cys

His

Iso

Leu

Lys

Met

Phe

Thr

Trp

Tyr

Val

Ala

Asp

Glu

Gly

Pro

Ser

Tau

2.64 3.74 3.77 3.75 3.67 3.15 3.23 3.25 3.12 3.03 3.04 2.81 2.86 2.82 2.86 3.04 3.13 2.12 2.29 2.78 2.70 3.02 3.05 2.62 2.63 2.25 2.27 3.39 3.40 3.14 3.05 2.27 2.42 2.88 2.83

0.65 1.79 1.80 1.91 1.78 0.82 0.83 0.71 0.65 0.63 0.60 0.63 0.58 0.47 0.46 0.54 0.52 0.43 0.45 0.47 0.44 0.68 0.71 0.75 0.78 0.66 0.68 0.71 0.73 0.45 0.45 0.39 0.45 0.50 0.48

1.28 1.19 1.16 1.29 1.26 1.37 1.42 1.34 1.39 1.30 1.30 1.21 1.22 1.53 1.38 1.64 1.51 1.26 1.35 1.32 1.27 1.21 1.22 1.11 1.13 1.02 1.04 1.18 1.19 1.40 1.38 1.07 1.12 1.40 1.40

2.13 2.75 2.78 2.81 2.75 2.27 2.36 2.33 2.29 2.01 2.01 2.14 2.06 2.15 2.11 2.38 2.35 1.91 2.08 2.05 1.95 2.25 2.27 1.97 2.01 1.82 1.86 1.96 2.00 2.04 1.99 1.74 1.95 2.28 2.31

4.40 5.14 5.21 5.36 5.28 4.42 4.56 4.47 4.33 4.11 4.08 4.18 4.07 4.78 4.68 4.63 4.59 4.03 4.00 4.08 3.88 4.38 4.35 3.88 3.98 4.03 4.11 3.95 4.04 4.03 4.02 3.40 3.66 4.65 4.72

2.71 2.44 2.46 2.70 2.70 3.05 3.13 3.17 3.09 2.85 2.81 2.38 2.43 3.15 3.15 3.39 3.47 2.25 2.29 2.69 2.67 2.92 2.96 2.63 2.64 2.18 2.22 2.55 2.54 2.27 2.25 2.01 2.12 2.73 2.68

1.05 0.91 0.90 0.96 0.94 1.08 1.13 1.15 1.10 0.98 0.96 1.12 1.02 1.30 1.23 1.38 1.43 0.95 1.05 1.21 1.16 0.97 0.98 1.01 1.01 0.91 0.94 0.99 0.99 0.92 0.81 0.79 0.94 0.89 0.96

2.42 3.02 3.05 3.04 2.98 2.42 2.51 2.47 2.49 2.24 2.38 2.35 2.27 2.38 2.34 2.54 2.53 2.02 2.22 2.29 2.14 2.54 2.56 2.17 2.22 2.11 2.16 2.47 2.51 2.39 2.34 1.90 2.02 2.47 2.49

1.82 2.44 2.49 2.58 2.52 2.03 2.08 2.05 1.96 1.81 1.77 1.94 1.92 2.13 2.09 2.28 2.29 1.73 1.84 1.96 1.88 1.95 1.94 1.83 1.85 1.65 1.69 1.77 1.77 1.83 1.81 1.65 1.77 2.22 2.20

0.45 0.54 0.52 0.56 0.56 0.53 0.62 0.50 0.58 0.41 0.51 0.34 0.30 0.30 0.31 0.25 0.23 0.25 0.20 0.20 0.20 0.60 0.56 0.51 0.46 0.34 0.38 0.48 0.43 0.28 0.29 0.29 0.29 0.48 0.51

1.70 1.93 1.92 1.97 1.94 1.84 1.89 1.87 1.74 1.63 1.66 1.32 1.31 2.60 2.62 1.54 1.60 1.43 1.47 1.37 1.34 1.89 1.88 1.60 1.61 1.58 1.51 1.68 1.72 0.81 0.81 0.75 0.75 1.03 1.01

2.38 3.76 3.79 3.77 3.65 2.64 2.77 2.68 2.61 2.40 2.39 2.54 2.48 4.11 3.95 2.72 2.64 3.54 2.37 2.34 2.25 2.42 2.43 2.25 2.26 2.06 2.09 2.28 2.30 2.37 2.33 2.00 2.14 2.66 2.62

2.82 3.17 3.22 3.28 3.24 3.14 3.19 3.29 3.15 3.12 3.12 3.25 3.15 3.05 3.01 3.41 3.42 2.61 2.95 3.14 3.01 2.70 2.67 2.50 2.54 2.59 2.65 2.54 2.59 2.69 2.67 2.46 2.63 3.36 3.33

3.92 4.47 4.57 4.61 4.54 4.22 4.35 4.36 4.23 3.94 3.94 4.10 4.06 4.62 4.56 4.96 4.98 3.73 4.03 4.26 4.12 4.60 4.65 3.68 3.73 3.36 3.45 4.05 4.08 4.65 4.57 3.63 3.84 4.71 4.67

9.47 9.29 9.36 9.56 9.45 9.02 9.19 9.12 8.70 8.70 8.70 8.78 8.63 8.79 8.73 9.65 9.82 7.60 8.44 8.68 8.38 9.53 9.60 8.54 8.78 7.83 8.16 9.32 9.49 9.98 9.96 7.84 8.24 10.2 10.2

2.52 3.95 4.00 3.79 3.69 3.47 3.47 3.74 3.56 3.80 3.80 3.87 3.82 2.93 3.01 3.39 3.47 2.21 2.53 3.55 3.40 2.48 2.47 2.44 2.45 2.18 2.23 2.37 2.42 2.72 2.67 2.18 2.30 2.77 2.70

3.30 5.00 5.04 5.00 4.80 3.62 3.72 3.63 3.56 3.62 3.62 2.16 2.95 3.74 4.12 2.88 2.92 3.77 3.30 1.92 2.60 3.07 3.19 2.93 3.29 2.90 3.23 2.76 3.10 2.48 3.43 2.72 1.95 2.24 2.26

2.06 4.29 4.37 4.25 4.05 2.40 2.47 2.20 1.92 2.06 2.06 2.63 2.59 2.37 2.33 2.54 2.54 2.03 2.19 2.38 2.24 2.16 2.17 1.87 1.92 1.82 1.93 1.98 1.98 2.32 2.30 1.92 2.04 2.54 2.56

0.28 0.22 0.21 0.21 0.21 0.29 0.30 0.32 0.31 0.23 0.23 0.22 0.21 0.39 0.30 0.41 0.33 0.18 0.19 0.33 0.33 0.28 0.27 0.28 0.28 0.26 0.26 0.27 0.27 0.19 0.20 0.16 0.17 0.21 0.23

ingredients, relative to fishmeal, and the lower ADCs have a negative correlation with high fiber or anti-nutritional factors (Zhou and Yue, 2012; Luo et al., 2008). Energy, protein, and amino acid ADCs generally followed the same pattern as dry matter ADCs without protease supplementation. ADC (without protease supplementation) of protein and energy ranged from 55.4–84.5% and 58.1–90.2%, respectively, for animal products, and 70.0–83.8% and 32.9–76.0%, respectively, for plant products. In the present study, the value for ADCs of each ingredient are relatively lower compared to others previously reported for this species (Bureau et al., 1999; Cheng and Hardy, 2003; Gomes et al., 1995). In addition to genetic and environmental differences between studies, fecal collection methods play an important role. There have been various studies examining the effectiveness of fecal collection methods, such as manual stripping, dissection, anal aspiration and several settlement techniques, for the determination of nutrient digestibility in fish (Austreng, 1978; Percival et al., 2001; Stone et al., 2008). Using a fecal settlement technique avoids the unwanted addition of undigested feed or sloughed cellular components associated with manual stripping, dissection and aspiration. However, when using settlement techniques, nutrient leaching from feces to the water is linked to exposure time, and this problem becomes more obvious when ingredients and feces contain high levels of water-soluble components such as free amino acids, peptides, water-soluble proteins, carbohydrates and minerals (VensCappell, 1985; Stone et al., 2008). Percival et al. (2001) compared collection of feces by manual stripping and dissection in Atlantic salmon, Salmo salar, and reported significantly lower protein digestibility from fecal samples collected by dissection, suggesting contamination of the feces with undigested food. In the current study, we

protein (P = .034). The apparent amino acid digestibility coefficients of the test ingredients in rainbow trout are presented in Table 7. Although no significant differences in ADCs for crude protein were observed with the protease treatments, amino acid digestibility results were ingredient specific. For all ingredients except cottonseed meal, canola meal, sunflower meal, meat and bone meal-2, distiller's dried grain and feather meal, there were significant improvements in apparent digestibility of different amino acids with protease treatment (P < .1). Soybean meal showed the greatest improvement in overall amino acid apparent digestibility, with protease treatment increasing ADC for 13 of the 18 amino acids analyzed (cysteine, P = .046; histidine, P = .044; isoleucine, P = .020; lysine, 0.016; methionine, P = .086; phenylalanine, P = .076; threonine, P = .015; valine, P = .034; aspartic acid, P = .001; glutamic acid, P = .056; glycine, P = .068; proline, P = .022; serine, 0.021).

4. Discussion Not surprisingly, our results show a consistent trend toward higher dry matter digestibility among animal protein sources and lesser for several plant ingredients in rainbow trout without protease supplementation. Dry matter ADCs ranged from 33.1% (sunflower meal) to 70.1% (peanut meal) for plant ingredients fed to rainbow trout, generally lower than values for animal protein ingredients which ranged from 51.0% (meat and bone meal-1) to 86.6% (sardine and menhaden meal. The low dry matter ADCs generally indicate a higher quantity of indigestible materials in the feedstuff (Li et al., 2013). Likewise, several previous studies have reported lower digestibility for plant protein 4

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collected samples through manual stripping from the ventral fin area to the anus which resulted in lower ADC values compared to previous studies, likely due to sloughed cellular components. Source of ingredient and associated differences in processing methods also affect ADC results. This can be seen in the present study where we compared the same feedstuffs (2 feather meals, 2 poultry by-product meals, and 2 meat and bone meals) acquired from different sources. The supplementation of protease has been considered to improve nutrient digestibility in low fishmeal diets (Dalsgaard et al., 2012; Drew et al., 2005; Kumar et al., 2009; Lin et al., 2007; Shi et al., 2016; Yigit et al., 2018; Li et al., 2019). Overall, supplementation of 175 mg of the protease complex/kg of feed resulted in ADC increases for dry matter, energy, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, alanine, aspartic acid and glutamic acid associated with a variety of protein feedstuffs. Particularly, protease supplementation increased ADCs for dry matter of soybean meal and one of the feather meals, ADCs for protein of one of the feather meals, one of the poultry by-product meals and a Methanococcus maripaludis single cell protein. The most common improvements in ingredient digestibility with the addition of protease were for individual amino acids. Among the 17 ingredients evaluated, 12 showed improved apparent digestibility of at least one amino acid. However, for the other six ingredients (cottonseed meal, canola meal, sunflower meal, meat bone meal-2, DDGS, and feather meal) protease had no effect on ADC for any of the nutrients, dry matter, or energy digestibility, with the exception of improved dry matter digestibility of feather meal-2. The greatest improvements in apparent digestibility were observed when the protease complex was added to soybean meal. Apparent digestibility of dry matter and several individual amino acids (cysteine, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, valine, aspartic acid, glutamic acid, glycine, proline, serine) were significantly improved. In trout diets high in soybean meal, the first limiting amino acids are typically methionine, lysine, and threonine and such diets require synthetic amino acid supplementation to meet the dietary requirements (NRC, 2011). Our observations showed that by adding protease, the required levels of supplementation of these essential amino acids could be reduced. Furthermore, the overall improvement in total amino acid digestibility may improve amino acid metabolic efficiency. These results may be attributed to not only the protease activity of the serine endopeptidase complex on protein hydrolysis in soybean meal but also the breakdown of protein components present in ANFs such as trypsin inhibitors and antigenic soybean proteins (Ao et al., 2010; Caine et al., 1998). Previous studies have mixed results of protease supplementation in rainbow trout feed (Dalsgaard et al., 2012; Drew et al., 2005; Farhangi and Carter, 2007; Ogunkoya et al., 2006) and other aquaculture species, including common carp (Leng et al., 2008), tilapia (Li et al., 2016), and white shrimp (Li et al., 2016). Differences in feed processing and heatstability of the protease used in each study may have influenced the outcomes observed. Efficacy and practical utilization of proteases and other enzymes in commercial extruded feeds primarily depends upon their stability under various processing conditions, such as heat, pressure and moisture (Shi et al., 2016). Since cooking-extrusion is the preferred pelleting method for fish feeds, the fate of feed ingredients, micronutrients and enzymes depend on their stability under extrusion conditions. The protease used in the current experiment is an alkaline serine endopeptidase with an optimal pH of 8.5 (JEFO Nutrition, Inc.), and has been demonstrated to have better heat-stability, maintaining a portion of its activity after extrusion at temperatures as high as 130° (Leng et al., 2008; Li et al., 2016). The high heat stability coupled with the results from the current experiment demonstrate the potential of this protease to improve apparent digestibility of several ingredients, most notably soybean meal, in extruded feeds fed to rainbow trout. Unpublished results from our laboratory also suggest a higher inclusion of protease supplement can further improve amino acid ADCs for ingredients such as DDGS and cottonseed meal. As the experiment

Table 6 Apparent digestibility coefficients (%) of dry matter, crude protein and energy for different ingredients fed to rainbow trout without and with the protease complex (+P). P < .1 is indicated with bold font (n = 2 tanks per diet). Diets

Dry matter

Crude protein

Gross energy

Feather meal-1 Feather meal-1 + P Pooled SE P value Feather meal-2 Feather meal-2 + P Pooled SE P value Poultry by-product-1 Poultry by-product-1 + P Pooled SE P value Poultry by-product-2 Poultry by-product-2 + P Pooled SE P value Meat and bone meal-1 Meat and bone meal-1 + P Pooled SE P value Meat and bone meal-2 Meat and bone meal-2 + P Pooled SE P value Sardine meal Sardine meal +P Pooled SE P value Menhaden meal Menhaden meal +P Pooled SE P value Black soldier fly larvae Black soldier fly larvae +P Pooled SE P value Single cell protein Single cell protein +P Pooled SE P value Soybean meal Soybean meal +P Pooled SE P value Canola meal Canola meal +P Pooled SE P value Distiller dried grains with soluble Distiller dried grains with soluble +P Pooled SE P value Cottonseed meal Cottonseed meal +P Pooled SE P value Peanut meal Peanut meal +P Pooled SE P value Sunflower meal Sunflower meal +P Pooled SE P value Algae meal Algae meal +P Pooled SE P value

67.6 61.2 2.18 0.272 52.4 59.7 1.82 0.003 59.1 61.5 2.29 0.737 61.9 61.8 1.89 0.989 51.0 55.6 1.55 0.360 63.5 63.5 0.75 0.991 86.6 85.1 1.54 0.751 86.6 86.8 0.83 0.959 66.9 66.7 1.74 0.969 81.3 84.7 1.43 0.402 54.5 63.9 2.57 0.082 42.0 51.9 3.17 0.216 52.0 48.5 1.37 0.368 38.2 38.8 1.39 0.888 70.1 68.9 1.17 0.739 33.1 32.4 0.46 0.633 66.3 71.2 1.57 0.221

70.6 65.5 1.61 0.214 64.1 65.8 1.08 0.606 68.5 69.1 1.19 0.868 70.5 69.9 1.07 0.876 68.4 71.2 0.89 0.221 55.4 58.5 1.03 0.250 84.5 83.3 0.40 0.256 83.6 83.3 0.37 0.803 68.8 70.0 0.44 0.294 81.0 83.4 0.71 0.154 83.8 86.7 1.61 0.558 76.8 81.6 1.53 0.210 70.0 68.1 1.08 0.575 78.9 77.2 0.89 0.531 81.9 81.5 0.26 0.618 73.1 74.5 0.78 0.544 75.8 78.4 1.09 0.393

69.5 65.9 1.89 0.527 59.1 64.9 1.59 0.095 70.1 71.5 1.33 0.742 74.4 80.5 1.60 0.041 72.3 73.9 1.65 0.752 58.1 58.2 0.71 0.976 87.0 91.3 1.52 0.309 90.2 87.6 1.00 0.344 61.8 63.6 0.64 0.295 78.8 83.4 1.19 0.034 63.2 73.2 2.81 0.103 54.8 62.6 2.26 0.136 60.6 59.9 0.90 0.808 52.9 57.1 1.67 0.379 76.0 74.4 1.17 0.665 32.9 36.7 1.40 0.332 70.1 72.0 1.56 0.689

5

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Table 7 Apparent digestibility coefficientsa (%) of amino acids for different ingredients fed to rainbow trout without and with the protease complex (+P). P < .1 is indicated with bold font (n = 2 tanks per diet). Diet

Feather meal-1 Feather meal-1 + P Pooled SE P value Feather meal-2 Feather meal-2 + P Pooled SE P value Poultry by-product-1 Poultry by-product-1 + P Pooled SE P value Poultry by-product-2 Poultry by-product-2 + P Pooled SE P value Meat and bone meal-1 Meat and bone meal-1 + P Pooled SE P value Meat and bone meal-2 Meat and bone meal-2 + P Pooled SE P value Sardine meal Sardine meal +P Pooled SE P value Menhaden meal Menhaden meal +P Pooled SE P value Black soldier fly larvae Black soldier fly larvae +P Pooled SE P value Single cell protein Single cell protein +P Pooled SE P value Soybean meal Soybean meal +P Pooled SE P value Canola meal Canola meal +P Pooled SE P value Distiller dried grains with soluble Distiller dried grains with soluble +P Pooled SE P value Cottonseed meal Cottonseed meal +P Pooled SE P value Peanut meal Peanut meal +P Pooled SE P value Sunflower meal Sunflower meal +P Pooled SE P value Algae meal Algae meal +P Pooled SE P value a

Essential and semi-essential amino acids

Non-essential amino acids

Arg

Cys

His

Iso

Leu

Lys

Met

Phe

Thr

Trp

Tyr

Val

Ala

Asp

Glu

Gly

Pro

Ser

82.1 80.7 1.12 0.681 80.3 80.0 0.54 0.861 78.3 81.3 0.85 0.111 78.6 78.5 0.40 0.910 78.2 79.5 0.35 0.043 76.6 79.2 1.29 0.489 92.5 94.7 0.62 0.105 92.3 93.2 0.89 0.722 81.3 77.0 1.46 0.260 91.0 92.9 1.03 0.532 92.2 93.9 0.51 0.151 92.5 94.9 1.00 0.386 84.9

49.0 40.9 2.89 0.303 55.7 52.4 1.09 0.233 7.5 15.0 2.00 0.063 52.6 43.1 2.71 0.119 43.1 48.7 3.74 0.624 66.6 72.1 1.70 0.202 87.6 87.2 1.44 0.942 87.7 85.4 1.24 0.541 47.5 69.8 6.04 0.076 74.1 81.3 3.15 0.426 61.5 72.0 2.74 0.046 72.4 77.2 2.33 0.483 57.6

71.7 69.8 1.43 0.680 50.9 52.3 1.17 0.704 75.1 80.7 1.68 0.177 70.1 71.4 0.90 0.637 74.0 77.2 1.33 0.389 53.6 58.9 2.69 0.499 90.4 88.0 1.65 0.638 90.3 84.6 1.86 0.235 64.6 78.7 3.75 0.062 85.2 84.2 1.77 0.854 84.6 89.0 1.15 0.044 86.2 88.7 1.30 0.529 72.3

74.4 74.9 2.12 0.946 74.1 74.0 1.09 0.981 66.4 73.0 2.11 0.207 68.9 68.4 1.04 0.873 73.9 78.0 1.48 0.316 69.2 70.8 1.30 0.693 91.7 91.4 1.51 0.951 93.4 90.7 0.85 0.202 72.5 82.7 2.99 0.151 86.9 87.4 2.15 0.934 80.7 85.8 1.29 0.020 75.5 82.3 2.28 0.248 63.1

63.6 64.2 2.97 0.955 66.4 67.0 1.23 0.867 64.4 70.8 2.35 0.321 68.7 67.4 1.42 0.772 72.6 76.6 1.91 0.484 61.8 63.4 2.03 0.802 92.9 90.9 1.01 0.513 95.3 90.2 1.39 0.071 75.4 80.8 2.05 0.342 85.4 85.2 1.84 0.975 78.3 83.9 1.55 0.102 79.0 85.2 2.21 0.293 74.9

74.0 72.5 1.41 0.738 58.0 59.8 0.80 0.436 81.2 85.7 1.21 0.072 72.7 71.8 0.67 0.648 76.3 78.5 0.99 0.443 65.2 66.6 1.52 0.767 93.9 94.7 0.60 0.677 95.7 95.3 0.13 0.277 73.6 76.7 1.81 0.576 91.6 92.1 1.23 0.900 88.1 92.2 1.06 0.016 86.6 90.4 1.25 0.239 54.0

68.3 67.5 1.87 0.900 56.5 59.6 1.46 0.465 80.5 85.0 1.41 0.194 75.2 74.5 0.80 0.788 77.6 82.1 1.47 0.238 42.2 45.9 1.22 0.246 91.1 95.3 1.30 0.196 90.0 93.2 0.91 0.142 68.3 71.2 6.46 0.886 87.6 92.6 1.29 0.038 82.0 89.5 2.07 0.086 87.5 90.6 1.05 0.274 77.4

70.5 70.2 2.21 0.966 71.2 71.7 0.91 0.865 65.0 70.9 1.83 0.195 69.5 69.9 1.01 0.892 73.3 79.7 1.95 0.174 68.6 67.6 1.79 0.862 89.7 91.3 0.89 0.532 91.1 89.5 0.49 0.192 73.9 78.8 1.85 0.346 85.2 84.9 1.72 0.966 84.3 87.9 0.98 0.076 81.9 85.8 1.42 0.319 72.9

63.3 61.4 1.78 0.737 63.1 63.9 0.67 0.691 64.1 68.3 1.50 0.306 65.6 63.1 1.26 0.500 67.1 70.7 1.28 0.297 60.5 65.2 2.40 0.508 90.8 90.6 1.00 0.962 92.5 89.3 0.98 0.175 63.5 73.7 2.98 0.140 85.9 86.3 1.62 0.936 74.0 80.5 1.65 0.015 71.1 78.2 2.70 0.340 56.5

95.4 95.7 0.89 0.920 91.5 91.6 1.11 0.988 88.4 95.0 1.69 0.022 83.2 87.1 1.00 0.043 87.1 99.4 3.30 0.068 83.2 100 – – 61.8 37.8 20.0 0.700 77.6 48.7 12.5 0.420 0 0 – – 55.0 32.6 14.3 0.607 88.4 85.7 0.88 0.245 87.8 87.4 1.91 0.946 34.3

70.2 69.3 1.91 0.880 71.8 71.6 0.86 0.937 66.5 73.0 1.91 0.156 74.5 71.5 1.17 0.369 76.5 81.2 1.57 0.263 60.5 59.5 0.61 0.608 96.6 92.7 1.10 0.108 97.2 90.5 1.78 0.066 92.7 74.3 5.04 0.087 88.3 90.0 1.20 0.637 87.7 89.4 0.60 0.285 85.8 87.4 1.41 0.722 79.2

72.3 72.4 2.08 0.984 72.5 72.0 0.91 0.839 57.5 65.1 2.23 0.148 66.1 64.3 1.40 0.684 66.5 73.5 2.54 0.313 49.5 52.5 1.87 0.589 94.4 80.8 3.73 0.088 95.7 80.4 3.88 0.010 78.3 64.4 3.88 0.103 71.3 71.8 2.49 0.944 78.1 84.7 1.73 0.034 72.8 79.8 2.34 0.253 61.7

68.9 68.1 2.11 0.907 64.7 66.5 0.96 0.527 74.0 76.6 1.53 0.566 73.2 73.4 0.63 0.915 72.9 75.0 0.61 0.142 69.8 73.4 1.97 0.545 92.6 92.6 0.81 0.982 91.1 90.0 0.78 0.667 75.7 81.9 1.61 0.038 88.9 90.5 1.15 0.654 80.0 85.9 1.77 0.171 80.0 88.2 2.83 0.279 80.4

46.4 42.5 1.97 0.499 46.4 49.1 0.75 0.075 56.2 63.7 2.18 0.142 57.7 53.7 1.31 0.227 57.9 64.5 1.79 0.075 44.0 52.2 3.71 0.447 83.2 86.6 1.06 0.192 86.3 85.1 0.87 0.645 57.2 61.8 1.70 0.325 77.2 76.7 2.32 0.946 79.9 85.1 1.32 0.001 73.3 78.8 2.82 0.510 49.5

58.0 57.5 2.36 0.946 58.8 60.9 1.13 0.534 70.8 75.6 2.10 0.431 69.2 67.3 1.32 0.633 69.6 73.7 1.54 0.327 62.6 65.5 2.32 0.679 91.2 91.2 0.87 0.989 92.7 91.4 0.54 0.371 73.8 84.1 2.63 0.026 87.6 87.9 1.42 0.953 80.4 87.9 1.99 0.056 83.6 86.7 1.10 0.294 65.5

74.0 71.5 1.30 0.508 71.3 71.5 0.55 0.945 69.0 72.1 1.14 0.317 73.5 71.6 0.96 0.483 67.4 71.4 1.02 0.026 68.4 73.2 2.01 0.391 85.5 88.4 1.05 0.320 78.1 83.4 2.81 0.533 55.2 54.9 3.82 0.979 84.5 85.4 1.04 0.781 77.5 87.6 2.69 0.068 83.9 90.7 2.84 0.401 60.4

59.1 56.3 2.45 0.714 64.2 64.2 0.95 0.983 61.8 66.8 1.60 0.224 70.8 69.6 1.12 0.734 65.4 72.4 1.78 0.014 16.7 52.8 12.4 0.271 96.1 57.7 10.48 0.084 88.5 71.4 5.76 0.255 82.3 63.4 4.93 0.044 69.1 82.5 3.37 0.012 69.8 86.4 4.23 0.022 69.8 83.8 4.29 0.186 66.3

68.3 65.0 1.48 0.455 72.2 73.4 0.40 0.240 52.9 55.2 1.13 0.481 64.0 60.9 1.20 0.351 65.1 63.8 0.51 0.339 61.5 69.1 2.83 0.330 86.5 85.1 1.14 0.687 87.5 83.4 1.27 0.200 59.0 68.6 2.54 0.047 79.2 81.6 1.56 0.613 81.0 85.3 1.09 0.021 72.8 80.9 3.02 0.328 74.3

83.8

58.9

71.8

63.3

76.1

56.5

77.0

72.3

54.6

40.3

74.2

60.8

78.9

48.3

68.4

58.4

74.4

69.5

0.95 0.711 91.8 91.3 0.23 0.487 96.9 96.1 0.49 0.565 97.9 95.8 0.69 0.243 88.0 88.7 0.55 0.690

0.37 0.096 72.0 72.9 1.15 0.815 87.8 85.4 1.04 0.429 67.9 70.2 2.54 0.779 84.8 89.8 1.49 0.155

1.27 0.903 79.5 79.6 0.25 0.924 100 100 – – 78.7 78.6 1.34 0.967 80.9 83.8 1.02 0.279

2.57 0.983 69.6 70.4 0.36 0.427 100 100 – – 100 100 – – 89.1 90.4 0.50 0.377

2.40 0.879 69.7 70.9 0.38 0.264 89.4 91.2 0.76 0.410 87.2 89.8 1.57 0.578 75.4 78.4 0.87 0.124

1.90 0.673 68.7 68.4 1.26 0.956 81.6 83.8 1.79 0.697 89.1 91.1 1.18 0.567 86.8 87.9 0.67 0.598

2.51 0.961 74.2 72.4 0.55 0.155 98.5 100 4.14 0.238 97.5 97.8 0.33 0.773 82.8 94.9 3.70 0.182

2.17 0.924 80.7 81.0 0.36 0.817 94.4 94.9 0.56 0.755 95.6 94.6 0.87 0.714 82.7 86.2 0.90 0.045

1.99 0.757 70.0 67.9 0.85 0.380 81.2 82.7 1.97 0.816 80.0 78.0 2.05 0.757 76.9 80.8 1.05 0.070

6.06 0.753 88.7 82.6 1.80 0.147 95.7 77.7 7.77 0.422 66.9 78.4 5.06 0.432 96.3 79.4 6.24 0.321

1.77 0.305 82.7 82.4 0.36 0.822 89.1 74.8 4.20 0.148 58.6 70.3 3.80 0.228 74.0 74.0 0.18 0.932

2.24 0.904 73.4 71.7 0.49 0.126 71.1 72.0 1.84 0.882 59.1 62.5 2.40 0.637 55.3 59.6 1.22 0.120

1.34 0.719 74.5 74.1 1.38 0.934 89.9 91.7 1.12 0.600 91.8 90.0 0.84 0.481 78.8 81.5 0.80 0.172

2.12 0.854 75.8 74.6 0.39 0.250 80.2 82.5 1.61 0.645 76.9 72.3 2.75 0.584 71.9 77.3 1.48 0.077

3.45 0.790 83.6 83.3 0.23 0.650 90.8 92.3 0.96 0.598 90.2 88.5 1.28 0.658 82.0 85.5 0.96 0.089

3.18 0.839 79.1 77.8 1.17 0.726 75.9 78.0 1.69 0.687 77.6 73.3 2.00 0.466 75.5 79.9 1.15 0.053

3.25 0.377 73.9 76.0 1.80 0.707 85.0 93.6 2.19 0.011 84.4 93.1 3.74 0.417 72.1 59.4 6.69 0.527

2.04 0.412 79.9 74.7 1.79 0.266 84.0 85.2 1.34 0.778 83.6 81.5 1.15 0.545 74.3 78.7 1.20 0.076

Negative values were rounded up to zero and values above 100 were rounded down to 100. Many factors can influence the apparent digestibility coefficients for 6

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a nutrient and could lead to negative ADC values or values greater than 100. Nutrients that are very low in an ingredient or have a true digestibility near to either threshold can yield values less than 0 and greater than 100.

reported here was limited by the treatment level of protease enzyme, higher inclusion levels of the protease complex should be explored to further increase ADCs for plant feedstuffs.

Farhangi, M., Carter, C.G., 2007. Effect of enzyme supplementation to dehulled lupinbased diets on growth, feed efficiency, nutrient digestibility and carcass composition of rainbow trout, Oncorhynchus mykiss (Walbaum). Aquac. Res. 38, 1274–1282. https://doi.org/10.1111/j.1365-2109.2007.01789.x. Gatlin, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W., Herman, E., Hu, G., Krogdahl, Å., Nelson, R., Overturf, K., Rust, M., Sealey, W., Skonberg, D., Souza, E.J., Stone, D., Wilson, R., Wurtele, E., 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquac. Res. 38, 551–579. https://doi.org/10.1111/j.1365-2109.2007.01704.x. Gaylord, T.G., Barrows, F.T., 2009. Multiple amino acid supplementations to reduce dietary protein in plant-based rainbow trout, Oncorhynchus mykiss, feeds. Aquaculture 287, 180–184. https://doi.org/10.1016/j.aquaculture.2008.10.037. Gomes, E.F., Rema, P., Kaushik, S.J., 1995. Replacement of fish meal by plant proteins in the diet of rainbow trout (Oncorhynchus mykiss): digestibility and growth performance. Aquaculture 130, 177–186. https://doi.org/10.1016/0044-8486(94) 00211-6. Hardy, R.W., 2010. Utilization of plant proteins in fish diets: effects of global demand and supplies of fishmeal. Aquac. Res. 41, 770–776. https://doi.org/10.1111/j.1365-2109. 2009.02349.x. Kumar, S., Sahu, N.P., Pal, A.K., Sagar, V., Sinha, A.K., Baruah, K., 2009. Modulation of key metabolic enzyme of Labeo rohita (Hamilton) juvenile: effect of dietary starch type, protein level and exogenous α-amylase in the diet. Fish Physiol. Biochem. 35, 301–315. https://doi.org/10.1007/s10695-008-9213-6. Leng, X.J., Liu, D.Y., Li, X.Q., Lu, Y.H., 2008. Effects of adding protease AG on growth and digestive protease activities of common carp (Cyprinus carpio) fingerling. Chin. J. Ani. Nutr. 20, 3. Li, M.H., Oberle, D.F., Lucas, P.M., 2013. Apparent digestibility of alternative plantprotein feedstuffs for channel catfish, Ictalurus punctatus (Rafinesque). Aquac. Res. 44, 282–288. https://doi.org/10.1111/j.1365-2109.2011.03035.x. Li, X.Q., Chai, X.Q., Liu, D.Y., Chowdhury, M.A.K., Leng, X.J., 2016. Effects of temperature and feed processing on protease activity and dietary protease on growths of white shrimp, Litopenaeus vannamei, and tilapia, Oreochromis niloticus × O. aureus. Aquac. Nutr. 22, 1283–1292. https://doi.org/10.1111/anu.12330. Li, X.Q., Zhang, X.Q., Chowdhury, M.A.K., Zhang, Y., Leng, X.J., 2019. Dietary phytase and protease improved growth and nutrient utilization in tilapia (Oreochromis niloticus × Oreochromis aureus) fed low phosphorus and fishmeal-free diets. Aquac. Nutr. 25, 46–55. https://doi.org/10.1111/anu.12828. Lin, S., Mai, K., Tan, B., 2007. Effects of exogenous enzyme supplementation in diets on growth and feed utilization in tilapia, Oreochromis niloticus x O. aureus. Aquac. Res. 38, 1645–1653. https://doi.org/10.1111/j.1365-2109.2007.01825.x. Luo, Z., Tan, X. ying, Chen, Y. dong, Wang, W. min, Zhou, G., 2008. Apparent digestibility coefficients of selected feed ingredients for Chinese mitten crab Eriocheir sinensis. Aquaculture 285, 141–145. https://doi.org/10.1016/j.aquaculture.2008.08.004. Mahmood, T., Mirza, M.A., Nawaz, H., Shahid, M., Athar, M., Hussain, M., 2017. Effect of supplementing exogenous protease in low protein poultry by-product meal based diets on growth performance and nutrient digestibility in broilers. Anim. Feed Sci. Technol. 228, 23–31. https://doi.org/10.1016/j.anifeedsci.2017.01.012. Mokrani, A., Liang, H., Ji, K., Kasiya, H.C., Yang, Q., Ge, X., Ren, M., Zhu, X., Pan, L., Sun, A., 2019. Essential amino acids supplementation to practical diets affects growth, feed utilization and glucose metabolism-related signalling molecules of blunt snout bream (Megalobrama amblycephala). Aquac. Res. 50, 557–565. https://doi.org/10. 1111/are.13928. Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin, D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. Proc. Natl. Acad. Sci. 106, 15103–15110. NRC (National Research Council), 2011. Nutrient Requirements of Fish and Shrimp. National Academy Press, Washington, D.C. Ogunkoya, A.E., Page, G.I., Adewolu, M.A., Bureau, D.P., 2006. Dietary incorporation of soybean meal and exogenous enzyme cocktail can affect physical characteristics of faecal material egested by rainbow trout (Oncorhynchus mykiss). Aquaculture 254, 466–475. https://doi.org/10.1016/j.aquaculture.2005.10.032. Percival, S.B., Lee, P.S., Carter, C.G., 2001. Validation of a technique for determining apparent digestibility in large (up to 5 kg) Atlantic salmon (Salmo salar L.) in seacages. Aquaculture 201, 315–327. https://doi.org/10.1016/S0044-8486(01) 00506-3. Seong, M., Lee, Seunghyung, Lee, Seunghan, Song, Y., Bae, J., Chang, K., Bai, S.C., 2018. The effects of different levels of dietary fermented plant-based protein concentrate on growth, hematology and non-specific immune responses in juvenile olive flounder, Paralichthys olivaceus. Aquaculture 483, 196–202. https://doi.org/10.1016/j. aquaculture.2017.10.023. Shi, Z., Li, X.Q., Chowdhury, M.A.K., Chen, J.N., Leng, X.J., 2016. Effects of protease supplementation in low fish meal pelleted and extruded diets on growth, nutrient retention and digestibility of gibel carp, Carassius auratus gibelio. Aquaculture 460, 37–44. https://doi.org/10.1016/j.aquaculture.2016.03.049. Sookying, D., Davis, D.A., 2011. Pond production of Pacific white shrimp (Litopenaeus vannamei) fed high levels of soybean meal in various combinations. Aquaculture 319, 141–149. https://doi.org/10.1016/j.aquaculture.2011.06.049. Stone, D.A.J., Gaylord, T.G., Johansen, K.A., Overturf, K., Sealey, W.M., Hardy, R.W., 2008. Evaluation of the effects of repeated fecal collection by manual stripping on the plasma cortisol levels, TNF-α gene expression, and digestibility and availability of nutrients from hydrolyzed poultry and egg meal by rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture 275, 250–259. https://doi.org/10.1016/j.

5. Conclusion The study examined the digestibility of a wide range of commonly used protein ingredients, with or without protease supplementation. Although no improvements in crude protein digestibility were observed, supplementation with the protease complex (175 mg protease complex/kg of diet) resulted in ingredient-specific ADC increases for dry matter, energy, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, alanine, aspartic acid and glutamic acid, with most ingredients having improved digestibility of at least one amino acid. Protease supplementation had the most profound improvement on ADCs for soybean meal, including dry matter and the majority of individual amino acids. Overall, this research demonstrates the benefit of the evaluated protease supplementation on the digestibility of feed ingredients commonly used in rainbow trout and other commercially cultured fish feeds, although the degree of improvement in digestibility varied among ingredients. Further studies with this protease complex are recommended to evaluate its effects at higher inclusion levels and in other species of cultured fishes. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This research was supported by Jefo Nutrition Inc., Canada. The authors would like to thank Eli Gough and Carol Hoffman at HFCES for their assistance during the feeding trial and analysis. References Angel, C.R., Saylor, W., Vieira, S.L., Ward, N., 2011. Metabolism and nutrition: effects of a monocomponent protease on performance and protein utilization in 7- to 22-day-old broiler chickens. Poult. Sci. 90, 2281–2286. https://doi.org/10.3382/ps.2011-01482. Ao, T., Cantor, A.H., Pescatore, A.J., Pierce, J.L., Dawson, K.A., 2010. Effects of citric acid, alpha-galactosidase and protease inclusion on in vitro nutrient release from soybean meal and trypsin inhibitor content in raw whole soybeans. Anim. Feed Sci. Technol. 162, 58–65. https://doi.org/10.1016/j.anifeedsci.2010.08.014. AOAC (Association of Official Analytical Chemists), 2000. Chapter 4. In: Cunniff, P. (Ed.), Official Methods of Analysis of the Association of Official Analytical Chemists, 17th edition. Association of Official Analytical Chemists, Inc, Arlington, VA Pages 46. Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 13, 265–272. https://doi.org/10.1016/0044-8486(78)90008-X. Bureau, D.P., Harris, A.M., Cho, C.Y., 1999. Apparent digestibility of rendered animal protein ingredients for rainbow trout (Oncorhynchus mykiss). Aquaculture 180, 345–358. https://doi.org/10.1016/S0044-8486(99)00210-0. Caine, W.R., Verstegen, M.W.A., Sauer, W.C., Tamminga, S., Schulze, H., 1998. Effect of protease treatment of soybean meal on content of total soluble matter and crude protein and level of soybean trypsin inhibitors. Anim. Feed Sci. Technol. 71, 177–183. https://doi.org/10.1016/S0377-8401(97)00139-9. Cheng, Z.J., Hardy, R.W., 2003. Effects of extrusion processing of feed ingredients on apparent digestibility coefficients of nutrients for rainbow trout (Oncorhynchus mykiss). Aquac. Nutr. 9, 77–83. https://doi.org/10.1046/j.1365-2095.2003.00226.x. Dalsgaard, J., Verlhac, V., Hjermitslev, N.H., Ekmann, K.S., Fischer, M., Klausen, M., Pedersen, P.B., 2012. Effects of exogenous enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed diets with high inclusion of plant-based protein. Anim. Feed Sci. Technol. 171, 181–191. https://doi.org/10.1016/j. anifeedsci.2011.10.005. Drew, M.D., Racz, V.J., Gauthier, R., Thiessen, D.L., 2005. Effect of adding protease to coextruded flax:pea or canola:pea products on nutrient digestibility and growth performance of rainbow trout (Oncorhynchus mykiss). Anim. Feed Sci. Technol. 119, 117–128. https://doi.org/10.1016/j.anifeedsci.2004.10.010.

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Aquaculture 524 (2020) 735270

S. Lee, et al.

aquaculture.2010.09.021. Yao, W., Li, X., Chowdhury, M.A.K., Wang, J., Leng, X., 2019. Dietary protease, carbohydrase and micro-encapsulated organic acid salts individually or in combination improved growth, feed utilization and intestinal histology of Pacific white shrimp. Aquaculture 503, 88–95. https://doi.org/10.1016/j.aquaculture.2018.12.064. Yigit, N.O., Koca, S.B., Didinen, B.I., Diler, I., 2018. Effect of protease and phytase supplementation on growth performance and nutrient digestibility of rainbow trout (Oncorhynchus mykiss, walbaum) fed soybean meal-based diets. J. Appl. Anim. Res. 46, 29–32. https://doi.org/10.1080/09712119.2016.1256292. Zhou, Q.C., Yue, Y.R., 2012. Apparent digestibility coefficients of selected feed ingredients for juvenile hybrid tilapia, Oreochromis niloticus×Oreochromis aureus. Aquac. Res. 43, 806–814. https://doi.org/10.1111/j.1365-2109.2011.02892.x.

aquaculture.2008.01.003. Vens-Cappell, B., 1985. Methodical studies on digestion in trout. 1. Reliability of digestion coefficients in relation to methods for faeces collection. Aquac. Eng. 4, 33–49. https://doi.org/10.1016/0144-8609(85)90004-4. Walk, C.L., Pirgozliev, V., Juntunen, K., Paloheimo, M., Ledoux, D.R., 2018. Evaluation of novel protease enzymes on growth performance and apparent ileal digestibility of amino acids in poultry: enzyme screening. Poult. Sci. 97, 2123–2138. https://doi. org/10.3382/ps/pey080. Yamamoto, T., Iwashita, Y., Matsunari, H., Sugita, T., Furuita, H., Akimoto, A., Okamatsu, K., Suzuki, N., 2010. Influence of fermentation conditions for soybean meal in a nonfish meal diet on the growth performance and physiological condition of rainbow trout Oncorhynchus mykiss. Aquaculture 309, 173–180. https://doi.org/10.1016/j.

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