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Aug 15, 2016 - Abstract. Effects of graded dietary levels of fish meal (FM) replacement with meat and bone meal (MBM) with garlic powder (G) or without garlic ...
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Received: 24 January 2016    Accepted: 15 August 2016 DOI: 10.1111/anu.12491

ORIGINAL ARTICLE

Effects of fish meal replacement with meat and bone meal using garlic (Allium sativum) powder on growth, feeding, digestive enzymes and apparent digestibility of nutrients and fatty acids in juvenile rainbow trout (Oncorhynchus mykiss Walbaum, 1792) M. Esmaeili1 | A. Abedian Kenari1 | A. N. Rombenso2 1 Department of Aquaculture, Faculty of Marine Science, Tarbiat Modares University, Noor, Mazandaran, Iran 2

Abstract Effects of graded dietary levels of fish meal (FM) replacement with meat and bone

Department of Zoology, Center for Fisheries, Aquaculture and Aquatic Sciences, Southern Illinois University Carbondale, Carbondale, IL, USA

meal (MBM) with garlic powder (G) or without garlic powder (WG) garlic powder were

Correspondence Abdolmohammad Abedian Kenari, Department of Fisheries, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Noor, Mazandaran, Iran. Emails: [email protected], [email protected]

total of eight dietary treatments were evaluated: control with garlic powder

Present address Artur Nishioka Rombenso, Nutrition Laboratory, Institute of Oceanography, Autonomous University of Baja California, Ensenada, Mexico Funding information Tarbiat Modares University (Noor, Iran)

investigated on growth, digestive enzymes, apparent digestibility, body composition and fatty acid profile of juvenile rainbow trout (Oncorynchus mykiss; 8.26 ± 1.10 g). A (0 MBM+G), 250 MBM+G, 450 MBM+G, and 650 MBM+G (g MBM/kg diet; treatments with garlic powder; G treatments), control without garlic powder (0 MBM), 250 MBM, 450 MBM and 650 MBM (g MBM/kg diet; treatments without garlic powder; WG treatments). Our results demonstrated that increased dietary content of MBM impaired growth and production performance, body composition, digestive enzyme activity, tissue fatty acid profile and overall digestibility. Addition of garlic powder was able to correct fish performance, body composition, enzyme activity and digestibility to some extent. Dietary supply of garlic resulted in increased digestive enzyme activity improving fish performance. The current results indicated that fish fed 650 g MBM/kg feed exhibited impaired growth that could be attributed to reduce digestibility of protein, fat, energy and dry matter, PUFAs and n-­3 LC-­PUFAs in muscle. We suggest that 450 g FM per kilo feed can be successfully replaced with MBM supplemented with 30 g garlic powder per kilo feed without impairing fish performance and physiology, however distorting tissue fatty acid composition resulting in lesser levels of n-­3 LC-­ PUFAs compared to the 0 MBM+G diet. KEYWORDS

digestive enzymes, fish meal replacement, meat and bone meal, nutrient digestibility, protein utilization

1 |  INTRODUCTION

fish consumption. Presently, 181 countries culture more than 600 fish species (Mathiesen, 2012). Additionally, more than 70% of aquacul-

According to FAO, in the next few years the fisheries and aquacul-

ture production is based on feed input, which typically accounts for

ture production will level off (Mathiesen, 2012). Aquaculture is the

50%–60% of the total operating costs. To promote sustainable aqua-

fastest growing food industry, and it is responsible for half of total

culture practices, reduction in production costs is an ongoing priority.

Aquaculture Nutrition 2017; 1–10 wileyonlinelibrary.com/journal/anu

© 2017 John Wiley & Sons Ltd  |  1

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ESMAEILI et al.

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Finding a suitable replacement for expensive and finite ingredients

Rainbow trout is one of the most important aquaculture species

such as fish meal (FM) is one way to minimize costs (Webster, Tiu,

worldwide, in particular in Europe, North America, Japan and Australia

& Tidwell, 1997). FM and fish oil consist the main supply of secure

(FAO, 2014). According to Iran Fishery Organization announcement

protein and fat, especially in carnivorous fish feeds, which normally

in 2012, fish production was 131 thousand tones with rainbow trout

accounts for 30%–50% of the diet (Tidwell, Coyle, Bright, & Yasharian,

being the highest consumed species nationally. As this species is car-

2005). According to FAO report in 2012, FM and fish oil consumed in

nivorous, it exhibits higher protein requirement, and more aggres-

aquaculture were, respectively, 68% and 89% of the global production

sive and/or complete FM replacement rates with alternative protein

(Mathiesen, 2012). Hence, many studies worldwide are focused on

sources such as MBM are known to impair fish performance and phys-

FM replacement with alternative protein sources. FM is an important

iology. The searches for alternatives without these negative effects are

ingredient in rainbow trout diets. As a carnivorous fish, this species

valuable to the industry. Accordingly, to assess whether dietary garlic

require a nutrient-­dense diet and it is known to well utilize alterna-

exhibits the above-­mentioned benefits and the effect of FM replace-

tive animal protein sources such as MBM (meat and bone meal), PBM

ment with MBM in rainbow trout diets, we evaluated the performance,

(poultry by-­product meal) and BM (blood meal). These alternative

feeding, digestive enzymes and apparent digestibility of nutrients and

animal protein sources are considered promising feed ingredients

fatty acids of juvenile rainbow trout fed diets containing 0, 250, 450

because of reduced carbohydrate content in comparison with plant

and 650 g MBM/kg feed MBM with or without garlic supplementation.

proteins. Additionally, MBM has high amount of protein, relatively lower price and good amino acid profile and is also considered a promising alternative to replace FM in salmon diet (Meeker, 2006). Nonetheless, MBM has some constraints, such as lower digestibility in comparison with FM, lysine and methionine deficiency, and also pres-

2 | MATERIALS AND METHODS 2.1 | Diet preparation

ents elevated ash content (Bureau, Harris, & Cho, 1999). Accordingly,

Eight isonitrogenous (460 g crude protein/kg feed) and isoenergetic

in rainbow trout, red drum Sciaenops ocellatus and sea bream Sparus

(22 MJ/kg) diets were formulated and manufactured to achieve 0,

aurata diets up to 300 g FM per kilo feed is commonly replaced with

250, 450 and 650 g FM replaced by MBM/kg feed (Table S1). The

MBM (Bureau et al., 2000; Kureshy, Davis, & Arnold, 2000; Robaina

control diets with (0 MBM+G) or without (0 MBM) garlic contained

et al., 1997). It has been described positive protein digestibility of

460 g/kg of FM (Kilka fish meal, Pars Kilka Company, Iran). The experi-

MBM for rainbow trout (75%) and carp (54%). In this study, it was

mental diets were based on the 0 MBM+G and 0 MBM, but contained

demonstrated that rainbow trout exhibited greater protein digestibil-

increasing level of MBM (Babolsar Company, Iran) according to FM

ity than carp for being a carnivorous species and for presenting more

replacement (250, 450 and 650 g/kg feed) with (G) or without (WG)

protease in its digestive system in comparison with carp, omnivorous

garlic: 250 MBM+G, 450 MBM+G, and 650 MBM+G (G treatments:

species. Thus, it was suggested that rainbow trout could better digest

with garlic powder), 250 MBM, 450 MBM, and 650 MBM (WG treat-

MBM (Watanabe, Pongmaneerat, Sato, & Takeuchi, 1993). It is known

ments: without garlic powder).

that growth and feed efficiency in fish are related to physiological

Garlic powder was added to formulations at 30 g/kg feed according

and biochemical capacity of digestion and transmission of nutrients,

to optimum level previously reported in rainbow trout studies (Farahi

but many factors have a role in digestion, absorption and transfer of

et al., 2010; Mohebbi, Nematollahi, Dorcheh, & Asad, 2012). The garlic

nutrients in the intestine. The ability of fish to digest nutrients de-

was purchased from local market and after hulling, hashing and drying

pends on the adequate amount of enzymes presented in the diges-

in oven at 50°C for 48 hr; the dried garlic was turned to powder using

tive system (Suárez, Hidalgo, García Gallego, Sanz, & De la Higuera,

a mill (Moulinex AR1066Q, France). Fish meal, MBM and other macro-­

1995). Accordingly, the use of some ingredients, which stimulate di-

ingredients were mixed using a mixer (Philips HR7628, Finland) until all

gestive enzymes, may improve digestibility and as a result improve fish

dry ingredients were well homogenized. Then, kilka fish oil (Pars Kilka

growth. Spices, specifically garlic, are among these ingredients. Garlic

company, Iran), soybean oil (Kesht Va Sanat Shomal company, Iran) and

helps digestion and absorption, first by stimulating digestive enzymes

tepid water were added and mixed properly. Next, the paste was pel-

such as lipase, alkaline phosphatase and trypsin, which improves di-

leted using a meat mincer (CGT Company, 2mec, Italy) with 2-­mm die

gestion. Secondly, garlic contains bile acid that plays a vital role in fat

and then dried in oven by 50°C for 18–24 hr to >90% dry matter. Finally,

digestion and absorption (Platel & Srinivasan, 2004). In a study with

the prepared feeds were packed in nylon bags and stored in −20°C.

tilapia Oreochromis niloticus, the use of garlic improved digestion and energy utilization, and also increased growth (Khalil, Nadia, & Soliman, 2001). Many studies have described positive effects of dietary garlic

2.2 | Fish and experimental condition

on fish growth and health (Farahi, Kasiri, Sudagar, Iraei, & Shahkolaei,

A total of 672 juvenile fish with average weight of 8.26 ± 1.10 g were

2010; Guo et al., 2012; Lee & Gao, 2012; Talpur & Ikhwanuddin,

purchased from a hatchery in Amol and transported to the Aquatic

2012). Garlic has many effects on fish, it contains antibacterial and an-

Laboratory at Tarbiat Modares University. First, fish were acclimated

tifungal properties, and besides, it has proper effects on meat quality,

for a week to recover from the transport. Then, they were transferred

body metabolism and immune system, increase in feed consumption

to 24 fibreglass tanks (eight treatments in triplicate) of 150 L for the

and growth (Lee & Gao, 2012).

experiment composed by a semi-­recirculation system, with a stocking

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ESMAEILI et al.

density of 28 fish per tank. Once again fish were acclimated, this time

until infusion to GC mass instrument (Firestone, 1998). The result-

for 8 days. Daily, 30% to 40% of tank water was siphoned to remove

ant fatty acid methyl esters (FAMEs) were separated using GC (Varian

faeces and debris. Water quality parameters were closely monitored

Analytical Instrument, CP 3800, Walnut Creek, CA, USA) equipped

during the culture period: temperature was 17 ± 1°C (mercury ther-

with a flame ionization detector fitted with a permanently bonded pol-

mometer, Zomorodazma Company, Iran) being measured daily, while

yethylene glycol, fused silica capillary column (PBX70 SGE Analytical

dissolved oxygen was 8 ± 1 mg/L (by Cyberscan Eutech instruments,

Science; 120 m × 0.25 mm internal diameter, film thickness 0.25 μm,

DO 110, Singapore), pH was 7 ± 0.6 (Hanna instrument, 8314, USA)

Melbourne, Australia). The injection volume was 1.0 μl, and the car-

both being measured weekly and total ammonium was lower than

rier gas was helium. The injector and detector temperature was, re-

0.05 mg/L during the 60-­day experimental period. The photoperiod

spectively, 230°C and 260°C. A split injection approach of 20:1 was

was adjusted in 12 hr of dark and 12 hr of light. Fish were hand-­fed to

used, and the temperature was programmed to increase from 160°C

apparent satiation three times daily (the first 4 weeks of the experi-

to 180°C at a rate of 2°C/min, and held at 180°C for 85 min. Individual

ment) and then twice daily (during the last 4 weeks of the experiment).

FAMEs were identified using external standard as reference (Sigma-­ Aldrich, Steinheim, Germany) (Table S2).

2.3 | Evaluation of growth parameter After the feeding trial, fish were fasted for one day and were anes-

2.6 | Chemical analysis

thetized with clove oil stock solution (50 ppm) to determine growth

The proximate analyses of whole fish composition including lipid,

parameters including weight gain, feed intake and specific growth

protein, moisture and ash were analysed according to AOAC (1995).

rate (Cowey, 1992). Also six fish from each tank were individually

Samples were homogenized and then stored in freezer −20 ˚C until

dissected, and livers and visceral mass were collected and weighed

the analysis. Crude protein was determined by Kjeldahl method

to assess hepatosomatic index (HSI) and viscerasomatic index (VSI).

using an auto Kjeldahl system (Kjeltec analyser unit 2300, Sweden);

After that, these samples were frozen at −20°C for body composition

crude lipid was analysed by Soxhlet extraction method (Soxtec 2050

assay. Additionally, 12 fish were taken and frozen at −80°C from each

FOSS Model, Switzerland). A Nabertherm muffle furnace (Model: K,

treatment for assay on digestive enzyme assay and fatty acid analysis

Germany) was used for ash (550°C for 4 hr). Moisture was determined

(N = 6 for each analyses).

by an oven (105°C for 12 hr). Nitrogen-­free extract (NFE) plus fibre expressed as carbohydrate was calculated by the difference: 100 -­

2.4 | Faecal collection technique For digestibility assay, faecal collection was performed according to in-­house protocol adapted from Oujifard, Seyfabadi, Kenari, and Rezaei (2012). Briefly, daily faecal collection was performed 3–4 hr after feeding during 14 days (from day 30th to 44th of the feeding trial). Faeces were carefully collected from the tank floor by siphon-

(protein + fat + ash + moisture) (Aksnes & Opstvedt, 1998). The gross energy of the diet and faeces was calculated according to NRC (1993). (Energy (MJ∕ kg) = (Protein × 23.6 kJ∕ g) + (Fat × 39.5 kJ∕ g) + (Carbohydrate × 17.2 kJ∕ g)).

2.7 | Apparent digestibility

ing, and extra attention was intended to this procedure in order to

For determination of apparent digestibility of protein, lipid, dry matter,

avoid contamination or misleading samples. Additionally, extra and/or

energy, carbohydrate, phosphorus and energy, 5 g chrome oxide per

uneaten feed pellets were collected 30 min after feeding. Once faeces

kilo feed was added to the diets as an inactive marker (Maynard &

samples were completely collected, they were freeze-­dried and kept

Loosli, 1969). The quantity of the chrome in the diet and faeces sam-

in -­20°C until the assay day.

ples was analysed by atomic absorption spectrophotometry using the method of Williams, David, and Iismaa (1962). The level of apparent

2.5 | Fatty acid composition Total lipid of diets, white muscles and faeces was extracted by chloroform/methanol method (Folch, Lees, & Sloane-­Stanley, 1957). Afterwards, methanol was added to the lipid for methanolization with BF3. Then, fatty acids methyl ester was extracted by n-­Hexane,

digestibility coefficient was defined by the formula below: % digestibility = 100 × 100 ((Chrome in feed∕ Chrome in faeces) × (Nutrient in faeces∕ Nutrient in feed))

2.8 | Sample preparation and digestive activity assay

methanolic NaOH 2% (2 g of NaOH in 100 g of methanol) was added

Stomach enzyme activity (pepsin), pancreatic enzyme (amylase, lipase,

to the lipid, and the content was carefully mixed and stood in boiled

trypsin and chymotrypsin) and intestinal enzyme activity (leucine

water for 10 min. After cooling, 2.2 ml of BF3 was added to the com-

amino peptidase, alkaline phosphatase) assay were determined as fol-

position and mixed again and stood in boiled water for 3 min., and

lows: the collected digestive tracts from each fish were divided into

then, 1 ml of n-­Hexane was added to the mixture and carefully mixed,

two sections. Intestine with pyloric caeca was used to analyse pan-

and 1 ml of saturated salt was added to the blend. This solution was

creatic enzymes, and the stomach section was used to analyse pepsin

strongly shaken and put in a stable location. After the two phases

enzyme activity. All procedures were conducted on ice to inactivate

were clearly noticed, the upper layer was separated and kept in −20°C

enzymes during the samples’ preparation.

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ESMAEILI et al.

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For pepsin enzyme extraction, samples were mixed with HCl (9:1) and the homogenates were centrifuged at 23,000 g for 20 min at 4°C,

Trypsin: U mg per protein =

(Δ absorption in min) × 1,000 × reaction volume 8,280 × mg protein

Lypase: U mg per protein =

(balnk absorption − sample absorption ) × 1,000 16,500 × mg protein

and then, the supernatant was stored in −80°C (Worthington, 1991). Pancreatic enzyme (amylase, lipase, trypsin and chymotrypsin) extraction was performed according to Harpaz method (Harpaz, Eshel, & Lindner, 1994). In this method, samples with solution Tris–HCl 50 mm, CaCl2 20 mm and KCl 0.05 mm in pH 8 were mixed and homogenized for 1 min in cool state. Then, the solution was centrifuged at 4°C and stored in −80°C. For intestinal enzyme activity (leucine amino peptidase, alkaline phosphatase), extraction Mannitol buffer 50 mm, CaCl2 10 mm and Tris–HCl 2 mm were used (Crane, Boge, & Rigal, 1979). Trypsin activity was assayed using benzoyl-­arginine-­p-­nitroanilide (BAPNA, Sigma B3133) as substrate according to Erlanger, Kokowsky, and Cohen (1961). Homogenization of samples was mixed with Tris–

Amylase: U mg per protein =

(micromole maltose released) incubation time (3min) × mg protein

LA Peptidase: U mg per protein = (Δ absorption in min) × 1,000 × (reaction volume) 10.8 × mg protein Alkaline phosphatase: U per L = (Δ absorption in min) × 2,750

2.9 | Statistics

HCl buffer (0.1 m), pH 8.3 containing NaCl (1 m) and CaCl2 (10 mm), and

All production performance, body composition, digestive enzymes,

after, BAPNA (1.0 mm) was added to the solution. The reaction was

apparent digestibility and fatty acid data were analysed by one-­way

read every minute for 5 min and the absorbance recorded at A 415.

analysis of variance (ANOVA). Additionally, production performance,

Chymotrypsin activity was assayed using N-­Succinyl-­Ala-­Ala-­Pro-­

body composition, apparent digestibility and fatty acid data were fur-

Phe p-­nitroanilide (SAPNA, Sigma S7388) as substrate. The assays

ther analysed by two-­way ANOVA using SPSS software (release 19.0

were run at 25°C. Ten millilitres of enzyme preparation was mixed

for Windows) to determine the significance of garlic supplementation

with 410 nm 0.590 ml of 0.1 mm SAPNA solution in 50 mm Tris–HCl

and FM replacement levels as main effects, as well as their interaction.

buffer, 20 mm CaCl2, pH 7.5. The absorbance was recorded every min

The Duncan’s multiple comparisons test was used to determine the

for 5 min (Erlanger et al., 1961).

differences between the treatment means. Results were considered

Pepsin activity was assayed using 2% of haemoglobin (Sigma

statistically significant at the level of p