|
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
|
ESMAEILI et al.
2
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
|
3
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.
|
ESMAEILI et al.
4
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
