1. Introduction
Aquaculture production is growing faster than any other major food sector. Indeed, with 110.2 million tons harvested in 2016, it will provide the most reliable supply of seafood in the upcoming years [
1]. Fish diets, and those for carnivorous species in particular, have traditionally incorporated a large amount of fishmeal (FM), which represents a high-quality source of protein with a well-balanced essential amino acids (EAA) and fatty acid (FA) profile, high digestibility, and good palatability [
2].
FM production depends on the catches of small pelagic wild stocks such as menhaden, herring, anchovies and sardines, which are processed to obtain different products [
3]. Unfortunately, the unrestrained use of FM over the last few decades has put wild stocks under critical pressure with no prospect of rapid recovery [
4]. As a result, the aquaculture industry has to face the problem of a limited FM supply and a consequent increase in its cost. Over the last few years, researchers and feed manufacturers have focused their efforts on reducing FM inclusion levels in commercial fish diets while, at the same time, maintaining fish health and the nutritional quality of the final products.
Many advances have been made in the partial replacement of FM with alternative protein sources in aquafeeds [
5]. The amount of FM used in diets for carnivorous species has shown a clear decreasing trend toward a more selective use of FM as a strategic ingredient at lower levels, depending on the fish life-cycle stage and species of the fish [
6]. Likewise, the amount of FM in feeds for omnivorous fish has also been reduced, especially in grow-out feeds [
5]. However, overall, the use of FM use in the aquafeed sector has continued to increase as a consequence of the growth in aquaculture production and the related consumption of aquafeeds [
5]. A further reduction in FM inclusion in aquafeeds is thus mandatory.
Plant protein sources are the most common alternatives used to replace FM. Unfortunately, they have shown adverse effects, such as an extremely variable protein content, EAA imbalances, and anti-nutritional factors, which limit their use in diet formulations [
6]. Insects have recently been considered promising alternative protein candidates to substitute FM in aquafeeds, thanks to their interesting nutritional values, in terms of their balanced amino acid (AA) profile, and their lipid, vitamin and mineral contents [
7]. Interest in insects as an innovative aquafeed ingredients has grown rapidly within the scientific community and among stakeholders and their use in aquafeeds was approved by the European Commission (Annexe II of Regulation 2017/893 of 24th May 2017), which authorized the use of insect-derived processed animal proteins from seven insect species (two flies, two mealworms and three crickets). Compared to conventional livestock, the rearing of insects to produce animal feeds offers several ecological and economic advantages, because insects grow and reproduce easily, generate low greenhouse gas emissions and can be reared on discarded organic by-products [
8,
9]. Moreover, rearing insects on bio-waste and organic side streams meets the recycling principles of the circular economy, thus reflecting the efforts of the EU to develop a sustainable, resource-efficient, low carbon and competitive economy [
10,
11].
Yellow mealworm,
Tenebrio molitor (L.) is one of the seven insect species authorized by the European Union. It is a worldwide distributed beetle; and its larvae can easily be reared on low-nutritive plants and can efficiently convert food waste and agricultural by-products into high-quality biomass [
12]. They are rich in proteins (44.1%–60.3% on a dry matter (DM) basis) and lipids (16.6%–43.1% DM) and their AA and FA profiles make them suitable for their inclusion in animal feeds [
13].
The use of
Tenebrio molitor larvae meal (TM) as a partial substitute for conventional protein sources has been studied for different aquaculture species, and promising results have been observed for fish growth performance, diet digestibility, and immune system parameters [
14,
15,
16,
17,
18,
19,
20,
21]. However, in the majority of these studies, the experimental diets were characterized by just a few ingredients (fewer than those normally used in commercial fish diets formulations) and by high levels of FM inclusion (up to 75% as fed). Thus, these diets are not truly representative of the commercial diets currently used in aquaculture.
It should also be considered that, over the last few years, insect manufacturers have increased the production of defatted insect meals. The defatting process allows insect meals to be obtained with larger amounts of crude protein (CP) and better resistance to degradation than full-fat insect meals. Indeed, the latter contain a high lipid content, which in turn makes the extrusion process difficult. Therefore, a defatting process could provide a useful product to reach an adequate feed composition [
22]. To date, only a few studies have been performed to evaluate the use of defatted TM in the diets of different fish species [
23,
24]. Further investigations should be performed to assess the effects of partially defatted TM dietary inclusion in commercial diets.
The present research was designed to assess the effects of a progressive FM substitution (0%, 25%, 50% and 100%) with a partially defatted TM (corresponding to dietary inclusion levels of 0%, 5%, 10% and 20%) in commercial diets on the growth performance, somatic indexes, nutrient digestibility and liver activity of key enzymes of lipogenic and amino acid catabolic pathways in grow-out rainbow trout, Oncorhynchus mykiss (Walbaum).
2. Materials and Methods
A growth trial and a digestibility trial were conducted at the Experimental Facility of the Department of Agricultural, Forest and Food Sciences (DISAFA) of the University of Turin (Italy). The experimental protocol was designed according to the guidelines of the current European and Italian laws on the care and use of experimental animals (European directive 86 609/EEC, put into law in Italy with D.L. 116/92). The experimental protocol was approved by the Ethical Committee of DISAFA (protocol n° 143811).
2.1. Experimental Diets
The used TM was supplied by Ÿnsect (Evry, France). The larvae had been raised on plant by-products and partially defatted using a mechanical process. No other information was given by the producer about either the rearing substrate or the processing methodologies, as this information is considered confidential.
Four experimental diets were formulated, in accordance with SPAROS LDA (Olhão, Portugal) and the TM producer, to be isonitrogenous (CP: about 42.5% as fed), isolipidic (ether extract (EE): about 24.2% as fed), and isoenergetic (gross energy (GE): about 23.8 MJ/kg). The diets were prepared including, as fed basis, increasing levels of a partially defatted TM in substitution of 0% (TM0), 25% (TM25), 50% (TM50) and 100% (TM100) of FM, corresponding to dietary inclusion levels of TM equal to 0%, 5%, 10% and 20%, respectively. In order to ensure that the experimental diets remained isonitrogenous, isolipidic and isoenergetic, and because of the different chemical compositions of TM and FM, the amounts of some other dietary ingredients (i.e., wheat gluten, wheat meal and sardine oil) were modified slightly with the dietary increase in TM inclusion. Moreover, AA supplementation was included in the diet formulations to meet the EAA requirements of the fish.
In order to prepare the experimental extruded diets (SPAROS LDA), all the powder ingredients were mixed according to the target formulation in a double-helix mixer (500L, TGC Extrusion, France) and ground (below 400 µm) in a micropulverizer hammer mill (SH1, Hosokawa-Alpine, Germany). The diets (pellet size: 4.0 mm) were manufactured using a twin-screw extruder (BC45, Clextral, France) with a screw diameter of 55.5 mm. The extruded pellets were dried in a vibrating fluid bed dryer (DR100, TGC Extrusion, France). After cooling, oils were added by means of vacuum coating (PG-10VCLAB, Dinnissen, The Netherlands). Immediately after coating, the diets were packed in sealed plastic buckets and shipped to the research site.
The composition of the ingredients of the experimental diets is shown in
Table 1.
2.2. Chemical Analyses of Feed
Feed samples were ground using a cutting mill (MLI 204; Bühler AG, Uzwil, Switzerland) and analyzed for DM (AOAC #934.01), CP (AOAC #984.13) and ash (AOAC #942.05) contents according to AOAC International [
25]; EE (AOAC #2003.05) was analyzed according to AOAC International [
26]. The GE content was determined using an adiabatic calorimetric bomb (C7000; IKA, Staufen, Germany). The proximate composition of the experimental diets is shown in
Table 2. Chitin was estimated according to Finke [
27] by correction considering the AA content of the acid detergent fiber (ADF) fraction and assuming that the remainder of the ADF fraction was chitin. The FA composition analysis of the experimental diets was performed as reported in Renna et al. [
28]. The fatty acid methyl esters (FAME) were separated, identified and quantified as reported by Ravetto Enri et al. [
29]. The results reported in
Table 3 are expressed as mg/100 g DM.
All the chemical analyses of the feeds were performed in duplicate.
2.3. Growth Trial
2.3.1. Fish and Rearing Conditions
Two hundred and fifty-two grow-out rainbow trout purchased from a private fish hatchery (“Troticoltura Bassignana”; Cuneo, Italy) were used to carry out a 154-day trial after a two-week period of acclimation during which the fish were fed a commercial diet (42% CP and 22% EE, Skretting Italia Spa, Mozzecane (Vr), Italy).
At the beginning of the trial, the fish were anesthetized slightly (MS-222, PHARMAQ Ltd., UK; 60 mg/L), individually weighed (78.3 ± 6.24 g) and randomly distributed into twelve 400-L tanks (three replicate tanks per diet, twenty-one fish per tank). Artesian well water (constant temperature of 13 ± 1 °C) was supplied in a flow-through open system (tank water inflow: 8 L/min). The dissolved oxygen levels were measured every two weeks and they ranged between 7.6 and 8.7 mg/L. The fish were fed 1.6% of the tank biomass for the first 8 weeks and then, according to the fish growth and water temperature, the daily quantity of distributed feed was decreased to 1.4%. The fish were fed twice a day (08:00 and 15:00) six days per week and the feed intake was monitored at each administration. In order to update the daily feeding rate, the biomass tanks were weighed in bulk every 14 days. Mortality was checked every day.
2.3.2. Growth Performance
At the end of the trial, the fish were left unfed for one day, anesthetized slightly (MS-222, PHARMAQ Ltd., Sandleheath, UK; 60 mg/L) and weighed individually (KERN PLE-N v.2.2; KERN and Sohn GmbH, Balingen-Frommern, Germany; d: 0.001). The following performance indexes were calculated:
SGR, FCR, PER and FI were calculated per tank.
2.3.3. Condition Factor and Somatic Indexes
Fifteen fish per treatment (five fish per tank) were killed by overanaesthesia (MS-222; PHARMAQ Ltd., UK; 500 mg/L). The fish were weighed individually (KERN PLE-N v.2.2; KERN and Sohn GmbH, Balingen-Frommern, Germany; d: 0.001) and the total length of the fish was measured to determine the Fulton’s condition factor (K). The fish were slaughtered to calculate the hepatosomatic index (HSI), the viscerosomatic index (VSI), and the coefficient of fatness (CF). The condition factor and the somatic indexes were calculated as follows:
2.4. Digestibility Trial
An in vivo digestibility trial was performed to assess the apparent digestibility coefficients (ADCs) of the diets. One hundred and eighty rainbow trout (94.6 ± 7.31 g) were divided into twelve 250-L cylindroconical tanks connected to the same open water system as that of the growth trial (three replicate tanks per diet, fifteen fish per tank). After fourteen days of acclimation to the experimental diets, the fish were fed by hand to apparent visual satiety twice a day (08:00 and 15:00), six days per week. The ADCs were measured using the indirect acid-insoluble ash method. To this aim, 1% celite
® (Fluka, St. Gallen, Switzerland) was added to the diets as an inert marker in the substitution of 1% of wheat meal. The faeces were collected daily from each tank for four consecutive weeks, using a continuous automatic device (Choubert’s system), as described by Palmegiano et al. [
30]. The feces were freeze-dried and frozen (−20 °C) until analyzed. The ADCs of the DM (ADC
DM), CP (ADC
CP), EE (ADC
EE) and GE (ADC
GE) were calculated as reported by Caimi et al. [
31] and expressed as a percentage.
2.5. Hepatic Enzyme Activities
Liver samples were collected (nine replicates per treatment) and stored at −80 °C to measure the alanine aminotransferase (ALAT; EC 2.6.1.2), aspartate aminotransferase (ASAT; EC 2.6.1.1) and glutamate dehydrogenase (GDH; EC1.4.1.2) activities. The liver samples were homogenized (dilution 1:10) in an ice-cold buffer (30 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.25 mM saccharose, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM K
2HPO
4, 1 mM dithiothreitol (DTT); pH 7.4). After being centrifuged at 1000×
g for 10 min at 4 °C, the supernatants were sonicated for 1 min (pulse 1 s, amplitude 50) and centrifuged again at 15,000×
g for 20 min at 4 °C. The resultant supernatant was collected for enzyme activity measurements. GDH activity was measured using 10 mM of L-glutamic acid, as described by Bernt and Bergmeyer [
32]. ALAT and ASAT were assayed using Spinreact kits (ALAT/GPT, ref. 41282; ASAT/GOT, ref. 41272) according to the manufacturer’s instructions.
For glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), malic enzyme (ME; EC 1.1.1.40), and fatty acid synthetase (FAS; EC 2.3.1.38) activities, the liver samples were homogenized (dilution 1:5) in ice-cold buffer (0.02 M Tris-HCl; 0.25 M sucrose; 2 mM EDTA; 0.1 M sodium fluoride; 0.5 mM phenyl methyl sulphonyl fluoride (PMSF); 0.01 M β-mercapto ethanol; pH 7.4) and the homogenate was centrifuged at 30,000×
g for 20 min at 4 °C. G6PD activity was measured according to Bautista et al. [
33], ME activity was measured according to Ochoa [
34] and FAS activity according to Chang et al. [
35], as modified by Chakrabarty and Leveille [
36].
All the enzyme activities were expressed per mg of hepatic soluble protein (specific activity). The protein concentration was determined according to Bradford [
37] using the Sigma-Aldrich protein assay kit (ref. B6916) with bovine serum albumin as standard. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 µmol of substrate per min at the assay temperature (37 °C). All the enzyme assays were carried out using a Multiskan GO microplate reader (Model 5111 9200; Thermo Scientific, Nanjing, China). All the reagents used for the enzymatic analysis were purchased from Sigma-Aldrich (Química, S.L., Sintra, Portugal).
2.6. Statistical Analyses
The obtained data were analyzed by means of one-way ANOVA, using IBM SPSS Statistics v. 25.0 for Windows. The following model was used:
where Y
ij = observation; μ = overall mean; D
i = effect of diet (TM0, TM25, TM50, TM100); ε
ij = residual error.
The assumption of normality was checked using the Kolmogorov–Smirnov test. Levene’s homogeneity of variance test was used to assess homoscedasticity. If such an assumption did not hold, the Brown–Forsythe statistic was applied to test the equality of group means instead of the F one. Pairwise multiple comparisons were performed to test the difference between each pair of means (Tukey’s test and Tamhane’s T2 in the cases of assumed or not assumed equal variances, respectively). The results were expressed as the mean and pooled standard error of the mean (SEM). Significance was set at p ≤ 0.05.
5. Conclusions
This study has evaluated, for the first time, the effects of the dietary inclusion of a partially defatted Tenebrio molitor larva meal on the growth performance, diet digestibility, and hepatic intermediary metabolism of practical diets for on-growing rainbow trout. The obtained results show that, in the current typical commercial diets that contain about 20% of FM and a well-balanced EAA profile, FM could be substituted completely by TM, without any negative effects on fish growth, the condition factor or the activity of hepatic amino acid catabolizing and lipogenic enzymes. Among the digestibility coefficients, only ADCCP was shown to be to be negatively affected by the inclusion of dietary insect meal, but it should also be highlighted that, in absolute values, the ADC remained high in all the treatments.
These results are of practical application for feed manufacturers and farmers. The inclusion of insect meals in fish diets could lead to sustainable feeds that would be able to support an increase in aquaculture production without the massive use of conventional protein sources, which are characterized by strong environmental impacts.
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