1. Introduction
In many countries, a policy objective for aquaculture development is diversification. This term not only applies to searching for new species, but also to increasing the production of those species currently farmed in small quantities [
1] such as tench,
Tinca tinca. Tench is a freshwater fish belonging to the Cyprinidae family that originally occurred in the waters of Europe and Siberia and today is present in the inland waters of all continents [
2]. Tench is commonly cultured in extensive systems where growth is often limited and production usually poor [
3,
4]. Considering that this species is appreciated by consumers as a tasty fish with healthy meat [
5,
6] and attractive for anglers, the development of intensive rearing systems is required to satisfy the demand. A major obstacle to increasing tench production is the deficit of young fishes for further growth or to restock open waters for sport fishing [
7,
8]. Some relevant advances have been achieved in juvenile tench rearing techniques under controlled conditions, focusing mainly on feed as an essential factor. In this sense, González-Rodríguez et al. [
9] established the protein requirements for juvenile tench between 48 and 52% and later, García et al. [
10] proposed a practical diet which allows good survival and growth performance, setting the basis for further nutritional studies.
Fishmeal (FM) is the most nutritious and digestible source of protein in aquatic diets, which is the biggest consumer sector with a global use in 2019 of 78% [
11]. In 2031, world production of fishmeal is expected to reach 5.6 Mt of which 71% will come from forage species [
12]. The environmental footprint derived from its use in feedstuffs is a main concern to reach sustainable growth [
13], especially when production mainly depends on a finite wild-harvest capture [
14]. In addition to environmental concerns, the upscaling demand and price for FM have awakened the search for alternative ingredients, specifically byproducts and those obtained by recycling nutrients, to support a circular economy within aquaculture [
15].
Among different possibilities, there is a recent interest in insects as a food source for animal feeding. Insect culture is considered sustainable as insects do not need large areas or much water and they contribute to waste recycling [
16,
17]. The review by Henry et al. [
18] highlighted the good potential of insect meal to replace FM in fish diets due to their high content of protein and adequate profile of essential amino acids (EAA). However, only a few species of insects have the potential to be reared at a large scale [
19]. In addition, there are regulatory obstacles to use insect protein for animal feeding. The recently approved UE regulations, 2017/893/EC, 2017, and 2021/1925/UE [
20,
21] allow the inclusion of protein from eight species of insects in aquafeeds. Among them, the black soldier fly (
Hermetia illucens) shows major advantages derived from its ability to turn bio-waste into larvae with valuable nutrient content [
22].
Table 1 includes data on the possibilities to replace FM by black soldier fly meals (BSFLM) in freshwater and marine fish species without negative effects on growth performance. Except for the experiments performed in the turbot (
Scophthalmus maximus) [
23], in the yellowtail (
Seriola quinqueradiata) [
24], and in the gilthead seabream (
Sparus aurata) [
25], where the inclusion of BSFLM negatively affected growth performance, levels of FM replacement between 20% in the dusky kob (
Argyrosomus japonicus) [
26] and 100% were shown to be feasible. However, the dietary amounts of BSFLM usually did not reflect the percentages of FM substitution. Therefore, total replacement of FM was achieved with 600 g kg
−1 in diets for Atlantic salmon (
Salmo salar) [
27] and 106 g kg
−1 in diets for Jian carp (
Cyprinus carpio var. Jian) [
28]. In addition to interspecific differences, several factors must be considered to explain the variability of results, such as the nutritional value of BSFLM including insect growth substrate and further meal processing, life stage of fish, diet formulation, and nutritional composition [
29].
Regarding the effect of FM replacement with BSFLM on body composition, several studies concluded that the inclusion of BSFLM did not affect the final whole-body or fish fillet proximate composition. Meanwhile, others reported significant increases in dry matter and lipid [
40,
43] or protein content [
50].
To the best of our knowledge, no studies have reported the effect of replacing FM by BSFLM in the diet of juvenile tench; therefore, the present study aimed to evaluate the effects of increasing substitution level of FM with BSFLM on survival, growth performance, incidence of externally visible deformities, and whole-body composition.
2. Materials and Methods
2.1. Ethics Statement
According to Spanish law (RD 53/2013) and an EU directive (2010/63/EU), the Ethic Committee of the University of León approved the experiment conducted in this study (Approval reference ULE_16_2015). Fish health, welfare, and the environmental conditions in the experimental tanks were checked twice daily by visual observation of animal behavior. Water quality parameters, such as oxygen saturation, temperature, and water flow were periodically measured (see
Section 2.2.). The necessary number of fish to analyze whole-body composition were euthanized with an over-dose of tricaine methanesulfonate (MS222, Ortoquímica S.L., Barcelona, Spain) by prolonged immersion. At the end of the experiment, the remaining animals were transported to the fish farm where breeder fish come from.
2.2. Fish, Facilities, and Experimental Procedures
Tench larvae were obtained by hatching using artificial reproduction techniques [
59] and reared in outdoor tanks. After 4 months, 540 juvenile tench from a homogenous pool were randomly distributed as groups of 30 fish in 18 fiberglass tanks (0.5 × 0.25 × 0.25 m) containing 25 L of water to obtain replicates corresponding to the different feeding treatments. Prior to distribution, 100 juveniles were anesthetized with tricaine methanesulfonate (MS-222; Ortoquímica S.L., Barcelona, Spain) to measure initial total length (TL) and weight (W). Values of 30.7 ± 0.28 mm and 0.39 ± 0.02 g (mean ± SEM) were obtained. TL was measured with a digital caliper (to the nearest 0.01 mm) and, after removing excess water with tissue paper, W was determined by a precision balance (to the nearest 0.001 g). Total biomass of each tank was weighed. Following a monofactorial design, diet was the experimental factor with three replicates per level of treatment. The juveniles were previously acclimated to experimental conditions for 4 days.
Artesian well water was supplied in an open system (flow-through system) and each tank had a water inlet (inflow 0.30 L min−1) and outlet (provided with a 250 µm mesh filter) and light aeration. Measures of the incoming water quality, ammonia, nitrites, hardness, and total suspended solids were performed once a week with a spectrophotometer HACH DR2800 (Hach Lange GMBH, Vigo, Spain). Dissolved oxygen in tanks was measured with a multi-meter HACH HQ30d (Hach Lange GMBH, Vigo, Spain). Mean values of water quality were pH 7.6, hardness 5.3 German degrees (calcium 32.8 mg L−1), total suspended solids 34.0 mg L−1, dissolved oxygen ranged between 5.7 and 7.3 mg L−1, ammonia < 0.10 mg L−1, and nitrites < 0.010 mg L−1.
Water temperature (measured twice a day) was 26 ± 1 °C and a 16 h light:8 h dark photoperiod was maintained throughout the experiment. Tanks were cleaned of feces and uneaten feed every two days. The experiment lasted for 90 days.
2.3. Diets and Feeding
Based on the results of our research group [
9,
10], different diets (50% crude protein) were formulated and prepared to test the effects of different substitution levels of FM by black soldier fly (
H. illucens) meal. A partially defatted BSFLM from Hermetia Deutschland GmbH & Co. KG (Baruth/Mark, Germany), obtained by processing larvae reared on a vegetable byproducts substrate, was included. According to the producer, partial defatting was made by a mechanical process using high pressure and without any solvents. Proximate composition and amino acid profiles of FM and BSFLM are in
Table 2.
A total of 6 diets (nearly isonitrogenous and isoenergetic) with different replacement levels of FM by BSFLM were formulated: 0% (control), 15%, 30%, 45%, 60%, or 75%, corresponding to 0, 117, 232, 348, 464, or 579 g of BSFLM kg
−1 diet, respectively (
Table 3). Ingredients were ground in a rotary mill BRABENDER (Brabender GmbH & Co. KG, Duisburg, Germany), mixed in a mixer STEPHAN UMC5 (Stephan Food Service Equipment, Hameln, Germany) and extruded using a stand-alone extruder BRABENDER KE19/25D (Brabender GmbH & Co. KG, Duisburg, Germany) at a temperature range between 100 °C and 110 °C. Pellets (1 mm diameter) were dried during 24 h at 30 °C and after receiving a coating of cod liver oil. Fish were fed manually three times a day (at 10:00, 14:00, and 18:00 h) to apparent satiation.
2.4. Chemical Analysis of Diets and Fish
Juveniles were fasted for 14 h before sampling. Samples of diets and juveniles were stored at −30 °C at the beginning and at the end of the experiment. Analyses were performed in duplicate by Analiza Calidad laboratory (Burgos, Spain) following Commission Regulation (EC) 152/2009. Moisture was determined by drying at 105 °C, crude protein was determined according to the Kjeldahl method, crude lipid was determined by extraction with light petroleum and further distillation, ash was determined by calcination at 550 °C, and gross energy was determined according to EU regulation 1169/2011. The content of nitrogen-free extract was calculated by subtracting moisture, protein, lipid, and ash content from the wet weight.
Amino acid profiles were analyzed by HPLC using AccQTag method from Waters (Milford, MA, USA). Amino acids were derivatized with 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate reagent (AQC) by the method of Cohen and Michaud [
60] and Cohen and De Antonis [
61] and detected by Dual λ Absorbance Detector Waters 2487 from Waters (Milford, MA, USA) at 254 nm. Quantification was carried out with Empower Pro 2.0 software from Waters (Milford, MA, USA). All analyses were performed in duplicate.
2.5. Data Collection
Juvenile tench behavior was observed and registered after cleaning, feeding, and measuring the water quality parameters.
Every 30 days a sample of 15 fish per tank (45 per treatment, 50% of total) were anesthetized to be individually weighed and measured to have information about the growth performance evolution. TL and W were measured as described in
Section 2.1., and afterwards juveniles were gently returned to their tanks.
At the end of the experiment, surviving fishes were anesthetized and observed one by one using a magnifying glass to detect externally visible deformities affecting spinal axis, operculum, mouth and tail fin. W and TL were measured individually and total biomass per tank was weighed. The following indices were calculated:
- -
Survival rate (%) = (final number of juveniles/initial number of juveniles) × 100;
- -
Specific growth rate, SGR (% d−1) = [(ln final W − ln initial W)/days)] × 100;
- -
Fulton’s coefficient or condition factor, K = 100 × [final W/(TL3)];
- -
Biomass gain, BG (g) = (final biomass/tank − initial biomass/tank);
- -
Feed conversion ratio, FCR = (total feed provided per tank/BG [
62].
All treatments were replicated three times and the experimental unit was a tank.
2.6. Statistical Analysis
After confirmation of normality and homogeneity of variance, statistical analysis of growth performance and whole-body composition data were conducted by one-way analysis of variance (ANOVA) and polynomial contrasts with the SPSS16.0 computer program (SPSS, Chicago, IL, USA). Significant differences between means were estimated by Tukey’s multiple range test. p < 0.05 was used for rejection of null hypothesis.
4. Discussion
Insects are part of the natural diet of many freshwater fish species and, thus, the inclusion of insect meals could be advantageous to ease feed intake [
63]. Tench feed on zooplankton and other small invertebrates in natural habitats, such as some insect larvae [
64,
65,
66]. Although black soldier fly is a terrestrial insect, the natural feeding habits of tench would help voluntary ingestion of diets independently of the different content of BSFLM, indicating that substitution of FM did not affect its palatability. This agrees with most experiments on partial or total FM replacement by BSFLM, where diets including insect meal were attractive to fishes. However, Kroeckel et al. [
23] reported a reduction in growth in turbot which was partially attributed to a decrease in feed intake due to a lower palatability of diets including BSFLM.
In their review, Barragán-Fonseca et al. [
22] reported that composition of BSFLM depends on body composition of fly larvae, which varies among the rearing substrates, but also on further processing. Black soldier fly larvae have a high fat content, reaching in non-defatted meals a lipid content average of 353.2 g kg
−1 [
67] making their inclusion in aquafeeds difficult. Thus, BSFLM is processed to obtain partially defatted meal with high protein content, which allows high inclusion levels of insect meal in fish diets without reducing the technical quality of extruded diets [
41]. According to the producer, the BSFLM of this experiment was partially defatted and its high protein content (54.7%) makes possible dietary amounts of up to 579 g kg
−1 (75% FM replacement). As the high content of saturated and monosaturated fatty acids in black soldier fly larvae is associated to a decline in growth of aquatic species [
68], the defatting process could have beneficial effects in the nutritional composition of diets.
Considering that aquaculture is diverse in terms of cultured species, production systems, and culture conditions, it is difficult to establish accurate comparisons between studies of FM replacement by BSFLM. In most cases, relative data (% FM replacement) are provided but correspond to different inclusion amounts. Therefore, total FM replacement were achieved in Jin carp, with 106 g kg diet
−1 [
37] and 140 g kg diet
−1 [
38], whereas in rainbow trout [
42] and Atlantic salmon [
24], amounts were 450 and 600 g kg
−1, respectively. In this study with juvenile tench, which have similar protein requirements than rainbow trout and Atlantic salmon, high amounts of BSFLM (579 g kg diet
−1) did not affect survival and growth performance compared with the control diet.
The nutritional quality of black soldier fly products must be considered to accurately interpret the response of fish. Available data on the nutritional value of
H. illuscens larvae meal showed that the EAA profile, an important indicator of protein quality [
69], does not differ much between studies [
22]. In agreement with Henry et al. [
18] and Maurer et al. [
70], the profile of EAA of BSFLM in the practical diets was similar to the FM (
Table 1), with the exception of arginine, lysine, phenylalanine, and methionine (60%, 49%, 69.2%, and 33.6% less than FM, respectively). Despite the reduction in the mentioned amino acids in BSFLM diets (
Table 4), juvenile growth was unaffected by inclusion of BSFLM, leading us to consider that EEA requirements were fully covered. According to Hua et al. [
29], the relationship between BSFLM incorporation level and the growth response is best described by a simple negative linear equation. In this experiment, we found a cubic relationship, showing an increasing growth (TL, W, SGR, FCR, and BG) from the control diet to 45% BSFLM and, with inclusion levels above this threshold, a trend to growth reduction. The negative effects of insect meal on fish growth could be partly due to a lower nutrient digestibility [
29]. Some authors speculated that the content of chitin in insect meals is closely related to a reduction in protein and fat digestibility [
27,
40,
71]. Considering this, the reduction in growth observed in juvenile tench from inclusion BSFLM over 348 g kg
−1 could be associated to an increasing content of chitin.
Despite the defatting process, the lipid content of BSFLM was higher than in FM, determining an increase in fat in diets from 107.5 g kg
−1 (control diet) to 123 g kg
−1 (75% BSFLM diet). The increase in lipid content in diets is usually correlated with an increase in fish body fat [
72,
73], but in opposition to this statement and similarly to results reported on turbot [
23] and Jian carp [
28,
56], whole-body tench lipid content decreased with the increase in dietary BSFLM. Kroeckel et al. [
23] and Li et al. [
28] hypothesized that this fact could be related to a reduction in lipid digestibility due to the content of chitin provided by the insect meal. In agreement with Ferrer-Llagostera et al. [
74], the information on the effects of chitin are still discussed and further research is required to clarify its role in aquatic species.
Kamisnki et al. [
3] reported one of the highest values of SGR in juvenile tench, 3.69% day
−1, with initial weight (W) of 0.24 g and at five-months old. In our study, SGR’s ranged between 2.20 and 2.45% day
−1 using six-month old juveniles with an initial W of 0.46 g. Considering that SGR slows down with age [
74], the lower values could be partially attributed to differences in age and initial weight.
As in former experiments performed by our research team, the control diet enabled acceptable growth and high survival and no external visible deformities. External visible body deformities are mainly associated to inadequate feeding, especially during early development [
75,
76]. In juvenile tench, a relationship between feeding commercial diets for other species and fish with elevated condition coefficients (between 1.3 and 1.4) and presence of body deformities has been suggested [
3,
77,
78,
79]. Although the K factor for some diets have shown values above 1.3, the percentage of deformed fish was insignificant (<0.05) showing that diets were well balanced for tench.
From this preliminary study, BSFLM should be regarded as a good protein source for these critical early stages of tench, being feasible for inclusion in high levels of FM dietary replacement without negative effects on survival and growth. Since the high protein quality requirements during this early growth stage were covered by BSFLM, it would be expected that FM saving would increase through inclusion of this insect meal in feedstuffs for further outgrowing phases, whenever the development of efficient insect rearing and processing systems allow for market availability at reduced cost. Under this consideration, further research on the effects of dietary BSFLM in nutritional and sensory quality in tench reared to commercial size should be performed.