1. Introduction
Aquaculture’s goal is to achieve sustainable development in all three pillars, that is, to be economically, socially, and environmentally sustainable [
1]. The correct formulation of feeds for farmed fish is key to maintaining the sustainable growth of the aquaculture sector within the vision of a circular bioeconomy, without compromising the nutritional quality of the product [
2]. Although fishmeal (FM) is an ideal protein source for carnivorous fishes [
3], it is a finite resource whose high price and impact on natural ecosystems have led to its use in aquaculture being increasingly reduced and replaced by plant protein sources [
4,
5]. Despite their high potential for aquaculture development, plant proteins face feed-food competition and have been shown to adversely affect fish growth performance and welfare in carnivorous species [
6,
7,
8].
Protein meals from terrestrial animals, such as black soldier fly (
Hermetia illucens) meal (HIM) and poultry by-product meal (PBM), appear to be promising replacements for conventional raw materials in diets for carnivorous fishes [
9,
10]. Both HIM and PBM are not directly intended for human consumption, and their production has a low environmental footprint [
11,
12]. In addition, the nutritional profile of HIM is similar to that of FM [
13,
14], and PBM is readily available on the market [
15]. Recent results on gilthead seabream (
Sparus aurata) show that HIM can be included at 11% [
16] and 15% [
17], partially replacing FM, without compromising fish growth performance, blood biochemistry, or stress parameters; however, the integrity of the intestinal mucosa and submucosa decreased with increasing levels of HIM in the diet [
16]. Similarly, the inclusion of up to 19.5% of HIM in FM-based diets for European seabass (
Dicentrarchus labrax) did not compromise the zootechnical parameters of fish and nutritional characteristics of the fillets and could also contribute to reducing their lipid oxidation [
18]. With regard to PBM, several studies demonstrated that it could partially replace FM in feed for juvenile black seabream (
Spondyliosoma cantharus) [
19], gilthead seabream (
Sparus aurata) [
20], and juvenile red porgy (
Pagrus pagrus) [
21] without negative effects on growth performance, survival, or intestinal digestive and absorptive functions.
Although the output of using either HIM or PBM individually is promising, in the frame of sustainable aquaculture intensification, a single protein source is unlikely to meet the essential nutritional requirements of fish and at the same time provide the best quality end-product [
10]. During the last five years, the national project “SUstainable fiSH feeds INnovative ingredients–SUSHIN” funded by the AGER2 Network Foundation has evaluated the potential of different unconventional and underused ingredients, tested singly or in combination, as alternative protein sources for aquafeeds, generating new information on the environmental footprint of feeds [
12], on fish growth and welfare, and on the nutritional traits of carnivorous fish species economically important for the European aquaculture [
22,
23,
24]. In addition, results obtained under experimental conditions show that feeding gilthead seabream for 18 weeks with diets containing a negligible amount of FM and 40% plant protein replacement by PBM and HIM alone or in combination (30% and 10%, respectively) improved the zootechnical performance of fish and the nutritional characteristics of the fillets, also ensuring physiological well-being and liver health [
24,
25]. Pleić et al. [
26] found that plant-based diets supplemented with HIM in combination with PBM resulted in the highest specific growth rates and lowest feed conversion ratios for European seabass while maintaining the nutritional value of the fillets for human consumption.
Based on the results obtained in other studies under laboratory conditions, the present study aimed to evaluate the effects on growth performance and food quality attributes of European seabass farmed under commercial conditions and fed a diet poor in marine protein, rich in plant protein, and including a combination of HIM and PBM.
4. Discussion
Aquaculture is striving toward the circular economy concept in its production process, and the path to sustainable, nutritious, and nonconventional aquafeed ingredients has been extensively investigated in controlled trials over the last few decades. However, little is known about research conducted under routine commercial farming conditions.
In the present study, after 66 days of feeding in a commercial farm, moderately higher growth and better zootechnical indices (K, SGR, FCR), although not statistically significant, were observed in fish fed the SSH diet. The eviscerated weight of SSH fish was also about 10% higher than that of fish fed CG diet, resulting in relevant commercial implications. No effect on growth performance was noticed in previous experimental studies in which European seabass were fed diets containing 19.5% HIM [
18], or gilthead seabream fed diets containing 32.4% HIM and 27.5% PBM [
6,
24,
25]. Thus, the present results confirm the possible use of HIM and PBM in a plant-rich diet for marine species previously observed on an experimental scale [
24,
25]. Aligning with the aforementioned findings, it was shown that European seabass fed the SSH diet improved its FCR, as reported in gilthead sea bream when fed a similar diet that was previously tested in an experimental setting [
25], supported by the fact that a partial replacement of the plant mixture with HIM and PBM could also activate brush border membrane enzymes [
26].
In the present study, the skin lightness (
L*) of fish fed the SSH diet was lower than that of fish fed the commercial diet, but the relative difference between the two values was subtle. Similarly, the inclusion of PBM in a vegetable-based diet was not able to pigment the skin of gilthead seabream [
43]. Future studies on consumer preferences for fish with different skin colors are envisaged to clarify whether changes such as those found in this study are perceived positively or negatively.
Diet can significantly impact fillet FA composition. In the present study, the sum of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid contents indicated that, independently of the diet, one serving portion of the fillets of this trial (150 g) would provide the consumer with 419.27 mg of EPA and DHA, an amount above the recommended daily intake [
44]. On the other hand, the fillets of fish fed the SSH diet had higher levels of lauric acid (C12:0), most likely originating from the dietary inclusion of HIM. It has been widely reported that this insect species has a specific ability to convert other FAs into C12:0 [
45], resulting in particularly high levels of this SFA, which may also impair the overall nutritional quality of fish fillets. However, previous studies have demonstrated that the inclusion of HIM up to 25%, 40%, or 50% of the total protein content in the diets for gilthead seabream and Siberian sturgeon, respectively, is associated with beneficial effects on fish gut health, such as immunostimulation and anti-inflammation [
6,
46]. These results are mainly attributed to the presence of bioactive compounds, including medium-short chain FAs, such as lauric acid [
6].
Marine fish are known to have minimal desaturase activity; nonetheless, gilthead seabream was proven to express a desaturase gene [
47,
48]. Based on the present study, it could be assumed that the diets modulated the estimated indices of the FA elongase and desaturase activities. The higher MUFA desaturase values in the SSH group hint that the estimated desaturase activity on MUFAs was higher in the SSH than in the CG, while it seemed that the SSH fish produced n-3 FAs to a lesser extent than the CG. In all probability, this is a direct consequence of the fact that C18:3n-3 content was higher, and EPA and DHA contents were lower in the CG diet in comparison with the SSH, stimulating the fish to elongate and desaturate C18:3n-3 to EPA and DHA. Besides, the Δ5 + Δ6 desaturase n-3 index of the CG fish was higher, suggesting that this group needed to produce n-3 FAs endogenously.
Liver plays a key role in the metabolism of nutrients in fish, and a wide range of enzymatic antioxidants protect against pro-oxidant species, such as reactive oxygen species. It is a fact that when investigating the use of new ingredients or searching for aquafeed formulations, an alteration in hepatic metabolic activities and liver oxidative status can be observed [
49], thus potentially indicating a health impairment. In the present study, there was an increased activity of G6PDH and HOAD in SSH fish, suggesting an increased β-oxidation and consequently increased utilization of FAs for energetic purposes. This could explain the significant reduction in C22:1n-11 fillet content in SSH fish despite its higher level in SSH feed. As verified by several authors, C22:1n-11 is largely used as a substrate for β-oxidation and is generally oxidized rather than stored in the body [
50,
51,
52,
53].
Another indicator of altered energy metabolism is the significant increase in AST in SSH fish. This enzyme, found in fish hearts, skeletal muscles, kidneys, and brains, assists in the transfer of the amino group from aspartic acid to α-ketoglutaric acid to form oxaloacetic and glutamic acids [
49,
54]. This pathway is well known in fish, and it is considered to be of paramount importance to maintain glucose homeostasis during periods of food deprivation [
55]; it is generally considered to be a good indicator of the utilization of amino acids as an energy source [
56]. In addition, as recently observed by [
57] in Chinese sturgeon (
Acipenser sinensis), AST amount in the liver increased as the specific growth and feeding rates increased. This could support the higher body weight (
p > 0.05) and lower FCR (
p > 0.05) of European seabass fed SSH, suggesting a better use of FAs and amino acids as energy sources. The factors determining this moderately positive effect remain unclear, even if the changes in the gut microbiome observed in diets containing HIM [
19] underline that this ingredient is able to increase the abundance of two interesting taxa in fish, such as Bacillaceae and Paenibacillaceae, involved in the production of short-chain FAs and other useful molecules able to improve fish health [
19].
While serum AST is frequently correlated with fish health [
58], the same increase was not observed in serum, gills, liver, and other tissues following toxicant exposure [
59]. This suggests that liver AST activity cannot be a reliable indicator of a diseased or stressful condition, which can induce oxidative stress in fish. The present study showed that the oxidative status of both fish liver and fillets was equivalent between the two dietary groups, in agreement with what was observed in rainbow trout fed diets containing HIM [
60]. A previous study performed on European seabass demonstrated that a dietary inclusion of 6.5 and 13 g/100 g of HIM decreased liver oxidative stress [
18], which was attributed to the presence of chitin. Indeed, chitin and its derivatives have been shown to act as antioxidants and prevent ROS formation in fish [
61]. Furthermore, in this study, the negative effect of dietary PBM on the activity of antioxidant enzymes was not observed, contrary to what was previously reported on barramundi
(Lates calcarifer) but at much higher levels, corresponding to the total replacement of FM [
62].