1. Introduction
Quails are birds of economic importance due to a series of positive features such as high meat quality and egg production, as well as fast returns on investment, which is possible due to their early sexual maturity, rapid growth, short generation interval, high laying rate, and limited feed and space required/bird [
1]. As for other poultry species, commercial feed formulations mainly rely on soybeans as protein and fat sources, which require a vast amount of water and land to grow and thus have a significant environmental impact [
2]. Due to growing demographic trends and the consequent augmented pressure on natural resources such as water and land (deforestation), as well as an ever-increasing feed–food competition, the search for novel feed ingredients for poultry diets with the potential to improve the sustainability of the sector is of utmost importance. From this perspective, insects have been recognized as one of the possible candidates to solve this issue. This was internationally emphasized for the first time with the first International Conference on insects for food and feed, organized by the Food and Agriculture Organization of the United Nations in 2014. In the following years, research studies on this topic as well as innovations and applications of techniques by the industry have grown tremendously [
3]. In addition, the European legislative framework regarding the use of insects in animal nutrition was updated, which led to the publication of Regulation No. 2017/893, which allows the use of seven insect species in aquaculture. Despite this key success, more research efforts are required to allow for their utilization in poultry feeding, where insect consumption already falls within their natural dietary habits, particularly in free-range production systems [
4].
One of the most promising insect species in this sense is the black soldier fly (
Hermetia illucens, HI), a Diptera of the Stratyomidae family. Together with their undoubtfully interesting nutritional profile [
5] and suitability for mass production,
H. illucens larvae can exploit a wide range of decomposing organic material for their growth, thus representing an opportunity to recycle organic matter into valuable nutrients [
6]. Independent of the inclusion level, when tested in the diets of different animal species (including fish [
7], poultry [
8,
9,
10,
11], pig [
12], and rabbit [
13,
14] species), overall results such as the health status of the animals, productive performances, and the overall quality of the derived animal products (i.e., meat and eggs) proved to be satisfactory. However, a constant nutritional drawback emerged related to the fatty acid (FA) profile of the animal product derived from animals fed with larvae of this Dipteran. In fact,
H. illucens larvae are naturally rich in saturated fatty acids (SFAs; mainly lauric, myristic, and palmitic) and poor in unsaturated FA [
15]. For this reason, they are suboptimal in providing healthy food for modern consumers. As previous research has demonstrated that the rearing substrate can have a great impact on the chemical composition of the larvae [
16], the hypothesis is that the FA profile of
H. illucens larvae could be improved by rearing them in a substrate enriched with a source of
n-3 polyunsaturated FA (PUFA), recycling a relevant nutrient source that is considered a byproduct: fish offal. The ultimate goal is the successful application of such larvae to poultry nutrition, aiming to obtain a meat with a healthy FA profile. The only research on this topic published to date found promising results [
17]. However, the study concerned a possible application of
n-3-enriched larvae in fish diets, whereas no scientific works are available on poultry species. Therefore, the present research project studied the inclusion of 10% HI larvae reared on a conventional substrate or on a substrate enriched with
n-3 PUFA in the feed of broiler quails, aiming to improve the FA profile of quails’ meat. In the first report of this study [
18] the total tract nutrients’ digestibility and feed-choice as well as the quails’ productive performance and carcass traits were studied: despite feed-choice and carcass weight were not in favor of the group including the
n-3 enriched larvae, nutrients’ digestibility, mortality and carcass yield were satisfactory. In this, the second part of the study, detailed meat quality aspects, including physical characteristics, proximate composition, cholesterol, heme iron and amino acid contents, the FA profile, sensory traits, and the storage stability of the quails fed these diets, were evaluated.
3. Results
Overall, quail breasts derived from birds fed with diets containing 0% or 10% dried
H. illucens larvae reared on two different growing media (HI1 and HI2) showed similar physical meat quality traits to those of the quails fed the control diet (
Table 2). Specifically, breasts displayed similar pH values (
p > 0.05), Commission Internationale de l’Éclairage L*a*b* color values (
p > 0.05), and thawing losses (
p > 0.05). The only exception was the cooking loss percentage, which was highest in quail breasts of the HI2 group (
p < 0.05); however, this did not affect the total loss percentage (
p > 0.05) nor the meat toughness (
p > 0.05), which was similar in the three experimental groups.
The chemical composition of quail breasts was significantly affected by the tested experimental diets (
Table 3). Breasts of the HI1 group displayed the highest lipid content (5.27% vs. 4.89% and 4.93% for HI1, C, and HI2 breast meat, respectively;
p < 0.01) to the detriment of the protein fraction (
p < 0.05), which was lower than that of C but not different from HI2. Independent of the growing substrate used to farm
H. illucens larvae, their inclusion in the quails’ diets increased the cholesterol content of the breast meat compared to the C group (
p < 0.01). Water, ash, and heme iron contents were not affected by the dietary treatment.
The protein percentage as well as the majority of the essential and nonessential amino acids (g/100 g meat) of quail breast meat was affected significantly by the dietary treatments (
Table 4). The 10% dietary inclusion of HI1 lowered the protein percentage (on a dry mass, defatted basis) of quails’ breast meat compared to C, whereas HI2 meat showed an intermediate result (81.4%, 83.4%, and 89.5% for HI1, HI2, and C breast meat, respectively;
p < 0.05). The above-mentioned result was mainly attributable to the low amounts of the essential amino acids histidine, leucine, methionine, and phenylalanine. The rearing substrate of the larvae played a role in determining the overall quality of the protein provided by the quail breast meat: in general, HI2 meat showed a slightly better amino acid profile than HI1, especially when considering arginine and threonine (
p < 0.05) of the essential amino acids and glycine, glutamic acid, and serine (
p < 0.05) of the nonessential amino acids.
The inclusion of fish offal in the conventional growing substrate for HI larvae influenced the main FA classes (
Table 1), but differently from the hypothesis, the saturated fatty acid (SFA) proportion increased (72.0% vs. 68.9% SFAs for HI2 and HI1 larvae, respectively), whereas total polyunsaturated fatty acids (PUFAs) decreased (6.99% vs. 12.6% PUFAs for HI2 and HI1 larvae, respectively). Such a reduction involved only the
n-6 PUFA fraction, whereas the
n-3 one showed a three-fold increase (1.57% vs. 0.53%
n-3 PUFAs for HI2 and HI1 larvae, respectively), thus resulting in a nutritionally valuable improvement in the
n-6/
n-3 ratio (3.45 vs. 22.9 for HI2 and HI1 larvae, respectively).
The FA profile of the quail breasts (% of total FAMEs) showed remarkable differences depending on the presence and type of HI included in the experimental diets (
Table 5). The inclusion of HI larvae increased the total SFA proportion compared to the C diet, with HI2 breasts showing a greater SFA proportion even compared to HI1 breasts (32.0% vs. 40.3% vs. 42.8% for C, HI1, and HI2 breast meat, respectively;
p < 0.001). The higher SFA content of quail breasts derived from HI-fed quails compared to C was attributable to the increasing percentages of C12:0, C14:0, and C16:0 (
p < 0.001) FAs (
Table 1). In addition, the monounsaturated FA (MUFA) proportion increased in the breasts of HI-fed quails, with the differences attributable to the different growing substrates used to farm
H. illucens larvae (18.8% vs. 22.7% and 22.2% for C, HI1, and HI2 breasts, respectively;
p < 0.001). The MUFAs affected by the dietary treatments to the greatest extent were the C14:1, C15:1, and C16:1 (
p < 0.01) FAs, although these specific FAs were present at low concentrations. As a result of the dietary treatment (
Table 1), the overall PUFA percentage differed in the C, HI1, and HI2 groups (
Table 5). Specifically, C showed the highest level compared to HI1 and HI2 breasts, which did not differ from each other (44.1% vs. 32.0% and 30.4% for C, HI1, and HI2 breast meat, respectively;
p < 0.001). Despite this, the two HI breasts showed different PUFA compositions: HI1 breasts highlighted a consistent decrease in both the
n-6 as well as the
n-3 PUFA fractions compared to the C group (
p < 0.001), whereas HI2 breasts exhibited the most intense lowering of the
n-6 PUFAs but a consistent increase of the
n-3 PUFA fraction, which was similar to that of the C group. The loss of
n-6 and the concomitant increase in the
n-3 PUFAs consistently reduced the
n-6/
n-3 ratio, which was the lowest in the HI2 breasts and the highest in the HI1 breasts (11.2 vs. 14.0 vs. 6.56
n-6/
n-3 for C, HI1, and HI2 breasts, respectively;
p < 0.001).
As a result of the changes in the FA profile of quail breasts belonging to the different dietary treatments, the health indexes of the quail breasts were also influenced (
Table 5): the atherogenicity index (AI) and the thrombogenicity index (TI) increased (
p < 0.001), thus worsening with the dietary inclusion of HI (both types). Furthermore, according to the different growing substrates used to farm larvae, different magnitudes in these changes were observed: HI1 breasts showed a lower AI but a higher TI compared to HI2 breasts. As a result of a growing SFA proportion in HI breasts compared to C, the hypocholesterolemic/hypercholesterolemic ratio (HH ratio) decreased, thus worsening, and the peroxidability index (PI) decreased, too, thus making the meat of HI1 and HI2 quails less susceptible to oxidative phenomena than that of the C group (61.7 and 66.5 vs. 81.9 for HI1, HI2, and C breast meat, respectively;
p < 0.001).
Overall, the dietary inclusion of HI larvae reared on different growing substrates into quails’ diets did not negatively impact the sensory profile of the breast meat, which showed similar traits in the three groups (
Table 6). The only exception was with regard to some specific textural attributes: juiciness and fibrousness. Specifically, the HI2 meat had the lowest juiciness compared to C and HI1 (4.53 vs. 5.01 and 4.90 for HI2, C, and HI1 breast meat, respectively;
p < 0.001). As a consequence, HI2 meat also exhibited the highest fibrousness, followed by HI1 and C meat (5.28 vs. 5.05 vs. 4.79 for HI2, HI1, and C breast meat, respectively;
p < 0.001). As indicated in
Table 7, the off-odors and off-flavors were not affected by the experimental diets.
The results of the retail display trial (
Table 8) showed that the dietary treatment did not play a role in affecting the broiler quail breast meat traits: Drip loss percentage, overall colorimetric characteristics, and oxidative status showed similar results throughout the retail display trial. The only exception was the lightness (L*) value measured at day 0 of the retail display, when HI2 meat had a higher value compared to C, with HI1 being intermediate (51.6 vs. 50.6 vs. 49.0 for HI2, HI1, and C breast meat, respectively;
p < 0.05).
4. Discussion
The physical breast meat traits observed in the present research (
Table 2) were overall satisfactory and in line with values reported for quails [
27]. The highest cooking loss shown by HI2 meat was similar to that observed by Cullere et al. [
8]. In the latter, breast meat derived from quails fed with the highest dietary inclusion level (15%) of a partly defatted
H. illucens larvae meal showed the highest cooking loss. In this case, a lower meat pH was also observed and it was hypothesized as causative agent of the higher cooking loss: as the pH value approaches the isoelectric point of proteins, their water holding capacity reduces thus generating a higher moisture loss. However, HI2 meat in the present trial had the same pH of the other experimental groups, and thus a causative agent for the observed different cooking loss percentage must be searched for elsewhere.
Even if the experimental diets HI1 and Control of the present experiment had slightly different absolute protein contents (23.9 and 24.3 g/100 g feed for HI1 and Control diets, respectively) [
18], the lower protein content observed in the breast meat of the HI1 quails compared to the Control group could not be directly attributable to this factor. It is now well established that not all nitrogen present in insects originates from protein: insects’ exoskeleton contain chitin, a polysaccharide containing N atoms, which typically falls within the amount of nitrogen quantified by the common Kjeldahl method. On the other hand, during the larvae’s sclerotization phase of development, different proteins harden the cuticle (exoskeleton) by linking the chitin fibers. This particular structural arrangement of the insect cuticle makes such proteins indigestible to animals [
28]. Up to a couple of years ago, these factors were ignored in the formulations of animal diets that included insects, thus technically resulting in diets that were only apparently isonitrogenous because the protein content of insects was generally overestimated [
29]. To solve this drawback, further research on this topic resulted in a recent recommendation of using a specific nitrogen conversion factor for insects (5.62) [
30]. This above-mentioned issue could be the main reason for the observed difference in the protein content of HI1 and Control quail breasts. Interestingly, for HI2 meat, the same finding was not applicable. It is known that different rearing substrates can determine diverse development rates in
H. illucens larvae [
31]: the HI2 larvae could have been slightly less developed than the HI1 larvae, and this could have led to different sclerotization stages of the cuticle in the two HI larvae. As a consequence, the amount of indigestible protein could have been different in the two HI larvae, thus leading to the observed results in breast meat protein content.
The results regarding the amino acid content of breast meat (
Table 4) further emphasized the above-mentioned speculations: HI1 meat had an absolute lower amino acid content compared to the Control meat (19.3 compared to 21.6 g/100 g meat), which determined the lower protein percentage in the HI1 group compared to the Control meat. However, in a previous study by Cullere et al. [
9], the same finding was not observed, as quails receiving increasing dietary inclusion levels (up to 15%) of a partly defatted
H. illucens larvae meal had similar protein and thus amino acid contents. This apparent discrepancy could be explained by the fact that, in Cullere et al. [
9], the tested insect source was a defatted meal derived from the same aged larvae that had been fed the same diets. Previous literature has shown that the defatting process concentrates protein and amino acids, therefore determining a higher amount of amino acids compared to full-fat larvae [
32]. When defatted
H. illucens larvae were incorporated into different avian species such as quail [
9], chicken [
33], and Barbary partridge [
10], the protein quantity of the derived meat was similar in the experimental groups.
The inclusion of dietary ingredients with cholesterol up to a certain threshold in poultry diets should only moderately affect meat because liver lipid metabolism can decrease cholesterol de novo synthesis as well as increase the transformation and transportation rate as a response to the dietary level: this mechanism is essential to guarantee optimal animal health, welfare, and growth [
34]. Despite this, independent of the growing substrate, the inclusion of 10% HI larvae into quail diets increased the cholesterol content of the breasts compared to the Control group. Literature studies considering this aspect are still limited, and available results are controversial: In a work by Cullere et al. [
35] testing
H. illucens fat as a soybean oil replacer in finisher broiler chickens, a 100% substitution produced breast meat with the same cholesterol content as the Control group. A similar finding was observed by Cullere et al. [
9] in broiler quails fed either a 10% or 15%
H. illucens meal. In the latter, however, a numerical but not significant increase in meat cholesterol contents going from the lowest to the highest inclusion level (71.6, 73.3, and 74.9 mg/100 g meat at 0%, 10%, and 15% inclusion levels, respectively) was noted. In past experiments studying quail metabolism, it emerged that these birds respond particularly quickly and intensely to dietary cholesterol content and that they can develop atherosclerosis relatively fast, with relevant differences depending on sex and genetics [
36,
37]. Based on such speculations, it would be interesting to investigate the effects of genetics, sex, and type of insect product fed to quails on their cholesterol metabolism.
The fact that HI1 meat showed the highest meat lipid content was unexpected, especially considering that the dietary fat content was similar in the three experimental groups and almost identical in the HI1 and HI2 diets [
18]. A possible reason to explain this could come from the results of the digestibility trial [
18], where the HI1 quails showed the highest apparent digestibility of starch and metabolizable energy, which could explain the higher meat lipid content observed in the present research. However, this finding was not confirmed by previous studies on quail [
9], chicken [
33,
38], or Barbary partridge [
10] fed with different inclusion levels of
H. illucens larvae protein meal or fat.
The results of the present research demonstrate once more that the FA composition of the
H. illucens larvae can only be moderately affected by the farming substrate. In this case, exploiting fish offal waste to modulate the larvae growing media improved the healthiness of their FA profile. In fact, the
n-3 proportion increased (+34% compared to HI1 larvae), almost exclusively due to eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, which are FAs typically found in marine fish sources that have important implications in human health mainly due to their positive effects on cardiometabolic risk factors [
39]. Despite this, the overall FA profile was similar in HI1 and HI2 larvae and coherent with the typical FA profile that has been reported in the literature for this insect species: rich in lauric (C12:0), myristic (C14:0), and palmitic (C16:0) FAs for the SFA fraction and with oleic (C18:1
n-9) and linoleic (C18:2
n-6) FAs as the main MUFAs and PUFAs, respectively [
10,
35]. The successful increase in the
n-3 proportions in the
n-3 HI-fed larvae was in agreement with previous studies testing the inclusion of fish offal [
17], fishmeal [
40], or brown algae (
Ascophyllum nodosum) in a growing substrate [
41]. The high SFA content of
H. illucens larvae is linked to their peculiar adult stage: In fact, after pupation, the buccal apparatus regresses, and then adults cannot feed anymore. For this reason, to survive, they rely on energy reserves accumulated during their larval stage. Furthermore, it seems that dietary PUFAs are primarily used by the larvae as an energy source (oxidation) and that extra energy is stored in their bodies mainly as SFAs and MUFAs [
40], thus explaining why their FA profile was poorly represented by PUFAs. As a direct effect of larvae FA profiles, when the 10% insect meal was included into the quails’ diets, their FA profiles were characterized by a 28% SFA increase and an overall reduction in MUFA (−5.5%) and PUFA (−57%) proportions. This had a direct effect on the FA composition of the meat obtained from quails fed the experimental diets. In fact, SFAs increased by 30% and PUFAs decreased by 29%. Even if dietary SFA intake in humans should be moderate, as they are associated with increased cardiovascular disease risk [
42], it must be stressed that SFAs play important roles in normal cellular and tissue metabolism and, among other things, they are essential constituents of cell membrane phospholipids, structurally part of important cell signaling molecules, and are involved in gene expression and in the modulation of cholesterol, fatty acid, and triacylglycerol synthesis and lipoprotein assembly, secretion, and clearance [
43]. Specifically, lauric acid, which accounts for the greatest part (about 65%) of larvae’s SFA percentage and for about 13% of the meat SFA proportion, is converted in human and animal bodies to monolaurin, which in turn aids in preventing viral, bacterial, and protozoal infections [
44]. Furthermore, 6–12 medium-chain FAs in general are efficiently absorbed, digested, and beta-oxidized in the gut and are thus very efficient energy substrates for both livestock and humans [
45]: they are reported to improve the gut’s health thanks to their intrinsic antibacterial and antiviral properties [
46]. The addition of HI to the quails’ diets reduced the overall dietary MUFA content, but the observed trend in the meat was not coherent in this sense: The meat of the HI1 and HI2 groups had about +15% MUFAs compared to the Control group. This outcome agreed with previous observations on quails fed increasing levels of a partly defatted
H. illucens larvae meal [
9] but not with those reported on broiler chickens whose dietary content of soybean oil was replaced by 50% and 100%
H. illucens fat [
35]. This could highlight, on the one hand, a particular intrinsic capability of quails to desaturate and elongate SFAs into MUFAs. On the other hand, it further corroborates the hypothesis that diets characterized by low fat and high carbohydrates have a role in the upregulation of the activity of the lipogenic enzyme ∆9 desaturase [
47]. In fact, the Control diet of the present study slightly differed in terms of fat and starch contents compared to those including the 10% HI larvae [
18]. Regarding meat PUFAs, it was observed that modulating HI larvae substrate with fish offal was a successful way to improve the
n-3 PUFA proportion in the meat of HI-fed quails and thus lower the breasts’
n-6/
n-3 ratio, which is considered a sort of target index for a meat to be considered as healthy [
48]. Specifically, as a result of the HI substrate modulation, in the present experiment a reduction of ~53% and ~41% of the
n-6/
n-3 ratio in HI2 quails compared to the HI1 and Control quails, respectively, was noted.
Research dealing with the effect of meat obtained from insect-fed poultry on its sensory characteristics is still limited. Despite the fact that
H. illucens larvae meals are characterized by a peculiar flavor [
49], existing knowledge seems to indicate that the impact of this emerging feed ingredient on the sensory traits of animal products is limited [
11]. The sensory profile of quail meat and that of eggs obtained from quails fed with
H. illucens larvae meal (up to a 15% inclusion level) did not differ from the Control group without insect meal [
9,
15]. In addition, the use of
H. illucens fat as an alternative fat source for broiler chickens did not affect the sensory traits of the derived meat compared to chicken fed with a conventional soybean oil diet [
35]. In this context, the present research is the first highlighting significant changes in the textural traits of quail meat: As meat of the HI2 treatment displayed the highest cooking loss, it was not surprising that meat juiciness and fibrousness were the lowest and the highest, respectively. This is because, regardless of the considered animal species, cooking loss has a great influence on the juiciness of meat [
50]. On the other hand, it was unexpected that higher fibrousness would be indicated for the HI1 meat compared to the Control. Furthermore, HI1 meat was also the richest in lipids, which should have had a positive effect on meat sensory characteristics, including textural and sensory perception [
51].
The results of the quail breasts subjected to the retail display trial are in agreement with Schiavone et al. [
38], who evaluated the impact of feeding
H. illucens fat to poultry on their meat quality, which was evaluated during refrigerated storage. Meat physical traits are responsible for its visual appearance, which has a key impact on consumer choice at purchase [
52]. In view of this, the fact that the meat of the three dietary treatments showed similar drip loss and comparable colorimetric characteristics is of utmost importance for marketing purposes. The highest L* value at day 0 of refrigerated storage observed in the HI2 meat was not expected, since the same result was not noted when studying the physical breast meat characteristics. In past studies, it was found that
H. illucens larvae contain carotenoids (about 2.00–2.15 mg/kg), which can positively affect the yolk yellowness of eggs produced by quails and hens fed with this larvae meal [
15,
53]. No mention of a possible effect on meat color/lightness, however, was found in literature that considered poultry species fed different inclusion levels and products derived from
H. illucens larvae. Interestingly, despite the intense modifications in the FA profile of the quail meat, its oxidative status remained unaffected throughout the retail display trial, ensuring that the meat, in this sense, was completely satisfactory from a qualitative point of view.