DO INSECTS AS FEED INGREDIENT AFFECT MEAT QUALITY?

The development of sustainable feed ingredients for monogastric livestock is nowadays considering insect meals and oils to replace or supplement conventional feedstuffs. Although the regulation on the use of insect products differs among countries resulting in restrictions on use in the diets of monogastric meat producers, global research is exploring all the strengths and weak-nesses of their inclusion. Therefore, whereas the scientific literature has extensively studied both the relationship between insect farming systems and safety (potential health risks), and between the dietary use of insects and the nutritional value of diets and production performance of farm animals (fish, poultry, swine, rabbit), the relationship between insect-containing diet and meat quality has only recently been considered. The present review therefore aims to collect the results of the studies that have related the dietary use of some insect species, such as the black soldier fly (Hermetia illucens),


Introduction
Statistics about demographic trends depict that by 2050 the World's population should reach 9.1 billion people. This scenario is putting pressure on the search for alternative and sustainable feed resources for the livestock sector. This was officially emphasised by the Food and Agriculture Organisation (FAO) of the United Nation in Report "The future of food and agriculture -Alternative pathways to 2050" [1]. Among the possible alternative feed (and food) sources, insects are seen as one of the most effective alternatives to improve global food and feed security, with remarkable potential sustainability [2]. In fact, insects generally reproduce quickly, have fast growth and high feed conversion efficiency, and can be reared on a wide range of bio-waste streams, thus becoming an effective natural tool to recycle waste into nutritionally rich feed/food ingredients. Compared with traditional protein production, that of insect has a really low hydric and ecological footprint, and its production requires small lands to be exploited for very high yield [3].

Why insects in animals' diet?
In addition to the above reasons, the use of insects in animal feeding is supported by the fact that numerous animal species among birds and mammals consume insects as part of their natural diet. About 80% of birds are reported to include insects in their diets [4], among which there are also the chicken (Gallus gallus), the turkey (Meleagris gallopavo), the guinea fowl (Numida meleagris), the quail (Coturnix coturnix), and the ostrich (Struthio camelus), which are species of interest for food production in different Regions of the World [5][6][7][8].
There are also some mammal species (such as the wild boar) that have a varied diets, in which insects are a part of them. Among the monogastric mammals used as a food source in many Countries, there is the farmed rabbit, which naturally does not include insects in its diet, but the lipid source generally consisting of sunflower oil and soybean oil could be partially replaced with insect oil, as alternative and sustainable feed resource.

Insects as feed ingredient
In 2014 the first international conference on "Insects to feed the world" was organized by the FAO [13] which proposed, from the first time, the use of insects as promising alternative food and feed sources as a possible solution related to the expected demographic growth. Since that time, a new research field has emerged and an impressive number of articles have been published (see reviews of [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]), with an exponential increase in scientific knowledge on insects as feed.
Most studies have focused on the potential industrial application of certain insect species and have shown that black soldier fly (Hermetia illucens) and yellow mealworm (Tenebrio molitor) have great potential in providing largescale and high-quality nutrients for aquaculture, poultry and pig diets, exploiting bio-waste and organic sidestreams [30].
In parallel, an increasing number of research is testing the feed use of insects by focusing on the circular economy of small and medium-sized livestock farm, with the aim of making them independent from the global market, thus differently sustainable, or focusing on niche products such as meat from native breeds to preserve the biodiversity, or raised according to the organic system [29].

Regulation
Geographically, there are three main legislative scenarios: 1) the Anglo-Saxon countries (Australia, Canada, New Zealand, UK, USA), for whom insects are not treated as unique/novel food and feed, and therefore the food and feed agencies have generally approved import and sales upon fulfilment of certain quality and safety requirements; 2) the Asian, centre-and south-American, and African countries, for which insects are usually considered a conventional food and feed; 3) the European Union, for which it is necessary to establish rules and supply approval before permitting any trade of a specific food/feed product.
The result from these extremely diversified scenarios, is that also the legislation structure of different areas around the World regarding insects' use as food and feed is extremely heterogeneous. To respond to the new food and feed trends worldwide, and thus market exigencies and perspectives, the legislative frameworks of countries are rapidly changing.
In the European Union (EU), there is no history related to possible use of insects as food. In 2015, the European Parliament established that insects could fall into the "novel foods" category, and consequently they are subject to the consequent approval processes. Recently, EFSA [31] approved the yellow mealworm as a novel food, and the European Commission is discussing a regulation authorising this insect as a food.
Likewise, there is no history in the EU regarding the use of insects as feed application. Only in 2014 the FAO conference triggered a gradual change in the EU legislative framework, which is still evolving. Currently, processed animal proteins (PAPs) extracted from insects are allowed to be used in aquaculture, laboratory, companion and fur animals (regulation (EU) no. 2017/893), whereas for poultry species and pigs discussion is still going on, thus legislation still needs to be updated. Conversely, the fat fraction extracted from insects is allowed as feed ingredient for any animal species. The regulation allows the use of seven insect species: the black soldier fly (Hermetia illucens), the yellow mealworm (Tenebrio molitor), the lesser mealworm (Alphitobius diaperinus), the house cricket (Acheta domesticus), the field cricket (Gryllus as-similis), the banded cricket (Gryllodes sigillatus), and the common house fly (Musca domestica), specifying also the substrates allowed to feed insects. Regulation (EU) 2017/1017 permits the use of live or dead terrestrial invertebrates with or without treatment as feed material, but not as processed as described in Regulation (EC) no. 106/2009. Thus, invertebrates are considered as a suitable material for a feed at all the stages of their lives, except for species that adversely affect plants, animal, or human health.
Within the EU, however, starting from the above-mentioned common legislative framework every country has adopted this legislative framework differently. As an example, Belgium, The Netherlands, Great Britain, Denmark and Finland, refer to national laws which allow the production, marketing and trade of insect-based products. Instead, Germany show a limited degree of tolerance, whereas other states (i. e., Italy), have close to zero tolerance.
Recently (on May 25 2021), delegates of the EU-27 in the EU Standing Committee on Plants, Animals, Food and Feed (PAFF Committee) backed draft regulation aimed at setting EU harmonised standards for insect frass and the addition of silkworm to the list of seven approved insect species for use as protein in aquaculture feed [32]. The silkworm species was already approved for use in the feed of non-food producing animals, and was evaluated by the EFSA in its insect protein risk profile opinion in 2015. On 22 June 2021 the ENVI (Committee on the Environment, Public Health and Food Safety of the European Parliament) backed the draft Commission Regulation amending Annex IV to Regulation (EC) No 999/2001 which would remove the ban on the use of PAPs (including insect-derived protein) in poultry and pig feed, which was approved by the European Commission on 17 August 2021, and it will enter into force on the twentieth day following that of its publication in the Official Journal of the European Union [32].

Insect nutritional sources and formulation
To exploit the maximum potential as feed ingredient, insects are processed to obtain whole insect meal (full-fat), protein meal (PAPs; only for aquaculture, pet and fur animals) which can be defatted or contain some proportion of lipids according to the extraction method, and fat/oil (for all animal species). In addition, there are also bioactive compounds, such as the chitin [33], the 1-deoxynojirimycin (1-DNJ, [34]), and the lauric acid [35] that can be extracted from insects and used for different industrial applications.
The high variability of the nutritional composition of insect species can be an issue when focusing on its use as feed ingredient. In particular, the quantity and quality of insect lipids play a leading role, since their nutritional contribution varies primarily according to the insect species, their living substrate and growth stage, and then, according to the degree of lipid extraction of the insect meal [2]. Therefore, the use of defatted insect meal ensures a more constant feed formulation.
The extracted insect fat/oil can serve as feed ingredient alternative to fish and other vegetable oils, but also as food ingredient, in the cosmetic industry and as biofuel. The fatty acid (FA) profile of insects lipids can be very extreme: two examples are the Hermetia illucens and the Bombyx mori. The fat extracted from Hermetia illucens larvae contains 60-79% SFA. Conversely, the Bombyx mori chrysalis is very rich in omega-3 PUFA [36] and its oil presents a favourable n-6/n-3 ratio of 0.17 (personal communication). Thus, combining full-fat or partially defatted insect meal from different species could help ensure the best FA profile for animal feed [3]. The amino acid profile also differs between the different species of insects; in the case of the Tenebrio molitor larva the content of all individual amino acids was found to be higher than that of barley, fish, brewer's yeast, beef/veal, and crustaceans, except for lysine, which was slightly higher in brewer's yeast [31].
Research studies conducted so far have tested a wide range of levels of substitution (5-100%, mainly with fishmeal or soybean meal) or inclusion (0.75-60%) of insect meal, to find the best level, to cover nutrient requirements and to maximise growth and health performance, and product quality from farmed aquatic and terrestrial animals (see review of [17]). Pioneering research is also testing the effect of feeding live larva on poultry, but has so far only focused on improving animal welfare [37] or laying hen egg production and quality [38].

Insects in feeds and meat quality
The effects of dietary insect products (larva meal, prepupa meal, oil) inclusion on the quality of food-producing terrestrial animals have been studied mainly in poultry, with sporadic and recent interest in porcine and rabbit species. The purpose of this review is to provide updated literature on the use of insect-based products as feed for meat-producing animals, detailing the effects on the physico-chemical-sensory quality of the meat obtained. The review will consider the black soldier fly (Hermetia illucens), the yellow mealworm (Tenebrio molitor), and the silkworm (Bombyx mori), the first two for the greatest commercial interest in the EU, the third because it is potentially interesting in improving the dietary-nutritional value of meat. To be used as feed compounds, insect products can be used partially processed (dried larva) or processed (partially or totally defatted meals, dechitinised meals, fats/oils). The use of live larva in livestock feed is in its infancy and currently poses some technical limitations. Regarding the inclusion/substitution level of insects and insect-derived products in feed, a wide range has been tested, and for each insect species the best inclusion/substitution range is being identified.

Dietary inclusion of black soldier fly (Hermetia illucens) and meat quality
The black soldier fly larva is one of the most used organism for aquaculture and one of the best studied for both aquaculture and poultry feeding. The black soldier fly larva averagely contains 43.1±5.05 g protein /100 g DM, and the amino acid profile is rich in leucine (6.72 g/100 g protein), lysine (6.22±1.08 g/100 g protein), and valine (5.38±0.82 g/100 g protein). Nutritionally important is also its contribution in calcium (24.1±12.8 g/kg DM) and phosphorus (6.01±1.77 g/kg DM) (see review of [29]). As aforementioned, the amount of larva fat and its FA profile are extremely variable and depend on the type of substrate. A description of the results obtained on the meat quality of poultry, pigs and rabbits is provided below.
Considering that the FA profile of the meat of monogastric animals is in line with the pattern of that of their diet, and that the black soldier fly (whatever its form) is rich in saturated fatty acids (SFA; approximately 70% of the total FAME, of which 43% is represented by the C12:0 [48] it follows that the proportion of SFA in meat increases as a function of the dietary inclusion level of the black soldier fly. In majority of the cases, this implies a worsening of the n-6/n-3 ratio [43], but either insect defatting or their food substrate, may not change [49] or improve [45] the omega-6/omega-3 ratio. If the FA profile of the meat is changed by the inclusion of the black soldier fly in the poultry diet, changes in the lipid oxidation of the meat is also expected. However, most of the studies did not observe changes in the oxidation of meat lipids in animals fed black soldier fly [40,51,53]. However it is interesting to note that Choi et al. [39] observed a significantly low TBARS value on fresh meat after 7 days of refrigerated storage, and authors attributed it to the improved antioxidant activity (measured through the DPPH radical scavenging activity) of the meat due to the inclusion of the black soldier fly in the diet.

Pig
The results until now obtained on feeding pigs with inclusion of black soldier fly meal highlighted any influence on the tested physical meat quality (pH, colour WHC, shear force; Table 2) [57,58]. As regards the meat proximate composition, Altmann et al. [57] did not observe differences, whereas a more recent study of Chia et al. [59] found higher protein content in groups fed with black soldier fly meal, but not in that with the highest substitution level.
The meat from pigs fed black soldier fly meal had higher concentrations of K, Fe and Zn [59], thus providing additional functional and nutritional minerals for humans. The dietary inclusion of 4% black soldier fly meal increased the marbling score of the Longissimus thoracis muscle, and upregulated the expression of genes related to lipid metabolism and to myosin heavy chain (MyHC-IIa) isoform, likely inducing the muscle fibre transition towards more oxidative fibres [58]; however the effect was not observed at the 8% inclusion level, and therefore further research should be provided to support this finding.
As expected, the lipids FA profile of the pork meat was affected by the dietary inclusion of black soldier fly meal, but to a lesser extent than in poultry. In fact, even if some single FA significantly differed (C12:0, C14:0, C16:1, C18:3 n-3, C20:4 n-6, C20:5 n-3, C22:6 n-3), no difference in FA classes or in n-6/n-3 ratio was observed [58]. The only sensory evaluation conducted so far, which considered 26 attributes, revealed statistically significant differences only in odour intensity and juiciness with the inclusion of insect meal in the diet, both considered as sensory improvements [57].
The exiguous number of research conducted until now on the use of black soldier fly meal in pig diets does not allow a detailed assessment of their effect on meat quality, but the response appears to be slightly more subdued than that observed in poultry, with positive implications especially in terms of FA profile.

Rabbit
For rabbit diets, the studies so far conducted only considered the fat obtained from the defatting of the black soldier fly meal (Table 3). Dalle Zotte et al. [60] evaluated the total replacement of the dietary fat source: linseed oil vs black soldier fly fat at 2 inclusion levels (3% and 6%), whereas [61] the partial (50%) or total replacement of soybean oil with black soldier fly fat.
The first study highlighted differences in rabbit meat colour (increased redness) and in oxidative stability (lower TBARS), whereas the second one did not find difference in meat physical traits, proximate composition and sensory evaluation. Both studies obtained the same response in terms of TBARS values, and similar response for the FA profile, that is oriented towards higher saturated FA proportion (C12:0 and C14:0).
These two studies provided new insights into the use of black soldier fly fat in rabbit diets, considering it an excellent substitute for commonly used oils and fats.

Dietary inclusion of yellow mealworm (Tenebrio molitor) and meat quality
The yellow mealworm larva is gaining attention as a source of protein for food purposes worldwide, and, recently, the EU included it in the Union list of authorised novel foods [62]. Yellow mealworm is also considered  a nutritionally suitable substitute for fishmeal and soybean for aquaculture and poultry diets, although its cost is currently too high and cannot financially compete with standard feed sources. It should be emphasised that the strength of this feedstuff therefore lies in the high protein content (Nx6.25: 56-61%), characterised by a high biological value, as it includes all the essential amino acids, in favourable proportions. Furthermore, it is a rich source of phosphorus [29] and potassium [31]. Fat (25-30%) contains approximately 24% saturated FA and polyunsaturated FA, and 50% monounsaturated FA, resulting in an omega-6/omega-3 ratio of 24 [31].

Poultry
Contrasting results were obtained for physicochemical traits of meat from chicken broilers, apparently not depending on the inclusion level of yellow mealworm meal (Table 4). However, the majority of the studies did not observe change in the meat pH, colour, moisture loss, shear force, and fatty acid profile [63][64][65]. Instead, [66] observed that the variables considered (WHC, lipids, ash, TVB-N, and sensory attributes) worsened as the level of yellow mealworm in the diet increased, and the authors partly attributed this trend to the possible presence of oxidised fat in dried insect meal. On the other hand, no other studies, testing higher yellow mealworm inclusion levels, found adverse effects on sensory traits; on the contrary, [67] observed an improvement in meat juiciness and tenderness. When other poultry species were considered, such as Barbary partridge [47] and quail [68], no substantial differ-ences in the meat physicochemical traits were observed due to the use of moderate to high levels of yellow mealworm in the diet. Only meat colour changed in both bird species, however with an unclear pattern, whereas the FA profile of the barbary partridge meat was significantly affected by the dietary yellow mealworm, particularly at the higher substitution level (50% of the soybean meal) [47]. FA changes resulted in the reduction of the C18:0 and omega-6/omega-3 ratio (P<0.05) and the increase of C14:0, C15:0, C16:1, and C18:1.

Pig
Only one research has considered the use of yellow mealworm meal as dietary protein source in growing pigs [69] (Table 5). Authors tested the hypothesis that partial or complete replacement of a conventional protein source by yellow mealworm larva meal can influence the intermediary metabolism of the pig.
The omics-techniques on key metabolic tissues (liver, muscle and plasma) demonstrated that the insect meal from yellow mealworm can be used as a dietary source of protein in pigs without strongly impairing their metabolism, and consequently their growth performance. In addition, the unaltered plasma and liver triglycerides and cholesterol concentrations rather indicated that the insect meal had no effect on lipid metabolism in pigs. Studies in this regard are in their infancy, and further research is desirable to evaluate pros and cons, in order to provide precise information on the possibility of using yellow mealworm meal in pig feeding.

Rabbit
The dietary inclusion of 4% yellow mealworm meal did not modify the proximate composition of the rabbit meat ( Table 6). The amino acids content was modified by the dietary inclusion of insect meal, but the pattern observed in the 2 meat portions (hind leg and saddle) was not unidirectional, suggesting going deeper into the study of this effect. Notably, phenylalanine and lysine significantly decreased in hindleg meat, whereas threonine, isoleucine and methionine significantly increased, and tryptophan decreased in saddle meat [70]. In the same study, the authors observed that the effect of the dietary inclusion of 4% yellow mealworm on the FA profile of rabbit meat was negligible, leading only to an increase in total MUFA (from 22.0 to 22.9% total FAME, for control and treated groups, respectively; P<0.05) due to the increase in C12:0, and a decrease in EPA and DHA (P<0.05); The other FA classes and the omega-6/omega-3 ratio remained unchanged.
Based on these first results, despite being derived from a single study, it appears that a low inclusion level of dried mealworm larva in the rabbit diet does not appear to have adverse effects on meat quality. However, future studies are needed to consolidate the results obtained so far.

Dietary inclusion of silkworm (Bombyx mori) and meat quality
The silkworm pupa is characterised by high protein content (53.9% in the full-fat meal, 66.7% in the defatted meal), by a variable amount of lipids (29% in the full-fat meal, 9.5% in the defatted meal), the latter able to provide an extremely healthy FA profile (omega-3 FA: 29.5% in the full-fat, and 31.5% total FAME in the defatted meal), suggesting that it is a valuable nutritional ingredient for feed of different monogastric livestock species [36].

Poultry
Silkworm pupa meal has been successfully included in chicken broilers diet (Table 7), as it produced no effect neither on colour values and lipids content [71] nor on sensory analysis [63] [67] of the meat. A slight effect has been observed for lipids (3.56 vs 4.48% for leg meat of control and treated, respectively) in the study of [63] and for protein and ash contents (however not coherent with the inclusion level; [71]). Meat pH increased with the silkworm meal inclusion level but it did not impair meat colour [71].
The best result of the dietary inclusion of silkworm pupa meal in chickens concerns the FA profile of the meat lipids: the PUFA n-3 increased, and the omega-6/omega-3 ratio decreased with the increase of the dietary silkworm meal substitution level (P<0.01). The C18:3 n-3 content in breast meat ranged from 6.75 to 15.0 to 28.4 mg/100 g meat, for control, 25% and 50% silkworm meal inclusion level, respectively (P<0.05; [71]).

Rabbit
The silkworm pupa has been tested on rabbits diet in two forms: meal [70,72] and oil [73,74] (Table 8). Meat proximate composition was not modified by the silkworm pupa products [70,72,74] as well as the meat physicochem-   The amino acid profile slightly changed with the silkworm pupa meal inclusion in the rabbit diet: tryptophan increased and lysine decreased (P<0.05) in hind leg meat, whereas isoleucine increased (P<0.05) in saddle meat [70]. Also the cholesterol content significantly decreased in both meat cuts of animals fed the silkworm pupa meal, thus reducing the already low level of cholesterol in the rabbit meat [70].
Similarly to what observed for the poultry meat, the greatest effect of the use of silkworm pupa products is on the FA acid profile of the rabbit meat obtained. The 5 and 10% inclusion level of silkworm meal considerably increased the levels of C18:3 n-3, C22:5 n-3 and C22-6 n-3 in hind leg meat [72], but a lower inclusion level (4%) obtained similar results for omega-3 FA, and favourably reduced the omega-6/omega-3 ratio of the same meat portion (ratio of 4.5, compared to that of 7.8 of the control group; P<0.01 [70]. The 100% substitution of sunflower oil with silkworm pupa oil significantly (P<0.01) lowered n-6 FA and increased n-3 FA, resulting in a omega-6/omega-3 ratio of 7.7 (compared to that of 18.9 of the control group) in hind leg [73].

Conclusion
The great economic impulse towards the use of insects as food and feed for the ecological-environmental sustainability purpose has generated new companies producing insect meal and derivatives. A flywheel of interest has therefore been generated on several fronts, and the use of these products increasingly requires confirmation of safety and efficacy. In the last 5 years, numerous research has been conducted relating to the use of insects for alimentary use. Many of these aimed at the feed sector, which however has mainly considered the effect of their use on animal ante-mortem variables, whereas the study of the effects on nutritional, rheological and sensory quality of the meat has only intensified significantly in the last 2 years. This review focused on collecting and describing the results of research conducted so far on the effect of insects as feed on the meat quality of terrestrial monogastric animals. The results showed different effects, more of these depending on the insect species than on the animal species that benefited from them. Overall, no adverse effects were observed on meat quality. Only the meat's FA profile was affected by the insect species included in the diet, suggesting its improvement through manipulation of the insect substrate, or the use of mixtures of insect meal or oil from different insect species.