1. Introduction
The EU approval for the use of the insect meals in poultry nutrition is “not so distant in the future”, and the approval by the EU Member States could be possible during the first quarter of 2019 [
1,
2]. Thus, it is mandatory to understand all the possible aspects related to the chemical-nutritional characteristics, the effects on the animal health and welfare, the impact of feed and food safety of the different meals deriving from insects. This goal is not easy to reach as some characteristics can be modified according to the species and, within the species, in relation to the harvesting stage, the growth substrate, etc. In recent years, several studies pointed out the attention on the use of insect meals in growing broiler [
3,
4,
5,
6], quail [
7], and barbary partridge [
8,
9]. In laying hens, the available literature on the effects of insect meals on laying performance, egg quality, metabolic and nutritional effects is limited and very often the results are conflicting due to the different species of insects used, the different strains and age of lay of the hens utilized in the trials [
10,
11,
12,
13].
An important aspect concerning the use of insect meal as a feed ingredient, related to human and animal health, is the possible accumulation of mineral elements in the insect body during the growing cycle. Some elements (Cu, Se, Cr, Fe, Mn, Ni) are essential for biological functions, but the heavy metals like Cd, Pb, Hg, and As can induce adverse effects due to their potential toxicity and bioaccumulation in the food chain [
14]. To regulate animal dietary exposure to heavy metals, the EU Commission established maximum levels (MLs) for different undesirable substances in animal feeds [
15]. Data on the transfer of chemical contaminants from different substrates to the insects are very limited [
16]. Therefore, monitoring of toxic elements concentrations, in the insect meal and diets, is necessary from the point of view of both nutrition and contamination. The few studies on the mineral profile of insect meals [
7,
17,
18] often showed different minerals with a very wide range of variation according to the composition of the substrate used for the insects growing. For example, increasing the inclusion level of the brown algae,
Ascophyllum nodosum, in the growing substrate led to increased calcium, sodium, and magnesium and decreased phosphorus, manganese, and copper concentrations in
Hermetia illucens larvae [
17].
This study represents the continuation and completion of a previous trial [
12] in which the laying performance, blood profiles, and nutrient digestibility of hens fed
Hermetia illucens (HI) larvae meal from 16 to 40 weeks of age were investigated. Thus, the aim of the present research was to evaluate the effect of the inclusion of a partially defatted meal from HI larvae in the diet on the volatile fatty acids (VFAs) production in the caeca, intestinal morphometry, and brush border enzymatic activity of 40 weeks old layers. In addition, the research aimed to contribute to the knowledge of the trace and toxic elements’ concentration in insect meals.
4. Discussion
The trace minerals content of the insect meals is still poorly investigated, but it is an important goal to formulate appropriate diets for poultry and avoid a possible excessive excretion into the environment. In addition, the knowledge of the potential accumulation of toxic elements is mandatory to guarantee humans and animals safety. A recent study showed that the concentration of many minerals (magnesium, calcium, iodine, iron, sodium, and potassium) in black soldier fly larvae increased linearly as the level of the correspondent mineral in the growing substrate increased, while manganese remained stable in the larvae, despite varying concentrations in the media [
17]. The data available in the literature on the mineral concentration of the black soldier fly larvae showed a high variability [
7,
17,
18], and thus it is difficult to compare our results to the available data. The discrepancy of the results is tied to the different kind of substrates used for the insects’ growth but also, for some other elements as Zn, to the technological process following the larvae harvesting. However, the concentrations of Co, Ni, and Se found in the edible insect of our trial were lower than those of the conventional food. The insect meal had a higher amount of Zn, considering the inclusion level, so the only insect meal supplied a daily amount of Zn (331.1 and 662.3 mg/kg, respectively, for HI25 and HI diets) exceeding the correspondent requirements indicated by Hy-Line Brown commercial layers management guide [
33] (minimum required 80 mg/kg in a complete diet during the laying period).
Concerning the toxic elements, the concentration of Cd, Pb, and Hg was negligible in all the analyzed samples. The mean value of As was 0.23 mg/kg in the HI meal, approximately comparable to the data reported in the literature and lower than in the complete diet samples.
Compared to the MLs of heavy metals set by the EU Commission [
15], Cd, Pb, Hg, and As levels in the diets and insect meal were always lower than the maximum values established for the feeding stuff and feed materials. In fact, the EU regulation establishes the following MLs of heavy metals content in mg/kg (ppm) relative to a feed with a moisture content of 12%: the Cd MLs in the feed materials and complete feed are 2.0 and 0.5 mg/kg, respectively; the Pb MLs in the feed materials and complete feed are 10.0 and 5.0 mg/kg, respectively; the Hg MLs in the feed materials and complete feed are both 0.1 mg/kg; the As MLs in the feed materials and complete feed are both 2 mg/kg.
The inclusion of an insect meal from HI larvae as 25 or 50% substitution of the soybean meal proteins had several effects on the small intestine morphometry and enzymatic activity, as well as on the caecal microbial activity. The morphometry changes mainly occurred in the duodenum and jejunum, but there were also some interesting modifications in the ileum. In general, the villi height decreased as the inclusion level of the insect meal increased in the diet, this is in accordance with the decreased nutrient ileal digestibility recorded in the first part of this trial [
12]. The crypt depth was unchanged among the groups, or it tended to decrease (in the jejunum) as the level of the HI increased in the diet. The small intestine is involved in the digestion and absorption of almost all the dietary nutrients [
34]: the duodenum digests around 95% of the fats [
35]; jejunum digests and absorbs fats, starch, and protein [
36,
37]; the ileum is mainly involved in water and mineral absorption, but it also digests and absorbs fats, proteins, and starch [
34]. The morphological studies of the small intestine are often used to assess its functionality and, in general, an increased villi height is indicative of an improved intestinal function [
38]. Another important consideration is that the ileal villi in chickens are smaller and lower than those of the previous tracts of the small intestine, as in hens fed corn-soybean based diets, very little amount of nutrients are available beyond the jejunum [
39,
40,
41]. In the present trial, the height of the ileal villi was lower than that of the duodenum, but higher compared to the jejunum for hens fed both the insect diets. The effect of the diets on the intestinal villi height can be affected by the nutrient digestibility of the diets. As recorded in the first part of this trial [
12], the dry matter digestibility of the SBM and the HI25 diets (75.0% and 70.3%, respectively) in hens was higher (
p < 0.01) than that of the HI50 group (64.3) and the result was mainly attributable to the crude protein digestibility (86.2 vs. 81.1 vs. 76.1%, respectively, for SBM, HI25, and HI50,
p < 0.01). The low nutrient digestibility in the hens fed the insect meal is tied to the chitin, present in the insect exoskeleton [
3]. Thus, our hypothesis is that higher availability of nutrients in the duodenum and jejunum of the hens fed the SBM, increased the intestine functionality, improving the villi height. In insect fed hens, the lower nutrient digestibility induced an increased amount of the potential digestible nutrients in the ileum. Yamauchi et al. [
42] stated that an increased load of nutrients deriving directly from the duodenum to the ileum (both for jejunum dissection or different diets) might stimulate the ileal absorptive function, resulting in a compensatory development of the villi. In general, longer villi are the result of activated cell mitosis in the crypts [
43]; thus, a larger crypt area indicates a more intensive cell production. In the present trial, the only crypt depth was recorded, and this was unchanged among the groups in the duodenum and ileum tracts. However, the villi height to crypt depth ratio is strongly related to the epithelial cell turnover [
44]. In our trial, the cell turnover was higher in the SBM than in the insect meal groups for the duodenum and jejunum, while no differences were observed among the groups in the ileum.
The presence of HI meals in the diet did not affect the activity of both the disaccharases except for the maltase in the duodenum of the hens fed the highest level of HI meal. Recently, Khol et al. [
45] showed that the activity of maltase in the small intestine of mallard, chicken, and quail varied depending on the species and, in the mallard, on the intestinal tract. Working with geese, they also demonstrated an effect of the interaction with the protein and the fiber content of the diet, with the highest activity of maltase registered in the Low Protein-Low Fiber group. Likewise, in the present study, the limiting action of the chitin inside the HI meal on the availability of starch during the digestion process led to lower availability of substrate for the maltase activity.
The linear decrease of IAP in the duodenum and jejunum of the hens as the dietary insect meal inclusion increased, shows that the SBM group presents the highest intestinal functionality. Similar results have also been observed by Cutrignelli et al. [
46], when the inclusion of HI larvae meal as 50% substitution of soybean meal protein in laying hens decreased the IAP levels in the jejunum and ileum. This enzyme is considered an excellent marker for the crypt-villus differentiation in chicken [
47], and, in the present study, the inclusion of HI meal resulted in a negative effect in the jejunum on both the villi/crypt ratio and the IAP specific activity.
The γ-glutamyl transpeptidase plays an essential role in the final digestion and absorption of the dietary proteins being involved in the amino acid transport in the intestine [
48,
49]. Overall, the effect of the inclusion of HI meal on the activity of the γ-GT in the ileum seems to be in contrast with the increased ileal villi height, while it is in agreement with the weight gain results reported by Bovera et al. [
12].
The inclusion of the insect meal in the hens’ diet induced several modifications in the microbial activity in the caeca, as showed by the VFAs production, but the effects were particularly evident with the highest inclusion level (HI50 group). The increased total VFAs production in the latter was mainly due to a higher production of butyrate (+21.5%) and acetate (+13.3%) than in the SBM group, while the valerianic acid decreased in the two groups fed the insect meal. These results completely agree with the finding of Cutrignelli et al. [
46], in which the hens fed an HI meal in total replacement of the soybean proteins showed an increased production of butyrate (+62.6%) and acetate (+36.1%) than the control. Similar to our results, Loponte et al. [
6] found an increased amount of the total VFAs (+45.6%), acetate (+40.3%), and butyrate (+64.6%) in broilers fed a
Tenebrio molitor larvae meal as a complete replacement of the soybean meal. The increased activity of the microbial population in the caeca can be related to the chitin level of the HI diet, confirming the hypothesis of Loponte et al. [
6] and Cutrignelli et al. [
46]. However, another important point emerging from our research is that the chitin needs to be at a sufficient level to act as “prebiotic”, stimulating the intestinal microbial activity. Based on our analysis and taking into account the formula proposed by Marono et al. [
23] for the estimation of the insect chitin from the chemical composition, the amount of the chitin in the
H. illucens larvae meal used in this trial was 6.64% as feed. The feed intake of the hens involved in this trial, and reported by Bovera et al. [
12], was 99.97, 97.69, and 101.9 g/day, respectively, for the SBM, HI25, and HI50 groups. Thus, considering the inclusion level in the diets, the HI25 group ingested around 0.47 g/d of chitin, while the HI50 group ingested around 0.99 g/d. Our hypothesis is that the lowest level of chitin is not sufficient to modulate the microbial population activity in the hens. The butyric acid is considered to be the main enterocytes energy source [
50], and it is also necessary for the proper development of the Gut-Associated Lymphoid Tissue [
51]. It is reported that the VFAs, in general, have a bacteriostatic effect against some enteric bacteria, including
Salmonella typhimurium, and, in particular, the butyrate is related to the decreased amounts of Enterobacteriaceae in chickens [
52]. Thus, both the increases in the total VFAs and butyric acid can improve, through different mechanisms, the health of the hens’ intestine.
Very interesting are the significant changes in the mutual proportions of the butyric, isobutyric, and valerianic acid observed, which might indicate that not only the activity but also the interactions among the different microbial species have been modified. This is in accordance with the findings by Borrelli et al. [
53], who observed changes in the gut microbiota of hens fed HI larvae meal. In particular, these authors found a strong correlation between levels of the bacteria strains
Flavonifractor plautii,
Christensenella minuta, and
Alkaliphilus transvaalensis and high production of propionate, butyrate, and total VFAs; these bacteria are the principal contributors to β-N-acetylhexosaminidases and N-acetylglucosamine 6-phosphate deacetylase production, and these enzymes represent the key enzymes responsible for a higher VFAs production.