1. Introduction
The production of broiler meat requires substantial amounts of grain feedstuffs [
1] including immense quantities of soybean meal (SBM), the primarily used protein source in poultry production. One problem is that about 98% of crude protein (CP) originating from SBM and used for feed in the European Union (EU) in 2017/2018 was imported mainly from South America [
2,
3]. This illustrates the severe “protein gap” regarding production and demand of SBM and revealing the strong dependency on imports of protein-rich feedstuffs in the EU. Considerable effort has been made in finding alternative and more sustainable protein sources with low feed-food competition. In this respect, an interest in insects as food and feed has evolved [
4,
5]. Particular scientific research interest regarding suitability of insects in broiler feed has been aroused in the EU [
6]. Currently only insect fat and live insects are permitted as feed for farmed animals, while non-hydrolyzed proteins are prohibited. Nevertheless, an exception was made in 2017, authorizing the use of seven insect species as feedstuff for aquaculture in the EU [
7]. However, application of insects in pig and poultry feeds might soon be possible since a new risk profile from EFSA is expected in the near future.
Among about 2000 edible insect species worldwide,
Hermetia illucens (HI), also known as black soldier fly, may possess a nutritional composition suitable for poultry nutrition as HI meal provides excellent apparent metabolizable energy corrected for nitrogen (AMEn) and digestible amino acids (AAs) to broilers [
8,
9,
10,
11]. However, the nutritive value of HI is highly dependent on the rearing substrate [
12,
13] as well as the developing state of larvae at harvest [
14]. Nevertheless, HI larvae are characterized by a high CP concentration ranging from 37 to 63% in dry matter (DM), combined with a crude fat content varying between 7 and 39% in DM. As the high fat concentration may impair digestibility, processed insect products, such as fat-reduced (protein) meals, are of greater importance for poultry nutrition [
10,
15].
Suggestions about the optimal inclusion rate of HI larvae meal in broiler feeds are yet inconsistent with recommendations ranging from a maximum of 15 to 50% in the diet [
6,
16,
17,
18,
19]. Higher dosages of HI seem to negatively affect the animal performance [
17], which may be related to the high amount of N as non-amino acid N in larvae compared to plant protein sources [
20]. Based on these foregoing observations, it seems that HI larvae meal in low rates can be included in poultry diets without hindrance. However, to the authors’ best knowledge, limited digestibility data of different larvae meals are available, and the results are rather inconsistent. In addition, the mechanisms involved in the digestion of HI larvae in diets of broilers is poorly documented. Regarding this, digestion and absorption of ingested feed, as the main functions of the intestine, have a direct or indirect impact on animal health [
21], and for the effective nutrient digestion and absorption, the absorptive epithelium of the small intestine is of particular importance [
22]. Its organization in villi-crypts units aims to optimize nutrient absorption by maximizing the absorptive area [
23]. Therefore, we expected a correlation for performance and gut morphology of the birds when increasing HI larvae meal in the diets.
Next to morphological changes in the gut epithelium, microbial activity in the gut may show differences in the nutritive value of diets. As already known, the ceca are the main site of microbial fermentation in broilers, due to their high bacterial density being 100–1000-fold higher compared to ileal digesta [
24]. Microbial metabolites are generated and arise from protein and carbohydrate fermentation. The generated microbial metabolites from protein fermentation comprise amines, indoles, phenols, cresols, and ammonia and altogether may have adverse effects on broiler growth and performance, when present in high concentrations [
25]. Apart from microbial and endogenous protein, also resistant protein of dietary origin and firmly bound nitrogen, like the N present in chitin, flows into the ceca. Hence, the amount of ileal undigested protein entering the ceca is determined by the ileal digestibility of dietary protein. This means the higher the digestibility, the lower the amount of resistant dietary protein entering the ceca and, therefore, also the putrefactive bacterial fermentation [
24]. It is therefore of particular interest to investigate the cecal fermentation processes and compare to apparent ileal digestibility (AID) data to gain knowledge of the nutritional-physiological background of larvae meal digestion.
Therefore, the present study aimed to investigate the suitability and optimal inclusion level of defatted HI larvae meal by substituting 15 or 30% of CP from SBM with HI defatted larvae meal, corresponding to 4–10% HI larvae meal inclusion in the diets. Additionally, it was an objective to generate AID data to enable better assessment of nutrient quality of HI larvae meal. We hypothesized that, based on equal ileal digestibility of HI larvae meal and SBM, not only substituting low, but also higher amounts of SBM CP will lead to similar broiler performance, without impairments on gut morphology or alteration in microbial hindgut fermentation.
2. Materials and Methods
The feeding trial was approved by the Federal Office for Food Safety (Austria) according to § 10 Abs 1 Futtermittelgesetz 1999, BGBl. I Nr. 139/1999 (FMG), with the reference number BAES-FMT-FV-2018-0001.
2.1. Birds, Housing, and Diets
In total, 216 chickens 1 day old (Ross 308) of both sexes with an initial body weight (BW) of 40.3 g (±0.41 g) were purchased from a commercial local hatchery. The trial was carried out at a poultry research station rented by the University of Natural Resources and Life Sciences, Vienna, Austria, and housing of animals on wood shavings as litter material was carried out under compliance with the 1st regulation of keeping of animals (BGBl. II Nr. 485/2004). The average ambient temperature at the beginning of the study was 29 °C and was gradually decreased to 20 °C until the end of the experiment. The lighting schedule was 18 h light, 6 h dark. In order to receive similar mean weights per pen, animals were weighed at day 1 and correspondingly assigned to treatments. Thereby, birds were allocated to 18 pens with 12 animals each, resulting in 6 replicates per treatment.
All diets were calculated to meet the Breeder’s nutritional specifications [
26] within a three-phase feeding program: starter diet was fed from day 1 to day 14, grower diet was fed from day 15 to day 28, and finisher diet was fed from day 29 to day 36. Due to scarce and inconsistent data concerning larvae meal digestibility, the present diets were calculated on the basis of Ross 308 Broiler Nutrition Specifications (2019) [
26] of total AA. The control diet (CON) was based on corn and SBM. For calculation of the two experimental diets, the amount of CP supplied by SBM in the respective control diet of each phase was replaced in graded levels (15, 30%) by CP of HI larvae meal.
Hermetia illucens larvae were reared on wheat bran, cracked rye, water, and fat-protein stillage. Following drying at 80 °C, the larvae were partly defatted with a screw press and afterwards ground into a meal. The composition of HI larvae meal is shown in
Table 1. Thus, three different treatments were finally prepared, which are referred to as CON, substitution level (SL) 15 (SL15), and SL30. During all phases, diets were calculated to be both iso-energetic and iso-nitrogenous (
Table 2), and diets were provided for ad libitum consumption. Moreover, diets were balanced for AA according to the Breeder’s nutritional specifications. All diets were expanded before pelleting. The starter diet was fed in crumbled form (granulation gap 1.7 mm), whereas grower (2.3 mm) and finisher (2.8 mm) diets were offered as pellets. Titanium dioxide was administered (3 g/kg fresh matter) to finisher feeds (29–35 d) prior to pelleting as external marker to determine AID. Animals had free access to water during the whole experiment.
2.2. Performance Parameters
Animal BW was determined pen wise on days 1, 14, and 28 and individually on day 35 for calculation of the performance parameters average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Parameters were determined for each feeding phase as well as the overall experiment. Mortality was recorded as it occurred, and dead birds were weighed to determine the losses. Feed conversion ratio was calculated from ADFI and ADG.
2.3. Carcass Traits
At the end of the experiment (day 36), all broilers were weighed individually, stunned, and killed by bleeding. Subsequently, the following traits were collected, and weights recorded: dressing, eviscerated carcass (as the weight of the slaughtered broiler without blood), feather, giblets and intestinal tract, chilled carcass (as the weight of the carcass after 16 h of storage at 3 °C in a cooling chamber), carcass for grilling (as the carcass chilled without head, neck and legs at the hock joints), giblets (heart, liver, stomach), and abdominal fat.
2.4. Sample Collection
Directly after slaughtering, the intestinal tract was removed and opened from four representative broilers per pen (n = 72), i.e., two males and two females closest to the median of BW. Digesta from two gut sections (ileum (Meckel’s diverticulum until colon) and ceca (whole contents of both ceca)) were collected. To obtain enough digesta, homogenously mixed samples of four animals were pooled per pen, put into narrow mouth bottles, and immediately frozen at −20 °C until further analysis.
Samples for histological analysis were taken from two representative broilers (one male and one female) per pen (=36 animals), which were already taken for digesta sampling. Tissue samples were taken from the jejunum, halfway between the duodenum and the Meckel’s diverticulum, and the ileum 3–6 cm proximal to the ileocecal junction. Samples were washed thoroughly with ice-cold phosphate-buffered saline to remove the entire digesta content, embedded in slotted cassettes, and immersed in 4% paraformaldehyde for 48 h.
2.5. Chemical Analyses of Feed and Digesta Samples
Ileal digesta samples were thawed at 4 °C and freeze-dried. All samples were ground through a 1 mm sieve and homogenized. Caeca digesta samples were analyzed in fresh matter.
The proximate composition of all diets and HI larvae meal was analyzed in duplicate according to the standard procedures [
27]: dry matter (DM; method no. 3.1.4), ash (CA; method no. 8.1.1), ether extract (EE; method no. 5.1.1), ether extract after acid hydrolysis (EEh; method no. 5.1.2), and crude fiber (CF; method no. 6.1.2). Nitrogen content of the diets was analyzed using Dumas combustion method (DuMaster 480, Büchi AG, Flawil, Switzerland) (method no. 4.1.2) [
28] and multiplied by 6.25 to calculate CP concentration. Acid-detergent insoluble nitrogen (ADIN) was measured according to Licitra et al. [
29]. Additionally, feed samples were wet-ashed in a microwave oven (CEM Mars 6, CEM Corp., Matthews, NC, USA) to analyze Ca, Na, and K by flame atomic absorption spectrophotometry (AAnalyst200, Perkin Elmer Inc., Massachusetts, USA), and P photometrically (Tecan Group Ltd., Männedorf, Switzerland) using the vanado-molybdate method at 436 nm [
27] (method no. 10.6.1).
Feed and digesta samples were also analyzed for titanium dioxide (TiO
2) concentration as described by Leone et al. [
30]. Briefly, 0.5 g of sample was weighed and, after addition of a catalyst tablet, digested in 25 mL concentrated sulphuric acid at 400 °C for 115 min on a block digesta. After removing and cooling the tubes, digestion was decanted to a volumetric flask, and the volume was made up to 100 mL with distilled water. Following filtration, 5 mL of each sample was mixed with 1 mL 1M sulphuric acid and 1 mL hydrogen peroxide (300 mL/L). Subsequently, mixtures were measured at 405 nm using a UV–vis spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan) and compared to a titanium sulphate standard. The gross energy (GE) content in feed was determined by bomb calorimetry (IKA C 200, IKA Werke GmbH & Co. KG, Staufen, Germany). Total AA analyses of finisher diets and ileal digesta samples were determined by ion-exchange chromatography with post-column derivatization with ninhydrin, as described in detail by Figueiredo-Silva et al. [
31]. The AA composition in insect meals was provided by the manufacturer, and the total content was used for diet formulation.
2.6. Microbial Metabolites
The concentrations of biogenic amines in cecal digesta were analyzed according to Saarinen [
32] using reverse-phase HPLC (Waters 2695e Separations Module, Waters, MA, USA). A RP-18 column (InertClone™ 5 μm ODS (2) 150 Å, 250 × 4.6 mm, Phenomenex, Torrance, CA, USA) was used, and the detection was performed by a UV detector (Waters 2489 UV-visible detector, Waters, Milford, MA, USA). Data calculation was done by the software Empower 3 (Waters, Milford, MA, USA). For eluents 1 and 2, 0.1 M ammonia-acetate buffer (pH 5) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and acetonitrile (HPLC grade; Carl Roth GmbH + Co. KG, Karlsruhe, Germany) were used, respectively. For determination of ammonia and total lactic acid content, approximately 1.0 g of digesta sample was vortexed with 1.0 mL perchloric acid (1 M), afterwards it was allowed to settle for 10 min, and then 8 mL of double-distilled water was added and vortexed again. Afterwards, samples were placed on a shaker (POLYMAX 1040, Heidolph Instruments, Schwabach, Germany) for 1 h and subsequently centrifuged for 10 min at 3215×
g (Centrifuge 5810R, Eppendorf, Wesseling-Berzdorf, Germany). The supernatant was immediately stored in 2 mL tubes at −20 °C. Thawed samples were centrifuged at 12,066×
g for 5 min (Minispin, Eppendorf, Wesseling-Berzdorf, Germany), and a supernatant fluid was used for the further analysis of lactic acid and ammonia.
To analyze the ammonia concentration, the samples were diluted, and a mixture of 0.5 mL of salicylate-nitroprusside color reagent (blend of equal parts of sodium hydroxide 0.3 M, ddest. H2O and salicylate-nitroprusside solution) and 0.25 mL of dichloroisocyanurate solution (0.050 g dichloroisocyanurate dissolved in 50 mL ddest. H2O) was prepared; 1.0 mL of sample extract or standard solution (Ammonia standard solution ROTI®Star, Karlsruhe, Germany) was added immediately for a proper coloring reaction. Afterwards, samples were incubated for 1.5 h in the dark at room temperature, and subsequently the concentration of ammonia was analyzed spectro-photometrically (Tecan Austria GmbH, Grödig, Austria) at 660 nm.
Total lactic acid content in cecal digesta was analyzed according to the procedure of Pryce et al. [
33] with slight modifications regarding sample preparation and amount of reagent. Briefly, 100 μL sample or standard solution (Lithium L-lactate, Sigma-Aldrich, Steinheim, Germany) and 3.9 mL of precipitating reagent were mixed and centrifuged for 5 min at 3215×
g. The following procedure was carried out with 0.5 mL of generated supernatant liquid, 3.0 mL sulphuric acid, and 50 µL p-hydroxybiphenyl. Absorbance at 565 nm was read using a spectrophotometer (Tecan Austria GmbH, Grödig, Austria).
2.7. Histomorphology
Gut tissue samples were dehydrated and embedded in paraffin wax blocks, sectioned at 5 μm thickness using a microtome (Leica RM2255, Leica Biosystems GmbH, Wetzlar, Germany), and mounted onto glass slides (Menzel-Gläser Superfrost-Plus, Thermo Scientific, Braunschweig, Germany). Afterwards, sections were stained (Leica Auto-Stainer XL ST5010, Leica Biosystems GmbH, Nussloch, Germany) following the standard protocol for Alcian blue-periodic acid–Schiff (AB-PAS). All morphometric indices examined were made on six well-orientated villi and crypts, respectively. Villus height was measured from the tip of the villus to the villus-crypt axis, villus width (Vw) (at the villus-crypt axis), villus area as cross-sectional area of a villus measured above the villus-crypt axis, and crypt depth from the base of the villus to the submucosa. Furthermore, the villus height-to-crypt depth ratio was calculated. Goblet cells were counted in six villi and are expressed as number of cells per 200 µm of villus epithelium. Furthermore, the thickness of the submucosa and muscularis circularis was determined in six randomly selected points. For visualization, a light microscope (Leica DM 6000 B, Leica) and the software Leica Application Suite (Leica, Version 4.13) were used.
2.8. Calculations
Feed conversion ratio was calculated according to the following equation:
The following equation was used to calculate the AID:
2.9. Statistical Analyses
The general model was:
where Y
ij is the dependent variable, μ is the overall mean, T
i is the treatment effect, and e
ij is the residual error, calculated using the Mixed procedure in SAS (Version 9.4; SAS Inst. Inc., Cary, NC, USA).
Pens are considered as the experimental unit for performance, AID and microbial metabolites, and animal for carcass yield and histological parameters. Significance was defined at p ≤ 0.05 and a statistical trend at p ≤ 0.10. Results are expressed as least-squares means, and differences between the least-squares means were tested post hoc using Tukey’s test.