Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 https://doi.org/10.1186/s40104-019-0325-x RESEARCH Open Access Partially defatted black soldier fly larva meal inclusion in piglet diets: effects on the growth performance, nutrient digestibility, blood profile, gut morphology and histological features Ilaria Biasato1, Manuela Renna2, Francesco Gai3, Sihem Dabbou2, Marco Meneguz1, Giovanni Perona4, Silvia Martinez5, Ana Cristina Barroeta Lajusticia6, Stefania Bergagna7, Luca Sardi8, Maria Teresa Capucchio2, Enrico Bressan1, Andrea Dama1, Achille Schiavone2,3* and Laura Gasco1 Abstract Background: The aim of this trial was to investigate the effects of different inclusion levels of a partially defatted black soldier fly (BSF, Hermetia illucens L.) larva meal on the growth performance, nutrient digestibility, blood profile, gut morphology and histological features of piglets. A total of 48 newly weaned piglets were individually weighed (initial body weight (IBW): 6.1 ± 0.16 kg) and randomly allocated to 3 dietary treatments (4 boxes as replicates/treatment and 4 animals/box). BSF larva meal was included at increasing levels (0% [BSF0], 5% [BSF5] and 10% [BSF10]) in isonitrogenous and isoenergetic diets formulated for two feeding phases: I (from d 1 to d 23) and II (from d 24 to d 61). The weight gain (WG), average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated for each feeding phase and for the whole trial. The haematochemical parameters and nutrient digestibility of the piglets were also evaluated. A total of 3 piglets per box were slaughtered on d 61 and the slaughtered piglets were submitted to morphometric investigations and histopathological examinations. Results: No overall significant differences were observed for growth performance (P > 0.05), except for the ADFI of phase II, which showed a linear response to increasing BSF meal levels (P < 0.05, maximum for the BSF10 group). Dietary BSF meal inclusion did not significantly influence the blood profile, except as far as monocytes and neutrophils are concerned, and these showed a linear and quadratic response, respectively, to increasing BSF meal levels (P < 0.05, maximum for the BSF10 and BSF5 groups, respectively). On the other hand, the nutrient digestibility, gut morphology and histological features were not affected by dietary BSF meal inclusion (P > 0.05). Conclusions: The obtained results show that a partially defatted BSF larva meal can be used as a feed ingredient in diets for weaned piglets without negatively affecting their growth performance, nutrient digestibility, blood profile, gut morphology or histological features. Keywords: Hermetia illucens L., Histopathology, Insect meal, Performance, Weaning pigs * Correspondence: achille.schiavone@unito.it 2 Department of Veterinary Sciences, University of Turin, Largo P. Braccini 2, 10095 Grugliasco, (TO) Turin, Italy 3 National Research Council, Institute of Science of Food Production, Largo P. Braccini 2, 10095 Grugliasco, (TO), Italy Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 2 of 11 Background Spranghers et al. [25] have recently reported gut antimicro- A recent FAO forecast has indicated that the world will be bial effects and unaffected gut morphology in weaned piglets inhabited by over 9.5 billion people by 2050 and that fed BSF prepupae, but no information related to BSF larva animal protein production will have to more than double utilization is currently available. Furthermore, there is still a to meet the increasing food demand [1]. Within the lack of data about the potential diet-related, histopatho- livestock sector, pigs constitute one of the fastest growing logical changes of organs that could be added to the know- sub-sectors. Furthermore, as a result of changes in con- ledge on blood parameters in animal health assessments. sumption patterns, the world’s pork meat production has On the basis of the above reported background, the increased from 95 to over 130 million tons in the last 15 present study has been aimed at evaluating the effects of a years, and currently represents the second most consumed partially defatted BSF larva meal on the growth perform- meat in the world [2]. Furthermore, it has been estimated ance, nutrient digestibility, blood profile, gut morphology that this production will increase by over 40% by 2050 [1]. and histological features of piglets, in order to increase Soybean meal (SBM) is the most frequently used protein knowledge and provide novel information on the potential source for pig compound feeds, with a recorded global use of insect meal in pig nutrition. world production of about 300 million tons in 2016 [3]. Compound feeds play a primary role in the world’s live- Methods stock production, with an overall annual amount of about The present trial was carried out at the Teaching Struc- 1 billion tons [4]. Plant proteins (e.g. SBM), fishmeal and ture of Veterinary Medicine of the University of Turin other processed animal proteins have been used extensively (Italy). in compounds feeds. However, these conventional sources are no longer considered sustainable and are limited by BSF larva meal and experimental diets European regulations and/or increasing prices [5]. There- A partially defatted BSF larva meal, obtained by process- fore, a crucial challenge for future years is the discovery of ing larvae reared on a vegetable by-products substrate, new and sustainable protein sources for animal feeds. was purchased from Hermetia Deutschland GmbH & Co. Insects are one of the most promising novel protein KG (Baruth / Mark, Germany) and used in the trial. The sources [6–8], since they are rich in proteins and have BSF larva meal was partially defatted by means of a mech- an adequate amino acid profile, lipids and other valuable anical process which was performed using high pressure nutrients such as vitamins and minerals [6]. Moreover, it and without any solvents. No other information was pro- has been claimed that they are highly sustainable, as vided by the producer regarding the substrate or process- some species can be mass reared in organic streams at a ing methodologies, as this information is considered much lower environmental cost than conventional pro- confidential. A total of 3 experimental diets were formu- tein sources [9, 10], thus exalting the concept of Circular lated to have the same net energy (NE) and crude protein Economy. Among the various suitable insects, black sol- (CP) levels. The diets for each dietary treatment were dier fly (BSF) (Hermetia illucens L., HI) is one of the divided into 2 phases: phase I (days of trial: 1 to 23) and most promising species. It is a cosmopolitan and world- phase II (days of trial: 24 to 61). The diets were prepared wide Diptera that belongs to the Stratiomyidae family to include, on a feed basis, increasing levels of BSF larva [11]. BSF larvae can be reared on several organic sub- meal in substitution of 0, 30% and 60% (BSF0, BSF5 and strates [12, 13] and can provide high-value meal that is BSF10, respectively) of SBM (Phase I) or 0, 32% and 65% rich in nutrients when processed [6]. Research on insect (BSF0, BSF5 and BSF10, respectively) of SBM (Phase II). meals as animal feeds has increased rapidly, with prom- The diets were formulated to meet the nutrient require- ising results having been observed for poultry [14–16] ments of piglets [30]. An aliquot of 0.5% of titanium oxide and fish [17–19]. Dietary insect meal inclusion for pigs (TiO2 – Nova Chimica Srl) was added to all the diets to has also attracted increasing interest, with the authors’ determine the nutrient digestibility at the end of the two attention being focused on growth performance [20–25], feeding phases, using the index method reported by nutrient digestibility [23, 25, 26] and blood profile modi- Zhang and Adeola [31]. Feed and fresh water were pro- fications [22, 23]. Recent studies have also investigated vided ad libitum. The ingredients of the experimental diets the potential gut health implications [24, 25] of using in- and their relative inclusion levels are reported in Table 1. sects in swine diets, as a consequence of the increasing importance of this research topic in animal nutrition Chemical analyses of BSF larva meal and experimental [27]. Within this scenario, insects could be particularly diets advantageous as a novel feed additive to improve gut The BSF larva meal and the experimental diets were health, because of the immune-system stimulating prop- ground using a cutting mill (MLI 204; Bühler AG, Uzwil, erties of chitin [28] and of the antibacterial/microbial ac- Switzerland) and analysed to establish the dry matter (DM; tivity of antimicrobial peptides [29] and lauric acid [25]. AOAC# 934.01), CP (AOAC# 984.13), acid detergent fiber Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 3 of 11 Table 1 Ingredients, proximate composition and net energy of the BSF larva meal and experimental diets Items BSF larva Phase I (1–23 d) Phase II (24–61 d) meal BSF0 BSF5 BSF10 BSF0 BSF5 BSF10 Ingredients, g/kg as fed Corn meal – 317.9 332.7 347.3 378.0 392.9 407.4 Barley meal – 220.0 220.0 220.0 220.0 220.0 220.0 Soybean meal – 200.0 140.0 80.0 185.0 125.0 65.0 Dried wheya – 140.0 140.0 140.0 100.0 100.0 100.0 Wheat meal – 60.0 60.0 60.0 60.0 60.0 60.0 Soybean oil – 25.0 21.0 17.0 20.0 16.0 12.0 BSF mealb – 0.0 50.0 100.0 0.0 50.0 100.0 Calcium carbonate – 6.0 7.5 8.0 7.0 8.5 10.0 L-lysine – 6.5 7.1 7.6 5.5 6.0 6.6 Titanium oxide – 5.0 5.0 5.0 5.0 5.0 5.0 Premix pigletsc – 5.0 5.0 5.0 5.0 5.0 5.0 Dicalcium phosphate – 5.0 3.0 0.0 6.0 3.0 0.0 DL-methionine – 3.0 3.0 3.2 2.4 2.4 2.6 Salt – 2.5 2.5 2.5 2.5 2.5 2.5 L-threonine – 2.5 2.6 2.8 2.0 2.1 2.3 L-tryptophan – 0.8 0.8 0.8 0.8 0.8 0.8 Pig aroma – 0.8 0.8 0.8 0.8 0.8 0.8 Proximate composition, g/kg as fedd DM 947.4 899.8 903.1 906.4 894.0 897.3 900.7 Ash 76.7 47.2 46.4 45.5 46.1 45.3 44.6 CP 559.0 178.8 178.9 179.0 170.4 170.4 170.6 EE 85.0 73.5 73.4 73.3 61.9 61.8 61.7 Starch – 346.8 354.3 361.7 384.2 391.8 399.1 NDF 192.3 109.8 115.2 120.7 114.2 119.6 125.1 ADF 87.0 40.5 42.1 43.6 41.4 43.0 44.5 Chitin 53.4 – – – – – – NE, MJ/kg – 10.6 10.6 10.6 10.4 10.3 10.3 Calcium 8.2 5.1 5.1 5.2 5.5 5.6 5.6 Phosphorus 13.1 5.1 4.9 4.9 5.1 4.9 4.7 Magnesium 4.5 1.7 1.8 1.8 1.7 1.8 1.8 Sodium 1.1 2.1 2.1 2.2 1.8 1.8 1.9 Potassium 18.7 10.1 9.6 9.1 9.2 8.7 8.2 Chlorine 4.3 5.2 5.5 5.8 4.5 4.8 5.1 Manganese, mg/kg 203.0 66.8 75.2 83.5 66.8 75.1 83.5 Zinc, mg/kg 172.0 128.6 134.1 139.7 129.2 134.6 140.1 Iron, mg/kg 154.0 184.7 178.4 174.1 186.6 180.3 174.0 Essential amino acids, g/kg as fedd Methionine 7.3 5.5 5.5 5.7 4.9 4.8 5.0 Methione + Cysteine 12.0 8.4 8.2 8.3 7.7 7.5 7.5 Lysine 26.6 14.0 14.0 13.9 12.6 12.5 12.5 Threonine 20.1 9.0 9.0 9.1 8.2 8.2 8.2 Arginine 25.6 10.3 9.4 8.6 9.9 9.0 8.2 Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 4 of 11 Table 1 Ingredients, proximate composition and net energy of the BSF larva meal and experimental diets (Continued) Items BSF larva Phase I (1–23 d) Phase II (24–61 d) meal BSF0 BSF5 BSF10 BSF0 BSF5 BSF10 Valine 21.9 8.0 8.1 8.2 7.6 7.8 7.9 Tryptophan 34.8 2.9 2.9 2.8 2.8 2.7 2.7 Isoleucine 30.2 7.1 6.8 6.6 6.7 6.5 6.2 Leucine 14.4 13.7 13.2 12.7 13.3 12.8 12.4 Histidine 31.3 4.3 4.2 4.2 4.1 4.1 4.0 Phenylalanine 7.3 8.2 8.2 8.3 7.9 7.9 8.0 BSF black soldier fly; DM dry matter; CP crude protein; EE ether extract; NDF neutral detergent fiber; ADF acid detergent fiber; NE net energy; BSF0 = control diet; BSF5 = 5% inclusion level of black soldier fly meal; BSF10 = 10% inclusion level of black soldier fly meal a Added with 20% of coconut oil b Black soldier fly larva meal purchased from Hermetia Deutschland GmbH & Co. KG (Baruth / Mark, Germany) c Premix: 16,000 IU vitamin A; 2,000 IU vitamin D3; 75.0 mg vitamin E; 2.94 mg vitamin K3; 3.0 mg vitamin B1; 6.0 mg vitamin B2; 4.0 mg vitamin B6; 0.05 mg vitamin B12; 98 mg vitamin C; 21.0 mg pantothenic acid; 40.0 mg vitamin PP; 1.20 mg folic acid; 0.25 mg biotin; 1,500 IU 6-phytase; 700 IU xylanase; 312.5 IU glucanase; 145.68 mg copper; 0.05 mg cobalt; 0.44 mg selenium d Values are reported as mean of duplicate analyses (ADF; AOAC# 973.18) and ash (AOAC# 942.05) contents Nutrient digestibility according to AOAC International [32]. The ether extract Faeces were collected for 4 consecutive days, twice a day (EE; AOAC# 2003.05) was analysed according to AOAC (09:00 and 15:00). Sampling was conducted in each box for International [33]. The neutral detergent fiber (NDF) was 2 consecutive hours (from 09:00 to 11:00 and from 15:00 analysed according to Van Soest et al. [34]; α-amylase to 17:00) as soon as the faeces were released by the piglets. (Sigma Aldrich, Saint Louis, MO, USA) was added, but no The faeces of each sampling moment were pooled per box sodium sulphite, and the results were corrected for the re- and immediately stored at − 18 °C. The feces samples sidual ash content. All the analyses were performed in du- were dried at 60 °C for 72 h, finely ground and pooled plicate. The NE values of the diets were calculated per box in equal proportions. Samples were analysed according to Noblet et al. [35]. The amount of chitin of for DM, CP and EE, according to the AOAC method- the BSF meal was estimated according to Finke [36]. Diet ologies described for feed analysis. The diet and feces samples were prepared using a 22-h hydrolysis step in 6 samples were analysed to establish the TiO2 content using mol/L HCl at 112 °C under a nitrogen atmosphere to de- a UV spectrophotometer (UNICAN UV-Vis Spectrometry, termine amino acids (AA). Performic acid oxidation oc- Helios Gamma, United Kingdom) according to the curred prior to acid hydrolysis for methionine and method of Short et al. [38]. cysteine. The AA in the hydrolysate were determined by Using the analysed values for nutrient concentration, as means of HPLC, after post-column derivatization, accord- well as the concentration of the TiO2 in the feed and fae- ing to the procedure described by Madrid et al. [37]. The ces, the nutrient digestibility was calculated as follows: Di- proximate composition and NE of the BSF larva meal and gestibility (%): 100 - [100 × (Nf × Td) / (Nd × Tf)] where of the experimental diets are shown in Table 1. Nf and Nd are the nutrient concentrations (% DM) in the faeces and diet, respectively, and Td and Tf are the TiO2 concentrations (% DM) in the diets and faeces, Growth performance respectively. A total of 48 commercial piglets (females, Topigs) were weaned at 20 ± 1 days of age, individually weighed (initial Blood and serum parameters body weight [IBW]: 6.1 ± 0.16 kg) and randomly allotted A total of 12 piglets per treatment (3 animals per box) were to the 3 previously described dietary treatments. Each diet randomly selected and slaughtered in the EC-approved was replicated in 4 boxes of 4 piglets with a homogeneous slaughterhouse of SDSV – Teaching Structure of Veterin- IBW per box. The piglets – all identified with ear tags – ary Medicine of the University of Turin (Italy) at 61 days of were individually weighed at d 23 (feed change from phase age. The animals were stunned, by electrocution, and ex- I to phase II) and at the end of the trial (d 61). The trial sanguinated. Sterile tubes containing heparin were utilized lasted 40 d. The weight gain (WG), average daily gain to collect blood samples for haematological analysis, which (ADG), average daily feed intake (ADFI) and feed conver- was conducted using an automated laser cell counter cali- sion ratio (FCR) were calculated for each feeding phase brated for swine (MS4-S Hematology Analyzer, Melet and for the whole experimental period. Mortality was Schloesing, Osny, France). A plain serum tube was instead checked daily and calculated as: (number of dead pigs / utilized as a container to obtain serum for biochemistry number of pigs at start) × 100. analysis and protein electrophoresis, which was conducted Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 5 of 11 using an automated photometer system (Screen Master tissue [GALT] activation) of each segment. The total score Touch, Hospitex Diagnostics, Sesto Fiorentino, Florence, of each gut segment was obtained by adding the mucosa Italy) and a semi-automated agarose gel electrophoresis and submucosa scores, while the total score of each piglet system (Sebia Hydrasys, Evry, France), respectively. was represented by the mean value of the duodenum, je- junum and ileum scores. All the slides were blind assessed Slaughter procedures and samples collection by 3 observers and the discordant cases were reviewed, The slaughtered piglets were submitted to anatomopatho- using a multi-head microscope, until unanimous consen- logical investigations. Intestinal segment samples (approxi- sus was reached. mately 5 cm in length) were excised and flushed with 0.9% saline to remove all the content. The collected segments of intestine were the tract after the pylorus (duodenum), the Statistical analysis mid jejunum (jejunum) and the tract before the ileocecal The data were analysed using the IBM SPSS Statistics junction (ileum). Samples of liver, spleen, lung, stomach V25.0.0 software (SPSS Inc., Chicago, IL, USA). An indi- and kidney were also collected. Gut segments and organs vidual pig was used as the experimental unit to analyse samples were fixed in Carnoy’s and 10% neutral buffered the blood parameters, gut morphology and histological formalin (4% formaldehyde) solutions for morphometric features (n = 12 per treatment), while each box was con- analysis (gut segments) and a histopathological examin- sidered as the experimental unit for the growth perform- ation (gut segments and organs). Tissues were routinely ance and nutrient digestibility (n = 4 per treatment). embedded in paraffin wax blocks, sectioned at 5 μm thick- The growth performance, nutrient digestibility and ness and mounted on glass slides. blood parameter data were tested by means of one-way analysis of variance (ANOVA), and the effect of dietary Histomorphological investigations BSF meal inclusion was evaluated by means of polyno- The Carnoy-fixed and paraffin-embedded intestinal sec- mial contrasts (linear and quadratic). The intestinal mor- tions were submitted to Haematoxylin & Eosin staining, phometric indices were analysed by fitting a general and one slide per each intestinal segment was examined by linear mixed model (GLMM). GLMM allowed the mor- means of light microscopy. Each slide was captured with a phometric indices (Vh, Cd and Vh/Cd, separately) to de- Nikon DS-Fi1 digital camera (Nikon Corporation, Minato, pend on three fixed factors (diet, intestinal segment and Tokyo, Japan), coupled to a Zeiss Axiophot microscope the interaction between the diet and intestinal segment). (Carl Zeiss, Oberkochen, Germany), using a 2.5× objective Animal was included as a random effect to account for re- lens. NIS-Elements F software (Nikon Corporation, Minato, peated measurements on the same piglet. The interactions Tokyo, Japan) was used for image capturing. Morphometric between the levels of the fixed factors were evaluated by analysis was performed by means of Image®-Pro Plus soft- means of pairwise comparisons. The histopathological ware, version 6.0 (Media Cybernetics Inc., Bethesda, Rock- scores were analysed by means of the Kruskal-Wallis test ville, MD, USA). The evaluated morphometric indices were (post-hoc test: Dunn’s Multiple Comparison test). the villus height (Vh, from the top of the villus to the The results were expressed as the mean (growth villus-crypt junction), crypt depth (Cd, from the villus-crypt performance, nutrient digestibility, blood parameter and junction to the base of the crypt) and the villus height to histopathological data) or least square mean (intestinal crypt depth ratio (Vh/Cd, calculated by dividing the villus morphometric indices) and standard error of the mean height by the crypt depth) [39]. Morphometric measure- (SEM). P values ≤0.05 were considered statistically ments were performed on 10 well-oriented and intact villi significant. A statistical trend was considered for 0.05 < and on 10 crypts chosen from the duodenum, jejunum and P ≤ 0.10. ileum [40]. Formalin-fixed and paraffin-embedded gut segments and organ sections were submitted to Haematoxylin & Results Eosin staining and examined by means of light micros- Growth performance copy. The observed histopathological alterations were The growth performance parameters of the piglets are evaluated using a semiquantitative scoring system as summarized in Table 2. The piglets readily accepted follows: absent (score = 0), mild (score = 1), moderate the experimental diets, with a survival ranging from (score = 2) and severe (score = 3). A single score was 98% (BSF10) to 100% (BSF0 and BSF5). Dietary BSF given for the histopathological alterations of the liver, meal inclusion did not significantly influence the spleen, lung, stomach and kidneys, while the gut histo- growth performance of the animals, except for the pathological findings were assessed separately for the ADFI of phase II, which showed a linear response to mucosa (inflammatory infiltrates) and submucosa increasing BSF meal levels (P < 0.05, with a maximum (inflammatory infiltrates and gut-associated lymphoid corresponding to the inclusion of 10% of BSF meal). Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 6 of 11 Table 2 Effects of dietary BSF larva meal inclusion on growth linear and quadratic response, respectively, to increasing performance of the piglets (n = 4) BSF meal levels (P < 0.05, with the maximum corre- Items Dietary treatments SEM P-value sponding to the inclusion of 10% and 5% of BSF meal, BSF0 BSF5 BSF10 Linear Quadratic respectively). BW, kg Initial 6.10 6.09 6.09 0.005 0.620 0.621 End Phase I 12.06 12.00 12.34 0.191 0.576 0.643 Histomorphological investigations The effects of the diet, intestinal segment and interaction End Phase II 31.88 32.23 33.06 0.457 0.708 0.587 between the diet and intestinal segment on the intestinal WG, kg Phase I 5.96 5.91 6.24 0.190 0.574 0.656 morphometric indices of the piglets are summarized in Phase II 19.81 20.23 20.72 0.498 0.399 0.894 Table 6. The intestinal segment significantly affected Vh, Total 25.78 26.14 26.96 0.581 0.361 0.791 Cd and Vh/Cd (P < 0.001, P < 0.05 and P < 0.01, respect- ADG, kg/d Phase I 0.26 0.26 0.27 0.008 0.574 0.656 ively). The duodenum and jejunum showed higher Vh and Phase II 0.52 0.53 0.55 0.013 0.399 0.894 Vh/Cd (P < 0.01 and P < 0.05, respectively) than the ileum, while a greater Cd (P < 0.05) was observed in the duode- Total 0.42 0.43 0.44 0.010 0.361 0.791 num than in the other gut segments. On the contrary, ADFI, kg/d Phase I 0.35 0.37 0.38 0.012 0.420 0.944 there was no influence of diet or interaction between the Phase II 0.94 0.95 0.97 0.005 0.016 0.709 diet and intestinal segment on the morphometric indices. Total 0.72 0.73 0.75 0.006 0.069 0.922 Histopathological alterations developed in the stomach, FCR Phase I 1.35 1.42 1.41 0.030 0.578 0.479 gut, liver and kidneys of the piglets for all the dietary treat- Phase II 1.81 1.79 1.76 0.038 0.718 0.976 ments (Fig. 1). On the contrary, the spleen and lung showed no alterations. The stomach and gut showed mu- Total 1.71 1.70 1.70 0.033 0.848 0.888 cosal/submucosal lymphoplasmacytic or eosinophilic in- BSF black soldier fly; BW body weight; WG weight gain; ADG average daily gain; ADFI average daily feed intake; FCR feed conversion ratio; SEM standard flammation, with or without GALT activation. Vacuolar error of the mean; BSF0 = control diet; BSF5 = 5% inclusion level of black degeneration of the hepatocytes and lymphoplasmacytic soldier fly meal; BSF10 = 10% inclusion level of black soldier fly meal inflammation with or without fibrous tissue deposition was observed in the liver. Finally, interstitial lymphoplas- Nutrient digestibility macytic inflammation and tubular steatosis were identified Dietary BSF meal inclusion did not significantly affect in the kidneys. However, dietary BSF meal inclusion did the piglets’ digestibility of the diets (Table 3). not affect the severity of the observed histopathological al- terations (Table 7). Blood and serum parameters The blood and serum parameters of the piglets are Discussion shown in Tables 4 and 5. Dietary BSF meal inclusion did Growth performance not significantly influence the haematochemical profile Overall, dietary BSF meal inclusion did not influence the or serum protein electrophoresis of the animals, except growth performance of the piglets involved in the present for the monocytes and neutrophils, which showed a study, although an increasing ADFI was identified in phase II for increasing levels of BSF meal inclusion. This could be attributed to an increased diet palatability, related to insect Table 3 Effects of dietary BSF larva meal inclusion on nutrient digestibility in the piglets (n = 4, end of both feeding phases) meal inclusion, as already reported by Jin et al. [23]. Fur- thermore, the higher feed assumption was only observed Items Dietary treatments SEM P-value in the second phase of the trial, thus suggesting a potential BSF0 BSF5 BSF10 Linear Quadratic tendency of piglets to eat the insect-based diets. However, DM Phase I 0.960 0.955 0.959 0.003 0.924 0.503 the overall growth performance observed in the current re- Phase II 0.958 0.954 0.958 0.003 0.926 0.534 search is in agreement with Driemeyer [22], who showed OM Phase I 0.965 0.960 0.964 0.003 0.857 0.524 no effects for the growth performance of piglets fed Phase II 0.964 0.960 0.964 0.003 0.913 0.518 maize-soybean and fish meal-based diets in which the CP Phase I 0.803 0.808 0.828 0.012 0.443 0.805 full-fat BSF inclusion level was 3.5% in partial substitution of fish meal. Similarly, Spranghers et al. [25] have recently Phase II 0.766 0.777 0.795 0.015 0.466 0.916 observed no effect on the growth performance of weaned EE Phase I 0.811 0.857 0.856 0.014 0.220 0.442 piglets fed diets including full-fat (4% and 8%) and defatted Phase II 0.853 0.828 0.865 0.012 0.716 0.291 (5.4%) BSF prepupae. More heterogeneous results were BSF black soldier fly; DM dry matter; OM organic matter; CP crude protein; EE previously reported, when other insect larvae were used as ether extract; SEM standard error of the mean; BSF0 = control diet; BSF5 = 5% inclusion level 5% inclusion level of black soldier fly meal; BSF10 = 10% protein sources in swine feeds. In fact, piglets fed diets in inclusion level of black soldier fly meal which HI replaced 75% of SBM tended to show a slightly Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 7 of 11 Table 4 Effects of dietary BSF larva meal inclusion on haematochemical parameters of the piglets (n = 12, end of the trial) Items Dietary treatments SEM P-value BSF0 BSF5 BSF10 Linear Quadratic White blood cells, 106/mm3 13.10 14.99 14.85 0.869 0.435 0.591 Lymphocytes, % 55.73 50.13 60.24 2.167 0.368 0.086 0.033 0.305 Monocytes, % 3.92 4.00 4.78 0.167 Neutrophils, % 36.16 41.72 26.12 2.485 0.076 0.035 Eosinophils, % 3.81 3.78 8.39 0.801 0.078 0.299 Basophils, % 0.382 0.383 0.30 0.041 0.421 0.630 Red blood cells, 106/mm3 7.57 7.69 7.89 0.306 0.681 0.953 MCV, fL 57.35 57.23 57.40 0.354 0.948 0.849 Haematocrit, % 43.43 43.91 45.47 1.812 0.658 0.892 MCH, pg 18.17 18.76 19.35 0.366 0.204 0.997 MCHC, g/dL 31.78 32.90 33.90 0.687 0.224 0.968 RDW, fL 14.13 14.15 14.15 0.159 0.956 0.974 Haemoglobin, g/dL 13.81 14.39 14.77 0.562 0.506 0.932 Platelets, 106/mm3 197.18 195.00 181.42 11.946 0.602 0.827 MPV, fL 11.98 11.74 12.10 0.081 0.518 0.081 Procalcitonin, % 0.24 0.23 0.22 0.015 0.598 0.978 PDW, fL 8.56 8.14 7.81 0.323 0.366 0.955 GOT, IU/L 111.21 126.78 143.31 12.808 0.324 0.986 GPT, IU/L 59.73 63.15 55.38 2.068 0.380 0.206 ALP, IU/L 28.52 31.19 60.49 5.952 0.082 0.880 GGT, IU/L 43.49 48.30 40.26 2.463 0.589 0.223 Total protein, g/dL 6.67 6.02 5.98 0.194 0.163 0.460 Phosphorus, mg/dL 14.65 17.10 14.94 0.966 0.929 0.272 Iron, μg/dL 118.39 146.99 130.32 8.975 0.617 0.242 Magnesium, mEq/L 1.65 2.09 1.93 0.116 0.335 0.230 Uric acid, mg/dL 1.66 3.03 2.71 0.576 0.481 0.502 Cholesterol, mg/dL 52.31 55.41 51.30 2.647 0.869 0.533 Triglycerides, mg/dL 58.93 47.94 55.28 4.444 0.764 0.342 Creatinine, mg/dL 1.44 1.68 1.61 0.058 0.242 0.212 BSF Black soldier fly; MCV mean corpuscular volume; MCH mean corpuscular haemoglobin; MCHC mean corpuscular haemoglobin concentration; RDW red blood cell distribution width; MPV mean platelet volume; PDW platelet distribution width; GOT glutamic oxaloacetic transaminasi; GPT glutamate-pyruvate transaminasi; ALP alkaline phosphatase; GGT gamma-glutamyl transferase; SEM standard error of the mean. BSF0 = control diet; BSF5 = 5% inclusion level 5% inclusion level of black soldier fly meal; BSF10 = 10% inclusion level of black soldier fly meal lower growth, feed intake and feed and protein conversion reported to influence the growth performance of sows and ratio than those fed a control diet [24]. Jin et al. [23] also their offspring [20] or early weaned piglets [21]. The het- found an improved growth performance in weaning pigs erogeneity of the results obtained from these previous fed corn-soybean meal-based diets in which increasing studies may be related to the nutritive value of the insect levels of a full-fat Tenebrio molitor (TM) larva meal (from meal that was used, which could have been influenced by 1.5% to 6.0%) was used to partially replace soybean meal the species, the insect life stage (adult, larva or pupa) and and soy oil, and they attributed this outcome to an increased the insect rearing substrate [7]. palatability of yellow mealworm. Chen et al. [41] also ob- served similar results for increasing dietary TM protein Nutrient digestibility concentrates of up to 6%. Improved weight gain was also Dietary BSF meal inclusion did not influence the nutri- found in weaning pigs fed Musca domestica (L.) [42] and ent digestibility of the piglets in the present study. This Bombyx mori (L.) [43] meal inclusion diets. On the other result is in agreement with the results of Spranghers hand, dietary house-fly larva meal inclusion has not been et al. [25], who observed a similar apparent total tract Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 8 of 11 Table 5 Effects of dietary BSF larva meal inclusion on serum Blood and serum parameters protein electrophoresis of the piglets (n = 12, end of the trial) All the haematochemical parameters recorded for the Items Dietary treatments SEM P-value piglets in the present study fell within the physiological BSF0 BSF5 BSF10 Linear Quadratic reference intervals reported for swine [47], thus suggesting Albumin, % 45.73 46.41 47.50 0.372 0.056 0.789 that the insect meal utilization did not negatively influence Albumin, g/dL 3.07 2.79 2.84 0.092 0.335 0.398 the health status of the animals. Moreover, the dietary BSF meal inclusion did not significantly affect the blood and Alpha globulin, % 25.00 25.63 25.01 0.280 0.988 0.307 serum traits of the pigs in the current research, although Alpha globulin, g/dL 1.67 1.55 1.50 0.054 0.209 0.746 increasing monocytes and neutrophils were identified for Beta globulin, % 17.02 16.43 16.41 0.186 0.198 0.467 increasing levels of BSF meal. This finding appears diffi- Beta globulin, g/dL 1.13 0.99 0.98 0.033 0.080 0.307 cult to explain, since none of the BSF piglets showed any Gamma globulin, % 12.26 11.53 11.08 0.381 0.227 0.868 signs of physical distress or inflammatory diseases. How- Gamma globulin, g/dL 0.80 0.70 0.66 0.034 0.106 0.673 ever, it is important to underline that the haematological and biochemical profiles of herds have rarely been used in BSF black soldier fly; SEM standard error of the Smean; BSF0 = control diet; BSF5 = 5% inclusion level 5% inclusion level of black soldier fly meal; BSF10 = pig veterinary practice. Therefore, further studies are rec- 10% inclusion level of black soldier fly meal ommended to assess their value as a diagnostic technique [22]. Similar findings, in terms of unaffected blood haema- tology and biochemical parameters (albumin, calcium, digestibility of nutrients in BSF prepupa- and basal phosphorus, iron, IgA, IgG and IgM), have been observed diet-fed weaned piglets. On the contrary, Newton et al. in piglets fed diets with a full-fat 3.5% BSF inclusion level [26] reported lower DM and higher EE digestibility in [22]. Jin et al. [23] instead reported that the dietary supple- piglets fed 33% dried HI larva meal than those fed mentation of up to 6% of dried full-fat mealworm in soybean meal, although the CP digestibility was similar. weaning pigs decreased blood urea nitrogen and increased With regards to other insect species, Jin et al. [23] ob- the serum insulin-like growth factor. However, no signifi- served a linear improvement in DM and CP digestibility cant effects were observed for the serum IgG and IgA for increasing levels of dried mealworm in diets for concentrations. weaning pigs. As already hypothesized for growth performance, these discrepancies may be related to the Histomorphological investigations different insect species and life stages, dietary inclusion Dietary BSF meal inclusion did not influence the gut levels and fat extraction methods. Furthermore, the het- morphology of the piglets in the present study. This re- erogeneity of the feed ingredients used in the present sult is in agreement with that of Spranghers et al. [25], and above-mentioned researches does not allow any who observed no effect on the jejunal morphology of reliable statement to be made about the effective role of BSF prepupa-fed weaned piglets. It is well known that BSF in nutrient digestibility modifications. However, the integrity of the intestinal structure is required for the identification of unaffected or even improved nutrient maintenance of intestinal nutrition [48]. Furthermore, digestibility may reflect a species-specific ability of chitin modifications of gut morphometric features have been digestion, as suggested by the high distribution and ac- reported to determine significant changes in the weight tivity of chitinase [44] and positive selection of chitinase gain of piglets [40, 49–51]. Since both the intestinal genes [45] previously reported in pigs. Feeding chito-oligo- morphology and the growth performance of the BSF-fed saccharide to weaning piglets has also been reported to im- piglets in the current research were unaffected, it is rea- prove DM and crude fat digestibility [46]. Finally, no data sonable to hypothesize that insect meal utilization does about organic matter digestibility have been reported for not negatively influence gut development or nutrient ab- insect-fed pigs, thus making the values obtained in the sorption. Another interesting aspect that should be consid- present study the first ones available. ered is that, independently of dietary BSF meal inclusion, Table 6 Intestinal morphometric indices in the piglets in relation to diet and intestinal segment (n = 12, end of the trial) Items Diet (D) Intestinal segment (IS) SEM P-value BSF0 BSF5 BSF10 Duodenum Jejunum Ileum D IS D × IS Vh, mm 0.38 0.36 0.38 0.41a 0.38a 0.34b 0.012 0.371 < 0.001 0.709 Cd, mm 0.11 0.11 0.10 0.12a 0.10b 0.11b 0.003 0.256 0.009 0.999 Vh/Cd 3.47 3.35 3.72 3.64a 3.71a 3.19b 0.126 0.159 0.007 0.566 BSF0 = control; BSF5 = 5% inclusion level 5% inclusion level of black soldier fly meal; BSF10 = 10% inclusion level of black soldier fly meal; Vh, villus height; Cd, crypt depth; Vh/Cd, villus height to crypt depth ratio The means with different superscript letters (a, b) within the same row per fixed effect (i.e. diet, intestinal segment) differ significantly (P < 0.05) Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 9 of 11 Fig. 1 Histopathological findings of the piglets. a BSF0 group. Mild and multifocal mucosal lymphoplasmacytic inflammation (arrow), as well as focal and moderate gut-associated lymphoid tissue (GALT) activation (arrowhead), are observed in the ileum. 5× haematoxylin-eosin stain. b BSF5 group. Stomach shows mild and multifocal mucosal lymphoplasmacytic inflammation (arrow) along with focal and moderate GALT activation (arrowhead). 5× haematoxylin-eosin stain. c BSF10 group. Liver, centrolobular zone. Moderate and multifocal vacuolar degeneration of the hepatocytes (arrowheads) is identified. 20× haematoxylin-eosin stain. d BSF0 group. Moderate and multifocal interstitial lymphoplasmacytic inflammation (arrow) and tubular steatosis (arrowhead) are observed in the kidneys. 10× haematoxylin-eosin stain the duodenum and jejunum showed greater morphomet- Dietary BSF meal inclusion did not affect the development ric indices than the ileum. Despite the heterogeneous or severity of the histopathological alterations in the piglets information about the morphological asset of the different in the current research. Gastric inflammation was attributed gut segments currently available in literature [52], the to the sensitiveness of pigs to damage of the gastric mucosa, identification of higher morphometric values in the which frequently occurs as a consequence of stress and feed- duodenum and jejunum appears to be rather constant for ing practices [55]. An analogous consideration can also be pigs [53, 54]. Therefore, the findings of the present study made for gut inflammation. The hepatic and renal alter- confirm what was previously reported in literature. ations varied from mild to moderate and from focal to multifocal in all the pigs, thus suggesting no clinical rele- Table 7 Effects of dietary BSF larva meal inclusion on the vance. Furthermore, no alteration of the serum chemistry histopathological scores of the piglets (n = 12, end of the trial) was observed in relation to the liver and renal functions. Items BSF0 BSF5 BSF10 SEM P-value Stomach 1.17 1.06 1.33 0.122 0.770 Conclusions Gut 2.79 3.26 3.08 0.26 0.933 The present study suggests that a dietary inclusion of up to Liver 0.64 0.53 0.68 0.115 0.576 10% of partially defatted BSF larva meal could be used for Kidney 0.82 0.96 1.00 0.064 0.381 weaning pigs without any adverse effects on the growth per- formance, blood profile, nutrient digestibility, gut morph- Spleen Absence of alterations Absence of alterations ology or histological features. These results confirm that Lung BSF has the potential to become a valuable source of BSF, black soldier fly; SEM, standard error of the mean; BSF0 = control diet; sustainable proteins in pig nutrition. However, since the BSF5 = 5% inclusion level 5% inclusion level of black soldier fly meal; BSF10 = 10% inclusion level of Black Soldier Fly meal price of insects is still not competitive, future studies should The data are expressed as the mean of the scores (0 = absence of alterations; be focused on the use of BSF as a potential feed additive to 1 = mild alterations; 2 = moderate alterations; 3 = severe alterations). The gut scores resulted from the mean of the three gut segments scores (in turn improve the gut health of pigs, in particular through the obtained from the adding of the mucosa and submucosa scores) modulation of the gut microbiota and mucin composition. Biasato et al. Journal of Animal Science and Biotechnology (2019) 10:12 Page 10 of 11 Abbreviations Received: 6 November 2018 Accepted: 24 January 2019 AA: Amino acids; ADF: Acid detergent fiber; ADFI: Average daily feed intake; ADG: Average daily gain; ALP: Alkaline phosphatase; BSF: Black soldier fly; BW: Body weight; Cd: Crypt depth; CP: Crude protein; DE: Digestible energy; DM: Dry matter; EE: Ether extract; FBW: Final body weight; FCR: Feed conversion References ratio; GGT: Gamma-glutamyl transferase; GOT: Glutamic oxaloacetic transaminase; 1. FAO. Food and Agriculture Organization of the United Nations. World GPT: Glutamate-pyruvate transaminase; HI: Hermetia illucens; IBW: Initial body Livestock 2011. Rome: Livestock in Food security; 2011. p. 117. weight; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular 2. OECD Agriculture Statistics. OECD-FAO Agricultural Outlook (Edition 2018). haemoglobin concentration; MCV: Mean corpuscular volume; MPV: Mean 2018. https://data.oecd.org/agroutput/meat-consumption.htm. Accessed 03 platelet volume; NDF: Neutral detergent fiber; NE: Net energy; PDW: Platelet Dec 2018. distribution width; RDW: Red blood cell distribution width; SBM: Soybean meal; 3. FAOSTAT. Food and Agriculture Organization of the United Nations SEM: Standard error of the mean; TM: Tenebrio molitor; Vh: Villus height; Vh/ Statistics Division. 2016. http://www.fao.org/faostat/en/#data/QC. Accessed Cd: Villus height to crypt depth ratio; WG: Weight gain 18 Sep 2018. 4. IFIF. International Feed Industry Federation. 2018. https://ifif.org/global-feed/ industry/. Accessed 18 Sep 2018. Acknowledgments 5. Veldkamp T, Bosch G. Insects: a protein-rich feed ingredient in pig and The authors gratefully acknowledge Mr. Dario Sola and Mr. Mario Colombano for poultry diets. 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AD, AS and LG collected the experiments data. IB and MTC performed the Forensic Sci Int. 2011;206:e76–8. histomorphometric investigations. SM and ACBL evaluated the nutrients 12. Meneguz M, Gasco L, Tomberlin JK. Impact of pH and feeding system on digestibility. SB investigated the blood parameters. IB and MR performed the black soldier fly (Hermetia illucens, L; Diptera: Stratiomyidae) larval statistical analysis. All authors interpreted the data. IB, MR and LG wrote the first development. PLoS One. 2018b;13:e0202591. draft of the manuscript. All authors critically reviewed the manuscript for 13. Jucker C, Erba D, Leonardi MG, Lupi D, Savoldelli S. Assessment of vegetable intellectual content and gave final approval for the version to be published. and fruit substrates as potential rearing media for Hermetia illucens (Diptera: Stratiomyidae) larvae. Environ Entomol. 2017;46:1415–23. 14. Biasato I, Gasco L, De Marco M, Renna M, Rotolo L, Dabbou S, et al. Effects Ethics approval and consent to participate of yellow mealworm larvae (Tenebrio molitor) inclusion in diets for female The experimental protocol was designed according to the guidelines of broiler chickens: implications for animal health and gut histology. Anim the current European Directive (2010/63/EU) on the care and protection of Feed Sci Technol. 2017;234:253–63. animals used for scientific purposes and approved by the Animal Welfare and 15. Biasato I, Gasco L, De Marco M, Renna M, Rotolo L, Dabbou S, et al. Yellow Ethical Committee of the Department of Veterinary Sciences of the University mealworm larvae (Tenebrio molitor) inclusion in diets for male broiler of Turin (Italy) (Ref. 2, 28/06/2016). chickens: effects on growth performance, gut morphology, and histological findings. Poult Sci. 2018;97:540–8. 16. Dabbou S, Gai F, Biasato I, Capucchio MT, Biasibetti E, Dezzutto D, et al. Consent for publication Black soldier fly defatted meal as a dietary protein source for broiler Not applicable. chickens: effects on growth performance, blood traits, gut morphology and histological features. J Animal Sci Biotechnol. 2018;9:49. 17. Gasco L, Henry M, Piccolo G, Marono S, Gai F, Renna M, et al. Tenebrio Competing interests molitor meal in diets for European sea bass (Dicentrarchus labrax L.) The authors declare that they have no competing interests. juveniles: growth performance, whole body composition and in vivo apparent digestibility. Anim Feed Sci Technol. 2016;220:34–45. Author details 18. Renna M, Schiavone A, Gai F, Dabbou S, Lussiana C, Malfatto V, et al. 1 Department of Agricultural, Forest and Food Sciences, University of Turin, Evaluation of the suitability of a partially defatted black soldier fly (Hermetia Largo P. 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