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Background In contrast to protein-rich insect meal, the feed potential of insect fat is generally less explored and knowledge about the suitability of insect fat as a fat source specifically in broiler diets is still limited. In view of this, the present study aimed to comprehensively investigate the effect of partial (50%) and complete replacement of soybean oil with insect fat from Hermetia illucens (HI) larvae in broiler diets on performance, fat digestibility, cecal microbi- ome, liver transcriptome and liver and plasma lipidomes. Thus, 100 male, 1-day-old Cobb 500 broilers were randomly assigned to three groups and fed three different diets with either 0 (group HI-0, n = 30), 2.5% (group HI-2.5, n = 35) or 5.0% (HI-5.0, n = 35) Hermetia illucens (HI) larvae fat for 35 d. Results Body weight gain, final body weight, feed intake, and feed:gain ratio during the whole period and apparent ileal digestibility coefficient for ether extract were not different between groups. Cecal microbial diversity did not dif- fer between groups and taxonomic analysis revealed differences in the abundance of only four low-abundance bacte - rial taxa among groups; the abundances of phylum Actinobacteriota, class Coriobacteriia, order Coriobacteriales and family Eggerthellaceae were lower in group HI-5.0 compared to group HI-2.5 (P < 0.05). Concentrations of total and individual short-chain fatty acids in the cecal digesta were not different between the three groups. Liver transcriptom- ics revealed a total of 55 and 25 transcripts to be differentially expressed between groups HI-5.0 vs. HI-0 and groups HI-2.5 vs. HI-0, respectively (P < 0.05). The concentrations of most lipid classes, with the exception of phosphatidyletha- nolamine, phosphatidylglycerol and lysophosphatidylcholine in the liver and cholesterylester and ceramide in plasma (P < 0.05), and of the sum of all lipid classes were not different between groups. Conclusions Partial and complete replacement of soybean oil with HI larvae fat in broiler diets had no effect on growth performance and only modest, but no adverse effects on the cecal microbiome and the metabolic health of broilers. This suggests that HI larvae fat can be used as an alternative fat source in broiler diets, thereby, making broiler production more sustainable. *Correspondence: Robert Ringseis Robert.firstname.lastname@example.org Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Journal of Animal Science and Biotechnology (2023) 14:20 Page 2 of 19 Keywords Broilers, Cecal microbiota, Hermetia illucens, Insect fat, Liver lipidome, Medium-chain fatty acids Introduction efficiency in broilers is affected by the gut microbiota com - In recent years, insect biomass has been increasingly rec- position through different mechanisms, such as a more ognized as an alternative and sustainable source of feed for complete digestion of substrates, an increased production monogastric livestock [1–3]. Consequently, the use of pro- of short-chain fatty acids (SCFA) or a decreased stimula- cessed insect biomass as a feed for poultry and pigs has been tion of the intestinal immune system [24, 25], an altera- authorized by the European commission in 2021 , with the tion of the gut microbiota might be of great relevance to aim of improving sustainability of food systems and securing broiler´s performance. In addition, convincing evidence food supply. Owing to its high protein content (40%–50% exists that the gut microbiota has also a profound influ - of dry matter (DM) depending on the insect species  and ence on energy metabolism and feeding behavior of the provided that the rearing substrate is suitable [6, 7]), research host due to the ability of the gut microbiota to communi- dealing with the feed potential of insect biomass has primar- cate with the host along the gut-liver axis via different gut- ily focused on its role as a source of protein. In this context, derived compounds . Moreover, MCFA-rich dietary a large number of studies in different monogastric livestock fats might also exert a direct effect on energy metabolism, species demonstrated that insect larvae meal obtained from because triglyceride (TG)-bound MCFA are considered to different edible insect species suitable for large-scale produc - be more efficiently absorbed than TG-bound LCFA due tion, such as Tenebrio molitor (TM) and Hermetia illucens to easier emulsification and less dependence on pancre - (HI), can replace conventional protein sources, like soybean atic lipase [27, 28], thereby increasing digestible energy meal, without impairing performance, metabolic health or intake to support a higher growth performance. Against product quality [8–11]. Apart from protein, insect larvae this background, the present study aimed to comprehen- contain a significant amount of fat (up to 47% and 43% of sively investigate the effect of partial (50%) and complete DM in HI and TM, respectively, depending on the rearing replacement of soybean oil, the most commonly used fat substrate ), which can be obtained by a defatting process source in commercial broiler diets, with HI larvae fat in during insect meal production in order to increase the pro- broiler diets on performance, fat digestibility, cecal micro- tein content and improve the storage stability of the insect biome, liver transcriptome and liver and plasma lipidomes. meal. In contrast to insect meal, the feed potential of insect fat is far less explored. Despite that several studies have dem- Methods onstrated that TM or HI fat has no negative impact on per- Animals and diets formance, gut morphology, selected blood parameters and The 35-d feeding trial was approved by the Animal Welfare product quality in broilers, laying hens and turkeys [13–21], Officer of the Justus Liebig University Giessen (approval knowledge about the suitability of insect fat as a fat source in no.: JLU 786_M). All experimental procedures described broiler diets is still limited. In particular, in-depth analysis of followed established guidelines for the care and handling the effects of insect fat on the gut microbiome and interme - of laboratory animals. The experiment included 100 male, diary metabolism is lacking. 1-day-old broiler chickens (Cobb 500, Cobb, Wiedemar, Despite that fatty acid composition of insect larvae is Germany), which were randomly assigned to three groups influenced by the fatty acids in the rearing substrate [5 ], (5 broilers/cage, group 1: 6 cages, group 2 and group 3: 7 the fatty acid composition of TM and HI fat are completely cages). The mean initial body weight (BW) (44.9 ± 2.4 g; different. While TM fat consists mainly of unsaturated mean ± SD) was similar across the groups. The broilers were fatty acids (up to 80%), most of which are long-chain fatty kept in 2.1 m cages equipped with nipple drinkers and feed acids (LCFA; C18:1 and C18:2; ), the majority of total automates and had free access to feed and water. At the fatty acids in HI fat are saturated fatty acids (SFA) [5, 16]. floor of the cages there were cardboards, which were cov - This explains that HI fat, but not TM fat has a hard con - ered with litter to allow scratching, pecking and dustbath- sistency at room temperature. A further characteristic of ing. Cardboards and litter were exchanged 2 times per week HI fat is its high proportion of the medium chain-fatty acid during the first two weeks and every 2 d during the last (MCFA) lauric acid (C12:0) (≈ 40%; ). Interestingly, three weeks of the trial. In addition, broilers were provided MCFA including lauric acid have been reported to exert with perches in elevated position for resting and sleep- antimicrobial effects (e.g., against Gram-positive cocci ing. Light intensity was constantly at 40 Lux and the light and Escherichia coli) in the gut of broilers [22, 23], and, regime was 24 h:0 h, 23 h:1 h, 22 h:2 h, 21 h:3 h, 20 h:4 h, thus, dietary inclusion of HI fat might alter the gut micro- 19 h:5 h (light:dark) at d 1, 2, 3, 4, 5, 6, and 18 h:6 h from d biota composition. Considering recent evidence that feed S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 3 of 19 7 onward, as recommended by the breeder . The room ether extract by the indicator method . The diets met temperature decreased from 28–29 °C on d 1, measured at the broiler’s requirements of nutrients and energy accord- pen height, to 23–24 °C on d 35. During the first 6 d, infra - ing to the breeder’s recommendations . For diet prepa- red lamps (Albert Kerbl GmbH, Buchbach, Germany) were ration, three different basal feed mixtures for the starter, used as additional heat sources in order to adjust the tem- grower and finisher period, respectively, were produced perature at the cage floor to 34 °C. Mean relative humid - from all feed components except the fat source using mixing ity was 60.0% ± 1.9%. The groups (group 1: HI-0, group 2: machines (V100 and V250, Diosna, Germany). The experi - HI-2.5, group 3: HI-5.0) were fed three different diets, which mental diets were obtained by mixing the different basal varied only in the fat source (HI-0: 0 HI fat and 5% soybean feed mixtures with the intended amounts of fat sources oil, HI-2.5: 2.5% HI fat and 2.5% soybean oil, HI-5.0: 5.0% (soybean oil, HI fat) using the mixing machine. Prior to add- HI fat and 0 soybean oil), in a three-phase feeding system ing the HI fat, the HI fat was melted in an electric oven at (starter diet form d 1 to 10, grower diet from d 11 to 21, fin - 50 °C. Subsequently, the experimental diets were pelleted isher diet from d 22 to 35). The HI fat was purchased from using a pelleting device (V3/30 C, Simon-Heesen, Boxtel, Madebymade (Pegau, Germany) and stored at −20 °C until Netherlands) and aliquots of all diets were stored at −20 °C diet preparation. Prior to diet preparation, the stability of for analysis of diet composition. Diets were fed in crumbled HI fat was assessed by determining the acid value and the form during the first 3 d, and in pellet form (2 mm diam - percentage of polar compounds . Low values for both eter) from d 3 until the end of the trial. Body weight (indi- parameters indicated no significant oxidation of the HI fat. vidually) and feed intake (per cage) were determined on d Company’s details on the rearing conditions (substrate, 1, 10, 21 and 35, and the feed:gain ratio was calculated from duration) and further processing of the HI larvae are not feed intake and body weight gains on cage basis. available for reasons of confidentiality. The composition of the three diets are shown in Table 1. The finisher diets con - Analysis of diet composition tained 0.5% titanium dioxide as indicator in order to cal- Concentrations of DM, crude protein, crude ash, ether culate the apparent ileal digestibility (AID) coefficient for extract, crude fiber and amino acids in the main diet Table 1 Composition of the broiler diets, g/kg Item Starter diets Grower diets Finisher diets HI-0 HI-2.5 HI-5.0 HI-0 HI-2.5 HI-5.0 HI-0 HI-2.5 HI-5.0 Maize 300 300 300 280 280 280 280 280 280 Soybean meal (44% CP) 380 380 380 320 320 320 300 300 300 Wheat 205 205 205 283.5 283.5 283.5 308.65 308.65 308.65 Soybean oil 50 25 - 50 25 - 50 25 - HI fat - 25 50 - 25 50 - 25 50 Mineral & vitamin mix – Starter 20 20 20 - - - - - - Mineral & vitamin mix – Grower - - - 20 20 20 - - - Mineral & vitamin mix – Finisher - - - - - - 20 20 20 Monocalciumphosphate 15 15 15 15 15 15 9 9 9 Calciumcarbonate 15.5 15.5 15.5 15.5 15.5 15.5 15 15 15 Sodiumchloride 4 4 4 4 4 4 4 4 4 DL-Methionine 3.2 3.2 3.2 3 3 3 2.6 2.6 2.6 L-Lysine 2.1 2.1 2.1 2.5 2.5 2.5 1.8 1.8 1.8 L-Threonine 1.1 1.1 1.1 0.9 0.9 0.9 0.4 0.4 0.4 L-Arginine 1.4 1.4 1.4 1.3 1.3 1.3 0.5 0.5 0.5 L-Valine 1.5 1.5 1.5 2.5 2.5 2.5 1.6 1.6 1.6 L-Isoleucine 1.2 1.2 1.2 1.8 1.8 1.8 1.45 1.45 1.45 TiO - - - - - - 5 5 5 The mineral & vitamin mix supplied the following minerals and vitamins per kg diet (starter/grower/finisher): Fe, 40/40/40 mg; Cu, 15/15/15 mg; Mn, 100/100/100 mg; Zn, 100/100/100 mg; I, 1/1/1 mg; Se, 0.35/0.35/0.35 mg; vitamin A, 10,000/10,000/10,000 IU; vitamin D , 5000/5000/5000 IU; vitamin K , 3/3/3 mg; 3 3 vitamin E, 80/50/50 IU; vitamin B , 3/2/2 mg; vitamin B , 9/8/6 mg; vitamin B , 4/3/3 mg; vitamin B , 0.02/0.015/0.015 mg; biotin, 0.2/0.18/0.18 mg; folic acid, 1 2 6 12 2/2/1.5 mg; nicotinic acid, 60/50/50 mg; choline chloride, 500/400/350 mg; pantothenic acid, 15/12/10 mg The experimental diets did not contain any technological (e.g., emulsifier) or zootechnical feed additives (e.g., feed enzymes) Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 4 of 19 components (wheat, maize, soybean extraction meal) titanium dioxide as an inert marker . Prior to analy- and the experimental diets were determined by official sis, ileal digesta samples were freeze-dried and ground methods . Concentrations of sugar and starch were using a centrifugal mill (Retsch, Haan, Germany). Ileal analyzed in the experimental diets by official methods digesta concentration of the indigestible indicator TiO (sugar according to method 7.2.1 and starch according to was determined by the method of Brandt and Allam with method 7.1.3 ). Total lipid fatty acid composition of slight modifications . Concentration of ether extract HI fat, soybean oil and diets were analyzed as described in the ileal digesta was determined by official methods below. Apart from total lipid fatty acid composition, the as described above. Based on the ileal concentrations of detailed composition (all major lipid classes and indi- indicator, the AID coefficient for ether extract was calcu - vidual lipid species) of the HI fat was comprehensively lated according to the following formula: analyzed using lipidomics, as described for liver and AID coeﬃcient (%) = 100 − [(TiO ∕TiO ) 2 Diet 2 Digesta _ _ plasma (see below). The apparent N-corrected metabo - ×(EE ∕EE )× 100], _ Diet lizable energy (AME ) content of the diets was calculated Digesta _ according to the formula of the World’s Poultry Science in which TiO is the TiO concentration in the diet 2_Diet 2 Association (WPSA) for poultry compound feed : (% DM), T iO is the T iO concentration in the ileal 2_Digesta 2 AME (MJ∕kg) = (0.01551 × crude protein) + (0.03431 × ether extract) + (0.01669 × starch) + 0.01301 × sugar Considering that the AME and A ME content of HI digesta (% DM), EE is the ether extract concen- N _Digesta larvae fat for broiler chickens was shown to be similar to tration in ileal digesta (% DM), and EE is the ether _Diet that of soybean oil , the formula is appropriate to cal - extract concentration in the diet (% DM). culate the ME contents of the experimental diets. Determination of microbiota composition and diversity Sample collection in the cecal digesta A total of 12 (group HI-0) and 14 (groups HI-2.5 and HI-5.0) Metagenomic DNA was isolated from approximately broilers per group (two broilers from each cage), whose 180–200 mg of cecal digesta using genomic DNA col- body weights represented the mean body weight of the umns (Macherey‐Nagel, Düren, Germany) according to whole group, were selected for sample collection in order to Lagkouvardos et al. . V3-V4 regions of the 16S rRNA avoid that effects were biased by random selection of broil genes were amplified using bacteria‐specific primers fol - - ers with very low or very high body weights. All analyses lowing a two-step procedure according to the Illumina described below were carried out in these animals. The ani - sequencing protocol as described . Amplicons were sequenced using a MiSeq system (Illumina, Inc., San mals were killed by bleeding (opening of Vena jugularis and Diego, CA, USA). Further processing of raw sequences Arteria carotis) under electrical anesthesia using a BTG- 40A stunning device (Westerhoff Geflügeltechnik, Hoogst was carried out as described recently . Finally, sequences with a relative abundance > 0.1% in at least one ede, Germany) in accordance with the European legislation sample were sorted, merged and operational taxonomic for euthanasia of animals . Whole blood was collected into ethylenediaminetetraacetic acid-coated polyethylene units (OTU) were picked at a threshold of 97% similar tubes (9 mL S-Monovette, Sarstedt, Nümbrecht, Germany). ity. Taxonomic classification to the OTU was assigned Plasma was prepared by centrifugation (1100 × g, 10 min) at using the SILVA database . Further downstream 4 °C and stored at −20 °C. The liver was excised, washed in analyses were done using Rhea (https:// lagko uvard os. ice-cold NaCl solution (0.9%), weighted and small aliquots github. io/ Rhea/). The differential abundance analysis of were snap-frozen in liquid nitrogen and stored at −80 °C. taxa was performed on the aggregated data at the differ The gastrointestinal tract was removed and digesta from ent taxonomic levels as described . For estimation of the ileum (segment between Meckel´s diverticulum and diversity within samples (α-diversity), the Shannon and the ileo-cecal junction) and the cecum was collected. Tissue Simpson indices, the most common indices to compare and digesta samples were snap-frozen in liquid nitrogen and diversity, were calculated and transformed to the corre stored at −80 °C pending analysis. sponding effective number of species according to Jost , because they are better suited at indicating the true diversity between samples and are minimally affected Determination of AID coefficient for ether extract by the number of rare species. To measure the similar The AID coefficient for ether extract was determined at - the end of the experiment by the indicator method using ity between different microbial profiles, the β-diversity S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 5 of 19 was determined by calculating generalized UniFrac dis- for calculation of summarized probe set signals (in tances with PERMANOVA statistical test as described log2 scale) using the Robust Multichip Analysis algo- previously . Visualization of bacterial profiles among rithm, comparison fold changes (FC) and significance different groups was done by computation of non-metric P-values (ANOVA). Annotation of the microarrays was multidimension distance scaling (NMDS) . performed with the “ChiGene-1_0-st-v1.na36.galgal3. transcript.csv” annotation file. The microarray data of Determination of SCFA concentrations in the cecal digesta this study have been deposited in MIAME compliant Cecal digesta SCFA concentrations were determined format in the NCBI´s Gene Expression Omnibus pub- as described previously . In brief, 50 mg aliquots of lic repository . Owing to the rather moderate dif- cecal digesta were mixed with 0.5 mL 5% o-phosphoric ferences in the hepatic transcriptomes between groups acid containing internal standard (0.15 mg/mL crotonic HI-5.0 vs. HI-0 and groups HI-2.5 vs. HI-0, the differen - acid). Extraction was carried out by vortexing for 3 min tially expressed transcripts were filtered based on a fold and subsequent centrifugation at 21,100 × g at 4 °C for change > 1.5 or < −1.5 and a P-value < 0.05. Identical or 10 min. Prior to injection, the supernatant was centri- similar filter criteria were also applied in several recent fuged again at 21,100 × g at 4 °C for 5 min. 1 μL of the studies [45, 46]. Filtering of differentially expressed tran - extract was injected into a gas chromatograph (Clarus scripts using the Benjamini & Hochberg false discovery 580 GC system, Perkin Elmer, Waltham, USA) equipped rate adjustment method could not be applied, because with a polar capillary column (10 m free fatty acid phase, the adjusted P-values for all transcripts were > 0.05. 0.32 mm internal diameter, 0.25 μm film thickness; Gene set enrichment analysis (GSEA) was performed Macherey and Nagel, Düren, Germany) and a flame ioni - with the identified differentially expressed transcripts in sation detector. order to identify enriched Gene Ontology (GO) terms within GO category biological process using the Data- RNA extraction and hepatic transcript profiling base for Annotation, Visualization and Integrated Dis- Total RNA from liver aliquots (20 mg) were isolated covery (DAVID) 6.8 bioinformatic resource [47, 48]. using TRIzol reagent (Invitrogen, Karlsruhe, Germany) Biological process and molecular function terms were according to the manufacturer’s protocol. RNA quantity considered as enriched if P < 0.05. and quality were assessed spectrophotometrically using an Infinite 200 M microplate reader equipped with a Validation of microarray data using qPCR analysis NanoQuant plate (both from Tecan, Mainz, Germany). Microarray data of 16 differentially expressed transcripts The average RNA concentration and the A /A ratio 260 280 were validated by qPCR. For qPCR analysis, total RNA of all total RNA samples (n = 40, means ± SD) were from all broilers (n = 12–14/group) was used. Synthesis 421 ± 48 ng/μL and 1.90 ± 0.02. For hepatic transcript of cDNA and qPCR analysis was performed with a Rotor- profiling, total RNA samples from six randomly selected Gene Q system (Qiagen, Hilden, Germany) as described broilers/group were sent on dry-ice to the Genomics recently in detail . Gene-specific primers were syn - Core Facility “KFB—Center of Excellence for Fluores- thesized by Eurofins MWG Operon (Ebersberg, Ger - cent Bioanalytics” (Regensburg, Germany). Following a many). Characteristics of primers are listed in Additional further RNA quality check using an Agilent 2100 Bio- file 1: Table S1. Normalization was carried out using mul- analyzer (Agilent Technologies, Waldbronn, Germany), tiple reference genes as described recently . which revealed an average RNA integrity number (RIN) value of 8.23 ± 0.19 for all samples (n = 18, means ± SD), total RNA samples were processed using an Affymetrix Tissue homogenization and lipid extraction GeneChip Array (Chicken Gene 1.0 ST), which covers Frozen liver tissue was homogenized in methanol/water 18,214 genes represented by 439,582 probes, according (50/50, v/v) with addition of 1% sodium laurylsulfate ™ ™ to the Applied Biosystems GeneChip Whole Tran- using bead-based homogenization at a concentration of script (WT) PLUS Reagent Kit User Guide (Thermo 0.05 mg/µL . Lipid class specific, non-endogenous Fisher Scientific, Waltham, MA, USA). Following scan - internal standards were added prior to lipid extraction. ning of the processed GeneChips, cell intensity files, An amount of 2 mg liver (wet weight) or a volume of 10 which provided a single intensity value for each probe µL plasma was subjected to lipid extraction according to cell, were generated from the image data using the Com- the protocol by Bligh and Dyer . A volume of 0.5 mL mand Console software (Affymetrix). The compressed of the chloroform phase was recovered by a pipetting array image files (CEL files) were imported into the robot and vacuum dried. The residue was dissolved in Applied Biosystems Transcriptome Analysis Console 1.2 mL chloroform/methanol/2-propanol (1:2:4, v/v/v) (TAC) (v. 4.0.2) software (Thermo Fisher Scientific) with 7.5 mmol/L ammonium formate. Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 6 of 19 Lipidomic analysis of major lipid classes by mass individual animal for all other data. All parameters were spectrometry tested with the Shapiro–Wilk test for normal distribution The analysis of lipids was performed by direct flow and with the Levene’s test for homoscedasticity. When injection analysis (FIA) using a high-resolution Fourier the normal distribution was followed only after a log Transform (FT) hybrid quadrupole-Orbitrap mass spec- transformation, the log transformed data were used for trometer (FIA-FTMS) . TG, diglycerides (DG) and statistical analysis. Differences between the three groups cholesteryl esters (CE) were recorded in positive ion were analyzed by one-way analysis of variance (one- mode as [M + NH ] in m/z range 500–1000 and a target way ANOVA) followed by a Tukey’s post-hoc test when resolution of 140,000 (at m/z 200). CE species were cor- the data were normally distributed and the variances rected for their species-specific response . Ceramides were homogeneous. If the data showed heterogeneity of (Cer), phosphatidylcholines (PC), ether PC (PC O), variance, the means of the three groups were analyzed phosphatidylethanolamines (PE), ether PE (PE O), phos- using Welch’s ANOVA in conjunction with the post-hoc phatidylglycerols (PG), phosphatidylinositols (PI), and Games-Howell test. If the normal distribution was not sphingomyelins (SM) were analyzed in negative ion mode followed, a Kruskal–Wallis one-way ANOVA was per- in m/z range 520–960; lysophosphatidylcholines (LPC) formed using the Mann–Whitney U test with Bonferroni and lysophosphatidylethanolamine (LPE) in m/z range correction as post-hoc test. For all tests, a P-value < 0.05 400–650. Multiplexed acquisition (MSX) was applied for was considered statistically significant. free cholesterol (FC) and the internal standard FC[D7] . Lipid annotation is based on the latest update of the Results shorthand notation . Lipid composition of the HI fat The datasets from liver and plasma lipidomes were sub - Analysis of the composition of major lipid classes jected to principal component analysis (PCA) using the of the HI fat revealed that HI total lipids consisted MetaboAnalystR 3.2 package for R version 4.2.1. For the almost completely of TG (99.2% of total fat). All other PCA, the relative metabolite composition of individual lipid classes detected (PG, PC, DG, PE and SM) made lipid species within the different lipid classes were used. up < 0.3% of total lipids (Table 2). Analysis of individual Prior to the PCA, variables with missing values were TG species demonstrated that TG 36:0, TG 38:0, TG either excluded from the analyzes if more than 50% of the 40:0 and TG 42:2 were the most abundant TG species, samples were missing or the missing values were replaced whereas all other TG species contributed < 5% of all TG by the limit of detection (1/5 of the minimum positive species (Table 2). The majority of TG species of the HI value of each variable). After normalization by log trans- fat was saturated (56.7%), while TG species with one, formation and autoscaling the remaining values were two and three or more double bonds made up 14.0%, used for the PCA. 17.3% and 12.0%, respectively. Due to the low propor- tions of PG, PC, DG, PE, PC-O, SM and LPC in the HI fat, the individual lipid composition of these non-TG Determination of fatty acid composition of total lipids lipid classes is not reported. Analysis of the fatty acid of the diets and the liver composition of HI fat by GC-FID revealed that SFA Fatty acid composition of total lipids of the liver and the were the dominating fatty acids, with C12:0 (57.2%), diets was determined by gas chromatography-flame C14:0 (8.7%) and C16:0 (10.9%) contributing to 76.8% ionization detection (GC-FID). Briefly, total lipids were of total fatty acids (data not shown). The essential fatty extracted from 75 mg liver aliquots with a 3:2 (v/v)- acids C18:2 n-6 and C18:3 n-3 made up 10.6% and 0.8%, mixture of n-hexane and isopropanol containing C19:0 of total fatty acids, respectively. (50 mg/mL) as internal standard. After extraction, samples were centrifuged (1200 × g, 10 min) and an aliquot of the Composition of the experimental diets supernatant was evaporated under a stream of N at 37 °C. The three experimental diets within each feeding phase Lipids were subsequently transmethylated using trimethyl- had similar concentrations of crude nutrients, sugar, sulfonium hydroxide solution (Sigma-Aldrich) and the starch and energy (Table 3) and amino acids (Addi- resulting fatty acid methyl esters (FAME) were separated tional file 1: Table S2), but substantially differed in the by a GC-FID system described in detail recently . fatty acid composition of dietary total lipids (Table 3). With increasing replacement of soybean oil by HI fat, Statistical analysis the proportions of C10:0, C12:0 and C14:0 markedly Statistical analysis was conducted with SPSS 27 software increased, while those of C18:0, C18:1, C18:2 n-6 and (IBM, Armonk, NY, USA). The cage served as the exper - C18:3 n-3 decreased. As a consequence, the dominat- imental unit for feed intake and feed:gain ratio and the ing fatty acids in the HI-5.0 diet were in decreasing S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 7 of 19 Table 2 Lipid composition of the Hermetia illucens larvae fat n-3; in the HI-0 diet the dominating fatty acids were in decreasing order: C18:2 n-6, C18:1, C16:0, C18:3 n-3 Item Content and C18:0. Monounsaturated fatty acids and polyunsat- Lipid class, % of total lipids urated fatty acids in the HI fat total lipids made up 9.4% TG 99.20 ± 0.02 and 11.4%, respectively. PG 0.28 ± 0.01 PC 0.18 ± 0.01 Performance and ether extract digestibility DG 0.17 ± 0.01 Performance data (body weight gain, final body weight, PE 0.12 ± 0.02 feed intake, and feed:gain ratio) during the whole period SM 0.02 ± 0.01 were not different between groups. However, body TG species, % of total TG species weight gain and feed intake during the starter period 34:0 2.06 ± 0.04 were higher in groups HI-2.5 and HI-5.0 than in group 36:0 30.06 ± 0.49 HI-0 (P < 0.05, Table 4). No differences of performance 37:0 0.60 ± 0.31 data between groups were found during the grower and 38:0 12.25 ± 0.51 the finisher period. AID coefficient for ether extract also 38:1 0.59 ± 0.01 did not differ between groups. 40:0 7.76 ± 0.39 40:1 2.40 ± 0.10 Cecal microbiota diversity and composition 42:0 2.11 ± 0.22 In order to identify alterations of the cecal microbiota 42:1 3.91 ± 0.08 structure of the broilers, 16S rRNA-based high-through- 42:2 6.04 ± 1.04 put sequencing was applied. Following quality check, 42:3 0.73 ± 0.07 chimera check and filtering, the high-quality sequences 44:0 0.92 ± 0.10 obtained from the cecum digesta samples of the 40 44:1 2.10 ± 0.21 broilers were delineated into 90 OTUs at 97% sequence 44:2 1.65 ± 0.09 identity (Additional file 1: Table S3). Treatment effect 46:1 2.54 ± 0.43 on microbial diversity was evaluated by the use of dif- 46:2 4.80 ± 0.21 ferent diversity metrics. None of the metrics used to 46:3 0.95 ± 0.03 describe α-diversity (effective species richness, shannon 48:1 0.92 ± 0.21 effective, simpson effective, evenness) differed between 48:2 1.79 ± 0.15 groups (Fig. 1a). In addition, β-diversity of cecal bacte- 48:3 1.78 ± 0.07 rial community calculated based on generalized UniFrac 48:4 1.89 ± 0.18 distances did not differ among groups. The MetaNMDS 50:2 1.21 ± 0.06 plot generated to visualize the difference in β-diversity of 50:3 0.86 ± 0.03 cecal bacterial community among groups shows that no 52:2 0.56 ± 0.09 clustering was most visible among groups (Fig. 1b). 52:3 1.13 ± 0.02 To analyze the effect on the microbiota composition, the 52:4 1.30 ± 0.17 microbial community was analyzed at different taxonomic Sum DB0 56.73 ± 1.16 levels (phylum, class, order, family, genus). In total, only Sum DB1 13.99 ± 0.98 the relative abundances of four different bacterial taxa dif - Sum DB2 17.29 ± 1.26 fered among groups. At the phylum level, only the relative Sum DB3 6.33 ± 0.16 abundance of the least abundant bacterial phylum, Act- Sum DB4 4.18 ± 0.39 inobacteriota, contributing less than 0.15% to total bacte- Sum DB5 0.94 ± 0.11 rial phyla was different among groups. While the relative Sum DB6 0.45 ± 0.08 abundance of this phylum was lower in group HI-5.0 than Sum DB7 0.09 ± 0.01 in group HI-2.5 (P < 0.05), it did not differ between group Abbreviations: DB Double bond, DG Diglycerides, LPC Lysophosphatidylcholine, HI-5.0 and HI-0 and between group HI-2.5 and HI-0 PC Phosphatidylcholine, PC O PC-ether, PE Phosphatidylethanolamine, PG (Fig. 1c). Amongst five different bacterial classes identified Phosphatidylglycerol, SM Sphingomyelin, TG Triglycerides in the cecal digesta of the broilers, only the relative abun- Only TG species > 0.5% are shown. Data are means ± SD, n = 4 samples dance of Coriobacteriia, which belong to the phylum Act- inobacteriota, was affected by treatment; while the relative order: C12:0, C18:2 n-6, C18:1, C16:0 and C14:0; in the abundance of this phylum was lower in group HI-5.0 than HI-2.5 diet the dominating fatty acids were in decreas- in group HI-2.5 (P < 0.05), it did not differ between group ing order: C18:2 n-6, C18:1, C12:0, C16:0 and C18:3 HI-5.0 and HI-0 and between group HI-2.5 and HI-0. At Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 8 of 19 Table 3 Concentrations of nutrients and energy in the broiler diets Item Starter diets Grower diets Finisher diets HI-0 HI-2.5 HI-5.0 HI-0 HI-2.5 HI-5.0 HI-0 HI-2.5 HI-5.0 Analyzed crude nutrient content Dry matter, % FM 87.5 87.5 87.4 87.6 87.3 87.7 87.5 87.4 88.1 Crude protein, % DM 21.6 21.6 21.6 19.8 20.0 20.0 19.1 19.0 19.3 Ether extract, % DM 7.3 7.1 7.2 7.6 7.4 7.4 7.6 7.4 7.2 Crude ash, % DM 5.6 5.9 5.9 5.5 5.5 5.6 5.5 5.4 5.5 Crude fiber, % DM 3.5 3.6 3.5 3.9 3.9 3.9 4.2 4.3 4.0 Sugar, % DM 3.3 3.0 2.7 3.8 3.0 2.7 2.9 3.0 3.4 Starch, % DM 35.7 35.9 35.5 37.2 36.4 37.7 36.4 38.6 38.3 AME , MJ/kg DM 12.2 12.2 12.1 12.4 12.1 12.3 12.0 12.3 12.3 Fatty acids , % of total fatty acids C10:0 - 0.41 0.82 - 0.46 0.81 - 0.44 0.80 C12:0 0.42 18.23 37.65 0.22 19.06 38.09 0.50 19.06 38.03 C14:0 0.18 2.93 5.89 0.15 3.09 5.97 0.20 3.08 6.05 C16:0 11.82 11.88 12.17 11.44 12.06 12.19 11.59 12.12 12.26 C16:1 0.22 1.00 1.90 0.16 1.02 1.92 0.16 1.07 1.92 C18:0 3.99 3.10 1.85 3.98 3.09 1.86 3.89 3.09 1.87 C18:1 24.00 19.44 12.42 25.57 18.65 12.60 25.00 19.00 12.56 C18:2 n-6 51.32 37.73 22.94 50.88 36.83 23.13 50.93 37.26 23.02 C18:3 n-3 5.66 3.74 1.65 5.48 3.55 1.64 5.55 3.56 1.62 C20:0 0.51 0.38 0.28 0.46 0.37 0.28 0.45 0.34 0.28 Abbreviations: AME Apparent N-corrected metabolizable energy, DM Dry matter, FM Fresh matter a # Only fatty acids > 0.5% of total fatty acids are shown. The AME content of the feed was calculated according to the formula of the World´s Poultry Science Association for poultry compound feed the order level, the relative abundance of Coriobacteriales downregulated: 24) in the liver between groups HI-5.0 (class Coriobacteriia) was identified as the only bacterial and HI-0. In Fig. 3a, the differentially expressed tran - order differing between groups; its relative abundance scripts between groups HI-5.0 and HI-0 are illustrated as was lower in group HI-5.0 than in group HI-2.5 (P < 0.05), red dots in the Volcano plot. Amongst the upregulated but was not different between groups HI-5.0 and HI-2.5 genes, only three genes (BDKRB1, XDH, IGJ) exhibited a (Fig. 1c). At the family level, the only taxon identified to regulation > 2.0-fold. The top 10 upregulated transcripts be altered was the Eggerthellaceae (order Coriobacteri- were in decreasing order of their fold change (in brack- ales), whose abundance was lower in group HI-5.0 than ets): BDKRB1 (2.57), XDH (2.36), IGJ (2.10), DPP4 (1.90), in group HI-2.5 (P < 0.05), but was not different between ENDOUL (1.83), UPP2 (1.82), EMB (1.81), BASP1 (1.76), group HI-5.0 and HI-0 and between group HI-2.5 and SIK1 (1.71) and B3GALT2. Amongst the downregulated HI-0 (Fig. 1c). No differences between groups were found genes, five genes (IL22RA2, ANKRD22, LOC101749538, regarding the relative abundances of bacterial genera. ATP2B2, SLC6A13) were regulated < −2.0-fold. The top 10 downregulated transcripts were in increasing order of their fold change: IL22RA2 (−5.09), ANKRD22 (−3.76), Cecal concentrations of short-chain fatty acids LOC101749538 (−2.55), ATP2B2 (−2.15), SLC6A13 The concentrations of total and individual SCFA [acetic (−2.12), DDO (−1.89), SCAP (−1.88), SPATA4 (−1.84), acid (C2:0), propionic acid (C3:0), isobutyric acid (iC4:0), CA4 (−1.82) and ULK1 (−1.81). The fold change and butyric acid (C4:0), isovaleric acid (iC5:0) and valeric acid P-value of all differentially expressed transcripts between (C5:0)] in the cecal digesta were not different between group HI-5.0 vs. HI-0 are shown in Additional file 1: the three groups (Fig. 2). Table S4. Considering the same filter criteria as for the compar - Liver transcriptome ison of groups HI-5.0 vs. HI-0, a total of 25 transcripts According to the filter criteria applied (P < 0.05; fold were identified as differentially expressed (upregu - change > 1.5 and < −1.5), a total of 55 transcripts were lated: 19, downregulated: 6) in the liver between groups identified as differentially expressed (upregulated: 31, S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 9 of 19 Table 4 Performance data and apparent ileal digestibility (AID) coefficient for ether extract of broilers fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d Item HI-0 HI-2.5 HI-5.0 P-value Whole period (d 1 to 35) Initial BW, g 44.8 ± 0.4 44.7 ± 0.9 45.1 ± 0.9 0.719 Final BW, g 2740 ± 139 2696 ± 237 2738 ± 92 0.860 BW gain, g 2696 ± 138 2651 ± 237 2693 ± 92 0.861 Feed intake, g 3745 ± 183 3696 ± 236 3719 ± 127 0.898 Feed:Gain ratio, g/g 1.39 ± 0.02 1.40 ± 0.11 1.38 ± 0.03 0.537 Starter period (d 1 to 10) b a a BW gain, g 272 ± 17 290 ± 10 287 ± 12 0.049 b a a Feed intake, g 290 ± 19 307 ± 8 308 ± 11 0.042 Feed: Gain ratio, g/g 1.09 ± 0.02 1.09 ± 0.01 1.08 ± 0.01 0.437 Grower period (d 11 to 21) BW gain, g 823 ± 37 830 ± 61 819 ± 34 0.901 Feed intake, g 1068 ± 53 1081 ± 61 1065 ± 43 0.842 Feed: Gain ratio, g/g 1.30 ± 0.01 1.30 ± 0.04 1.30 ± 0.03 0.926 Finisher period (d 22 to 35) BW gain, g 1601 ± 91 1522 ± 210 1587 ± 79 0.560 Feed intake, g 2387 ± 124 2309 ± 181 2346 ± 98 0.612 Feed: Gain ratio, g/g 1.49 ± 0.03 1.54 ± 0.23 1.48 ± 0.06 0.822 AID coefficient for EE, % 89.0 ± 1.6 88.6 ± 1.5 88.8 ± 2.8 0.950 Data are means ± SD, n = 6–7 cages/groups. Abbreviation: BW Body weight, EE Ether extract HI-2.5 and HI-0 (Fig. 3b). Only three genes (NSMF, In order to extract biological meaning from the tran- CHIA, BDKRB1) were regulated > 2.0-fold and no scripts differentially expressed between groups HI-5.0 genes were regulated < −2.0-fold. The 10 most strongly and HI-0, GSEA was performed using GO category bio- upregulated transcripts were in decreasing order of logical process. Due to the low number of differentially their fold change (in brackets): NSMF (2.29), CHIA expressed transcripts, GSEA was not conducted for the (2.16), BDKRB1 (2.04), ENPEP (1.80), PFKFB3 (1.80), comparison of groups HI-2.5 and HI-0. Within GO cat- MIR1790 (1.72), SIK1 (1.71), DPP4 (1.71), LOC395159 egory biological process, the most enriched biologi- (1.69) and CP (1.66). The 6 downregulated transcripts cal process terms assigned to the transcripts regulated were in increasing order of their fold change: GUCY2C between groups HI-5.0 and HI-0 were (in increasing (−1.53), ATP2B2 (−1.53), INDOL1 (−1.51), MIR17 order of their P-values): small molecule biosynthetic pro- (−1.51), DDO (−1.51) and PROM1 (−1.50). The fold cess, cholesterol metabolic process, secondary alcohol change and P-value of all differentially expressed tran - metabolic process, sterol metabolic process, lipid biosyn- scripts between groups HI-2.5 vs. HI-0 are shown in thetic process, steroid biosynthetic process, cholesterol Additional file 1 : Table S5. biosynthetic process and secondary alcohol biosynthetic Microarray data of 16 differentially expressed transcripts process (Fig. 3c). between groups HI-5.0 and HI-0 were validated by qPCR. As shown in Additional file 1: Table S6, the effect direction (pos Liver and plasma lipidomes itive or negative fold change) was the same between micro- The major lipid classes in the liver were in decreasing array and qPCR for all validated transcripts, whereas the order: TG, PC, PE, FC, PI, DG, SM, CE, PE O, Cer, LPC, effect size (value of fold change) differed to some extent for LPE, PC O and PG. Despite that the sum of total lipid the validated transcripts between microarray and qPCR. Sta- classes in the liver were not different between groups tistical analysis of qPCR data revealed that twelve transcripts (HI-0: 47.6 ± 8.9 µmol/g, HI-2.5: 42.4 ± 8.9 µmol/g, were regulated either significantly (CA4, DDO, IL22RA2, HI-5.0: 47.7 ± 6.5 µmol/g, mean + SD, n = 12–14/group, HMGCS1, SCAP, SH2D4A, ULK1, XDH) or at a P-value < 0.1 P = 0.170), hepatic concentrations of PE, LPC, LPE and (AKR1D1, ANKRD22, MTHFS, OGN), whereas four tran- PG differed between groups (P < 0.05), even though only scripts (DPP4, FANCL, SIK1, SPATA4) were not regulated. slightly (Fig. 4a). Hepatic concentrations of LPE and PG Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 10 of 19 Fig. 1 Analysis of the cecal microbiome. Indicators of α-diversity (effective richness, shannon effective, simpson effective, evenness) of the cecal bacterial community (A), visualization of the difference in β-diversity of cecal bacterial community between groups by non-metric multidimension distance scaling plot (B), and distribution of cecal bacteria at different taxonomic levels (phylum, order, family) (C) of broilers fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d. A: Box and whisker plots for n = 12–14 broilers/group; C: Data are means a,b for n = 12–14 broilers/group. Means without a common letter differ across the groups, P < 0.05 were higher in group HI-5.0 than in groups HI-0 and higher in group HI-5.0 than in group HI-0, while those HI-2.5 (P < 0.05). Concentrations of PE and LPC in the with five and six double bonds were lower in group liver were higher in group HI-5.0 than in group HI-2.5 HI-5.0 than in group HI-0 (P < 0.05, Fig. 4b). With regard (P < 0.05), but not compared to group HI-0. to PC, the second most abundant hepatic lipid class, In contrast to the slight or absent differences in the relative proportions of species with one and three dou- concentrations of the lipid classes in the liver between ble bonds were higher and those with four, five and six groups, pronounced differences between groups were double bonds were lower in group HI-5.0 than in group seen with regard to the composition of individual lipid HI-0 (P < 0.05, Fig. 4c). Within PE, the third most abun- species within the different lipid classes. Regarding TG, dant lipid class in the liver, relative proportions of species the dominating lipid class in the liver, relative propor- with two and three double bonds were higher and those tions of species with zero and one double bonds were with four, six and eight double bonds were lower in group S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 11 of 19 Fig. 2 Analysis of microbial fermentation profile in the gut. Concentrations of individual [acetic acid (C2:0), propionic acid (C3:0), butyric acid (C4:0), isobutyric acid (iC4:0), valeric acid (C5:0), isovaleric acid (iC5:0)] and total (= sum of individual) short-chain fatty acids (SCFA) in the cecal digesta of broilers fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d. Data are means ± SD, n = 12–14 broilers/ group HI-5.0 than in group HI-0 (P < 0.05, Fig. 4d). The propor - of species with zero, one, and two double bonds were tions of all individual TG, PC and PE species are shown higher and those with four, five, six, seven and eight dou - in Additional file 1: Table S7. ble bonds were lower in group HI-5.0 than in group HI-0 In plasma, the major lipid classes were in decreas- (P < 0.05, Fig. 5d). The proportions of all individual CE, ing order: PC, CE, FC, TG, PE, SM, LPC, PC O, PE O, PC and TG species in plasma are shown in Additional DG, LPE and Cer (Fig. 5a). Like the concentrations of file 1: Table S8. most lipid classes (PC, FC, TG, PE, SM, LPC, PC O, PE The pronounced differences among groups with regard O, DG, LPE), the sum of total lipid classes in plasma did to the composition of individual lipid species within the not differ among groups (HI-0: 7992 ± 1423 µmol/L, different lipid classes in liver and plasma were reflected HI-2.5: 7683 ± 1742 µmol/L, HI-5.0: 8550 ± 752 µmol/L, by the results from PCA analysis which was carried out mean + SD, n = 12–14/group, P = 0.358). In contrast, the with the relative proportions of individual lipid spe- plasma concentrations of CE and Cer differed among cies of all lipid classes. The dimensional reduction of groups (P < 0.05), but only slightly; plasma concentra- the lipidome datasets from the liver (Fig. 6a) and the tions of CE and Cer were higher in group HI-5.0 than in plasma (Fig. 7a) revealed a clear separation (rightward group HI-0. Plasma concentration of Cer in group HI-5.0 shift) between the three feeding groups with increasing was also higher than in group HI-2.5 (P < 0.05), whereas dietary levels of HI fat. For the liver lipidome the cumu- plasma concentration of CE did not differ between lative proportion is 54.6% with principal component group HI-5.0 and group HI-2.5. Similar as in the liver, 1 accounting for more than 40% of the variance of the pronounced differences among groups were observed dataset. In the plasma, the cumulative proportion adds regarding the composition of individual lipid species up to 51.8% with principal component 1 accounting for within the different lipid classes in plasma. With regard 38.8%. The loading plot for the liver lipidome dataset to CE, the dominating lipid class in the plasma, relative shows that the left shift of the HI-0 group was mainly proportions of species with zero and one double bonds caused by individual TG and PC lipid species with three were higher in group HI-5.0 than in group HI-0, while or more double bonds and the right shift of the HI-2.5 those of species with two, three, four, five and six dou - and HI-5.0 groups was mainly caused by individual TG, ble bonds were lower in group HI-5.0 than in group PC and PE lipid species with less than 3 double bounds HI-0 (P < 0.05, Fig. 5b). Regarding PC, the second most (Fig. 6b). In the plasma, the right shift of the HI-2.5 and abundant plasma lipid class, the relative proportions of HI-5.0 groups was largely driven by individual TG, PC PC species with zero, one and three double bonds were and PE species with less than three double bounds, while higher, whereas those with four, five, six and seven dou - the left shift of the HI-0 group is driven by individual ble bonds were lower in group HI-5.0 than in group HI-0 TG, PC and CE species with more than 3 double bounds (P < 0.05, Fig. 5c). With regard to TG, relative proportions (Fig. 7b). Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 12 of 19 Fig. 3 Differential transcriptome analysis of the liver. Volcano plot illustrating the differentially expressed transcripts in the liver of broilers between group HI-5.0 vs. HI-0 (A) and group HI-2.5 vs. HI-0 (B). The double filtering criteria are indicated by horizontal (P-value < 0.05) and vertical (fold change > 1.5 or < −1.5) dashed lines. Red dots in the upper left and the upper right corner represent the downregulated and the upregulated transcripts, respectively. C Most enriched gene ontology (GO) biological process terms associated with the transcripts differentially expressed between group HI-5.0 vs. HI-0. GO terms are sorted by their enrichment P-values (EASE score) (top: lowest P-value, bottom: highest P-value) Fatty acid composition of liver total lipids investigated using omics technologies. Since no study As expected, fatty acid composition of hepatic total lipids existed in the literature reporting the lipid composi- was affected by dietary inclusion of HI fat. Proportions of tion of HI fat in detail, with the exception of total lipid C12:0, C14:0 and C16:1 were higher in group HI-5.0 than fatty acid composition, lipidomics was applied for the in groups HI-2.5 and HI-0, whereas proportion of C18:3 first time to fully characterize the HI fat. Our lipidomic n-3 was lower in groups HI-5.0 and HI-2.5 than in group analysis showed that the HI larvae fat used consisted HI-0 (P < 0.05, Table 5). almost completely (> 99%) of TG, whereas PG, PC, DG, PE and SM were only minor lipid classes. Amongst the Discussion TG species, saturated ones (34:0, 36:0, 38:0, 40:0, 42:0) In the present study, the effect of replacement of soy - made up approximately 60% of all TG species. TG spe- bean oil with HI larvae fat in broiler diets on the cecal cies with one or two double bonds contributed to nearly microbiome and metabolic health was comprehensively 30% of all TG species, while those with three or more S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 13 of 19 Fig. 4 Analysis of the liver lipidome. Concentrations of the major lipid classes in the liver (A) and lipid species composition within the three most abundant lipid classes in the liver, TG (B), PC (C) and PE (D), of broilers fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d. Lipid species were grouped according to the number of double bonds (DB). Data are means ± SD, n = 12–14 broilers/group. Abbreviations: CE, cholesteryl ester; Cer, ceramide; DG, diglycerides; FC, free cholesterol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PC O, PC-ether; PE, phosphatidylethanolamine; PE O, PE-ether; PG, phosphatidylglycerol; PI, phosphatidylinositol; SM, sphingomyelin; TG, triglycerides double bonds contributed less than 10% of all TG species. during the finishing period did not differ among groups. In line with the high proportion of saturated TG spe- This indicates that soybean oil replacement with HI lar - cies and TG species with a low number of double bonds, vae fat has neither beneficial nor adverse effects on fat additional fatty acid analysis of HI larvae fat total lipids digestibility, feed utilization and growth performance of revealed that C12:0, C16:0, C14:0 and C10:0 in decreas- broilers. However, our study revealed that feed intake and ing order of their proportions made up nearly 80% of body weight gain, but not feed efficiency were increased total fatty acids. Our observation that saturated TG spe- by dietary inclusion of HI larvae fat during the starter cies made up 60% of all TG species, while saturated fatty period, whereas no effect was found during the grower acids made up 80% of all fatty acids in HI fat total lipids, and the finisher period. This suggests that feed accept - however, is not a discrepancy. This is likely attributed ance was obviously improved in broilers during the early to the fact that saturated fatty acids are not exclusively d of life by the use of HI larvae fat. The reason underlying found in TG species with zero double bonds, but are this observation is unclear, but we exclude the possibility also present in TG species with 1 or more double bonds. that the taste of the soybean oil used in the HI-0 diet was Regardless of this, our data from lipidomics and fatty acid impaired due to fat deterioration during storage or diet analysis clearly show that the HI fat used in the present preparation, because the same batch of soybean oil was study contained large amounts of saturated fatty acids, also used for the diet of group HI-2.5, despite feed intake which largely explains the hard consistency of the HI fat of this group was similar as in group HI-5.0. Unfortu- at room temperature. Despite the marked difference in nately, comparable data from other studies are not availa- the fatty acid profile between soybean oil, the commonly ble to explain our observation. While in two other studies used fat source in broiler diets, and HI larvae fat, partial dealing with the use of HI larvae fat as an alternative fat or complete replacement of soybean oil with HI larvae fat source in broiler diets performance data were reported had no effect on performance parameters (feed intake, either for the whole period  or only the finishing body weight gain, feed:gain ratio) of the broilers over the period , a further study did not provide any perfor- whole 35 d-period. In line with the unaltered growth per- mance data . However, the latter study is not suitable formance, ileal digestibility of ether extract determined due to inadequate control of nutrient composition of the Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 14 of 19 Fig. 5 Analysis of the plasma lipidome. Concentrations of the major lipid classes in the plasma (A) and lipid species composition within the three most abundant lipid classes in plasma, CE (B), PC (C) and TG (D), of broilers fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d. Lipid species were grouped according to the number of double bonds (DB). Data are means ± SD, n = 12–14 broilers/group. Abbreviations: CE, cholesteryl ester; Cer, ceramide; DG, diglycerides; FC, free cholesterol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PC O, PC-ether; PE, phosphatidylethanolamine; PE O, PE-ether; SM, sphingomyelin; TG, triglycerides diets as evident from the marked difference of crude fat phyla, Bacteroidota and Firmicutes, together account- content between the starter diets (3.6% and 5.9% in the ing for approximately 99% of all bacteria in the three control diet and HI larva fat diet, respectively). groups. Considering this and the rather small reduc- Despite studies demonstrating antimicrobial effects tion in the abundances of the abovementioned bacte- of MCFA in the intestine of broilers at dosages between rial taxa suggests that the impact of HI larvae fat on the 0.35% and 1.4% in the feed [22, 23], the present study, cecal microbiota structure was very low. One important in which the dietary concentration of MCFA (sum of reason might be that the amount of MCFA reaching the C10:0 and C12:0) was about 1.25% and 2.5% in groups cecum was not sufficient to exert pronounced antimicro - HI-2.5 and HI-5.0, respectively, showed only a very little bial effects. This assumption is supported by our observa - impact of dietary inclusion of HI larvae fat on the cecal tion that the ileal digestibility of crude fat was very high microbiome. This was evident from the observation that (approximately 90% in all groups) and the fact that TG- cecal microbial diversity (α and β) did not differ between bound MCFA are hydrolysed and absorbed more rap- groups and taxonomic analysis revealed differences in idly than TG-bound long-chain fatty acids due to easier the abundance of only four low-abundance bacterial taxa emulsification and less dependence on pancreatic lipase among groups; namely, the relative abundances of the activity [27, 28]. In agreement with this, an increased phylum Actinobacteriota, the class Coriobacteriia, the ileal fat digestibility has been observed in a recent study order Coriobacteriales and the family Eggerthellaceae with broilers by dietary substitution of soybean oil by HI (all belonging to Actinobacteriota) were lower in group fat . Nevertheless, reports exist demonstrating that HI-5.0 compared to group HI-2.5, but the abundances of short-term administration of specific MCFA, such as these bacterial taxa did not differ between groups HI-5.0 caprylic acid (C8:0) and 1-monoglyceride of capric acid and HI-0 and between groups HI-2.5 and HI-0. No dif- (C10:0), via the feed or the drinking water reduces cecal ferences at all were found between groups in the abun- colonization of pathogenic bacteria, such as Campy- dances of bacterial families belonging to the two main lobacter jejuni, after artificial infection [22, 23, 57]. S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 15 of 19 Fig. 6 Principal component analysis of liver lipidome. Scores plot with plotted 5% confidence interval (A) and associated loading plot (B) of principal component analysis (PCA) of the liver lipidome of the broilers. Broilers were fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d. Data are principal components (PC 1 or PC 2) and their loadings calculated based on the relative abundances of the lipid species within their lipid classes, n = 12–14 broilers/group. Abbreviations: Cer, ceramide; DG, diglycerides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PC O, PC-ether; PE, phosphatidylethanolamine; PE O, PE-ether; PG, phosphatidylglycerol; PI, phosphatidylinositol; TG, triglycerides However, contradictory results have been reported from groups are not surprising and are a further indication of another study, in which addition of either non-coated or the limited impact of HI larvae fat on the cecum micro- coated specific MCFA (C6:0, C8:0, C10:0) to the feed at biota of the broilers. In agreement with our results, a comparable concentrations as in the studies from Solis de recent study demonstrated that complete replacement of los Santos et al. [22, 23] did not reduce cecal Campylo- 5% corn oil in the diet with HI larvae fat did not affect the bacter colonization in broilers . The reason for these concentrations of total and individual SCFA and viable contradictory results is not clear, but differences between counts of Clostridium perfringens in the ileum and cecum studies in the formulation of the MCFA in the feed may of broilers during a 30-d-feeding period . In addi- explain that the MCFA were less absorbed in the small tion, feeding a diet containing about 1% MCFA as lau- intestine, thereby, reaching the ceca at higher concentra- ric acid had no influence on the DNA copy numbers of tions in the one study than in the other. Thus, it is possi - some beneficial bacteria (Lactobacillus and Bifidobacte - ble that the formulation of the diets containing HI larvae rium spp.), opportunistic pathogens (Enterobacteriaceae, fat in the present study allowed an efficient absorption of Escherichia coli) and the pathogen Campylobacter jejuni MCFA in the small intestine, but a low passage of MCFA in the jejunal digesta of broilers . into the ceca of the broilers. Consistent with our finding that the dietary fats Owing to differences between bacterial families of dif - including the HI larvae fat were readily absorbed in the ferent taxa with regard to the use of fermentation sub- small intestine, the present study revealed some effects strates and the metabolic pathways engaged in substrate of dietary inclusion of HI larvae fat on the hepatic and utilization, a shift in the gut microbial community is plasma lipidomes of the broilers. However, the effects typically accompanied by an altered profile of microbial of dietary inclusion of HI larvae fat were mainly related fermentation products, such as SCFA, in the gut. Since to the individual lipid species composition of the lipid dietary inclusion of HI larvae fat into the broiler diets did classes, whereas the concentrations of the different lipid not cause a substantial shift in the bacterial community classes in liver and plasma were either not or only mar- of the cecum, the unaltered concentrations of total and ginally (liver: PE, PG, LPC and LPE; plasma: CE and individual SCFA in the cecal digesta among the three Cer) affected. In line with this, the sum of total lipids in Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 16 of 19 Fig. 7 Principal component analysis of plasma lipidome. Scores plot with plotted 5% confidence interval (A) and associated loading plot (B) of principal component analysis (PCA) of the plasma lipidome of the broilers. Broilers were fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) Hermetia illucens (HI) larvae fat for 35 d. Data are principal components (PC 1 or PC 2) and their loadings calculated based on the relative abundances of the lipid species within their lipid classes, n = 12–14 broilers/group. Abbreviations: CE, cholesteryl ester; Cer, ceramide; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PC O, PC-ether; PE, phosphatidylethanolamine; PE O, PE-ether; SM, sphingomyelin; TG, triglycerides with zero or few double bonds (e.g., in the liver: TG spe- Table 5 Fatty acid composition of hepatic total lipids of broilers fed diets with either 0 (HI-0), 2.5% (HI-2.5) or 5.0% (HI-5.0) cies with 0 and 1 double bonds, PC species with 1 and Hermetia illucens (HI) larvae fat for 35 d 3 double bonds, PE species with 2 and 3; in plasma: TG * species with 0, 1 and 2 double bonds) were increased Fatty acid , HI-0 HI-2.5 HI-5.0 P-value g/100 g total but those with four or more double bonds (e.g., in the fatty acids liver: TG species with 5 and 6 double bonds, PC spe- c b a cies with 4, 5 and 6 double bonds, PC species with 4, 6 C12:0 0.56 ± 0.17 1.03 ± 0.2 1.53 ± 0.34 0.000 c b a and 8 double bonds; in plasma: TG species with 4, 5, 6, C14:0 0.48 ± 0.18 1.18 ± 0.41 1.89 ± 0.53 0.000 7 and 8 double bonds) were decreased in group HI-5.0 C16:0 22.0 ± 2.7 22.4 ± 2.1 22.3 ± 2.4 0.945 b b a compared to group HI-0. These alterations in the indi - C16:1 1.93 ± 0.68 1.81 ± 0.85 2.62 ± 0.55 0.012 vidual lipid species composition of lipid classes likely C18:0 21.5 ± 2.9 21.5 ± 2.4 19.7 ± 2.2 0.108 reflected the higher percentage of saturated fatty acids C18:1 n-9 21.6 ± 4.4 20.4 ± 4 23.1 ± 3.3 0.204 in the HI-5.0 broiler diets (59%–60%) than in the HI-2.5 C18:2 n-6 20.5 ± 2.6 20.4 ± 2.2 19.3 ± 2.9 0.436 a b b (37%–38%) and the HI-0 (16%–17%) broiler diets, C18:3 n-3 0.61 ± 0.09 0.50 ± 0.11 0.43 ± 0.10 0.000 thereby, resulting in a higher intake of saturated fatty C20:3 n-6 0.87 ± 0.31 0.97 ± 0.33 0.95 ± 0.15 0.352 acids from HI larvae fat. Although most of the altera- C20:4 n-6 8.52 ± 2.09 8.46 ± 1.74 7.10 ± 1.34 0.065 tions in the plasma and liver lipidomes among the treat- C22:6 n-3 0.92 ± 0.37 0.83 ± 0.34 0.67 ± 0.18 0.035 ment groups were probably attributed to the marked Data are means ± SD, n = 12–14 broilers/group. Only fatty acids > 0.5 g/100 g difference in fatty acid composition between soybean oil total fatty acids are shown and HI larvae fat, certain changes in the liver or plasma lipidomes might also reflect specific metabolic effects of the characteristic fatty acids, such as MCFA, contained liver and plasma were not influenced by dietary treat - in the HI larvae fat. For instance, the reason for the ment. With regard to the individual lipid species com- slight increase of CE by HI larvae fat might be based on position of the different lipid classes, the most striking the fact that MCFA, such as capric acid and lauric acid, observation was that the proportions of lipid species S chäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 17 of 19 and the long-chain saturated fatty acid myristic acid and these genes occurred either only at the transcriptional palmitic acid exert hypercholesterolemic effects and level or at a too weak level to substantially alter the increase blood levels of low-density lipoprotein (LDL) abundance of the encoded proteins. In any case, our [60–62], in which CE makes up approximately 50%, findings from hepatic transcriptome analysis indicate at least in humans. Considering this, it appears possi- that the effect of soybean oil replacement with HI lar- ble that the raise in plasma CE levels in group HI-5.0 vae fat on intermediary metabolism of broilers is rela- resulted from elevated LDL levels. The latter might tively weak. be explained by an effect of HI larvae fat on different aspects of lipoprotein metabolism, such as the catabo- Conclusion lism of very-LDL (VLDL) particles by lipoprotein lipase Comprehensive analysis of the effect of partial and com - in extrahepatic tissues (white adipose tissue, skeletal plete replacement of soybean oil with HI larvae fat in muscle), hepatic clearance of LDL particles from plasma broiler diets on growth performance, cecal microbi- or the exchange of CE between lipoproteins which is ome, liver transcriptome and liver and plasma lipidomes governed by CE transfer protein. revealed only very modest, but not any adverse effects In order to provide a deeper insight into the meta- of dietary HI larvae fat inclusion. Interestingly, growth bolic effects of HI larvae fat in broilers, differential performance of the broilers during the starter period transcriptome analysis of the liver was carried out. was even improved by dietary inclusion of HI larvae fat. The finding that only 55 annotated genes out of more The findings of this study suggest that HI larvae fat can than 19,000 genes screened were found to be differen- be used as an alternative fat source in broiler diets, which tially expressed between group HI-5.0 and group HI-0 makes broiler production more sustainable through the and the number of genes regulated greater twofold exclusion of soybean oil and the utilization of HI lar- was very low, suggests that the impact of HI larvae fat vae fat from regional production. inclusion on the hepatic transcriptome was modest. As expected, the impact of HI larvae fat inclusion on Abbreviations the hepatic transcriptome was less at the lower die- AID Apparent ileal digestibility tary inclusion level of HI larvae fat; i.e., only 25 anno- AKR1D1 Aldo –keto reductase family 1 member D1 tated genes were identified as differentially expressed CE Cholesteryl esters Cer Ceramides between groups HI-2.5 and HI-0, from which 7 genes DG Diglycerides were also differentially expressed between groups DM Dry matter HI-5.0 and HI-0. Bioinformatic GSEA of these 55 dif- FAME Fatty acid methyl esters FC Free cholesterol ferentially expressed hepatic genes revealed a particu- FDFT1 Farnesyl-diphosphate farnesyltransferase 1 lar involvement of the encoded proteins in biological GO Gene ontology processes dealing with sterol metabolism, such as cho- GSEA Gene set enrichment analysis HI Hermetia illucens lesterol metabolic process, cholesterol biosynthetic HMGCS1 3-Hydroxy-3-methylglutaryl-CoA synthase 1 process, sterol metabolic process, steroid biosynthetic HSD17B7 H ydroxysteroid 17-beta dehydrogenase 7 process and lipid biosynthetic process. This observa- LCFA Long-chain fatty acids LDL Low-density lipoprotein tion was likely explained by the finding that several LPC Lysophosphatidylcholines genes involved in sterol synthesis, such as HMGCS1 LPE Lysophosphatidylethanolamines (encoding 3-hydroxy-3-methylglutaryl-CoA synthase MCFA Medium-chain fatty acids NMDS Non-metric multidimension distance scaling 1), SC AP (encoding SREBF chaperone), AKR1D1 OUT Operational taxonomic units (encoding aldo–keto reductase family 1 member D1), PC Phosphatidylcholines FDFT1 (encoding farnesyl-diphosphate farnesyltrans- PC O Ether PC PCA Principal component analysis ferase 1) and HSD17B7 (encoding hydroxysteroid PE Phosphatidylethanolamines 17-beta dehydrogenase 7), were amongst the genes PE O Ether PE differentially expressed between groups HI-5.0 or PG Phosphatidylglycerols PI Phosphatidylinositols HI-2.5 and HI-0. Despite that the proteins encoded SCAP SREBF chaperone by HMGCS1, FDFT1 and SC AP are involved in cho- SCFA Short-chain fatty acids lesterol synthesis , the unaltered levels of free SFA Saturated fatty acids SM Sphingomyelins cholesterol and CE in the liver between groups sug- TG Triglycerides gests that the regulation of these genes by HI larvae TM Tenebrio molitor fat inclusion had no impact on hepatic cholesterol lev- VLDL Very low-density lipoprotein WPSA World´s Poultry Science Association els. Plausible reasons might be that the regulation of Schäfer et al. Journal of Animal Science and Biotechnology (2023) 14:20 Page 18 of 19 References Supplementary Information 1. van Huis A. Potential of insects as food and feed in assuring food security. The online version contains supplementary material available at https:// doi. Annu Rev Entomol. 2013;58:563–83. org/ 10. 1186/ s40104- 023- 00831-6. 2. Gasco L, Biasato I, Dabbou S, Schiavone A, Gai F. 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Journal of Animal Science and Biotechnology – Springer Journals
Published: Mar 1, 2023
Keywords: Broilers; Cecal microbiota; Hermetia illucens; Insect fat; Liver lipidome; Medium-chain fatty acids
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