WP1 – HI rearing optimisation
1) to adjust the nutritional composition of the HI-larvae and required rearing protocol based on the nutritional needs of chickens;
2) to formulate and define specific diets for HI larvae, reared for slow growing chickens, with residual streams based on objective 1;
3) to maintain continuous rearing system based on acquired knowledge during the project to deliver live HI larvae for feeding trials in WP2;
4) to assess agronomic possibilities of frass via plant growth trials.
This WP will also contribute to the ongoing discussion on organic insects on EU level, as it is unclear at this stage what is necessary to produce organic insects.
All trials planned are performed using 6-day-old HI larvae produced with the established insect colony present in Inagro facility under standardized circumstances (crates, climate control, machinery etc.). The standard feed trials protocols will be used. Microbiological and heavy metals analyses will also be performed on insect larvae and their rearing substrates (WP3).
Previous research projects resulted in standardized rearing protocol for HI larvae. Up to now the main goal was: rearing lots of big and healthy HI larvae in a short time for mass production. The composition of the larvae can be influenced by the feed. Therefor feed substrate is very important.
In case of lack of nutritional ingredients the lifecycle is prolonged. Live insects are not being processed and integrated in homogeneous mixed feed. This tasks adjusts the standard rearing protocol based on the needs of chickens.
Different organic side streams (vegetable and fruit market waste, agro-industrial, winery, brewery, eggs, bakery and milk by-products) are allowed by the EU as feed. A selection of those streams is made based on availability (amount and time), experience and knowledge and price tag (transport,
product itself). The combination of the nutritional composition (Task 3.1) and nutritional needs of HI larvae allows us to prepare dietary mixtures (later on “compound diets”). In the H2020 project ‘Susinchain’ Inagro is responsible for the standardisation of feed trials in different insect species,
In this process the standard protocol that Inagro developed during previous research projects will be further optimised and implemented internationally afterwards. 6 replicates of 10000 larvae per compound diet is prepared with the aim of rearing a sufficient amount of larvae to be analysed for their chemical composition and microbiological safety. In each feeding trial, 3 experimental compound diets are tested against the reference diet. For each replicate, the larvae are allocated in 60 by 40 plastic containers directly on the rearing substrate placed in the climatic chamber. To identify the best dietary mixture, different performance parameters are assessed. Larvae are then provided for the microbiological and chemical assessments planned in WP3.
Based on the results obtained in Tasks 1.1 and 1.2, the best compound diet is identified (= optimized diets) in terms of performance parameters and used for continuous small scale mass production. This is a challenge due to time-limited availability of residual streams, different composition of those streams and possible hygienical issues,. Briefly, the larvae rearing protocol utilized is the following: every 10 days, 6-day old larvae are allocated to containers (62 cm × 42 cm × 11 cm), grow for 10 days and stocked in a refrigerated room (T°: 15°C) in order to stop their growth, but keep them alive. The produced insects can be used for feeding trials with chickens in WP2 . In the H2020 project ‘Susinchain’ Inagro is looking into the most ideal transport conditions for live larvae. This knowledge will be used in this task as well.
This task aims to assess the potential use of insect frass as fertilizer. Frass is analyzed to determine macro- and micronutrients (total C, N, P, K, Fe, Ca, Mg, Mn, Cu, Zn, Ni, Pb, Cd, Cr), pH, moisture, and ash content. All these parameters are assessed according to standard procedures.
Frass is then used in plant pots in a controlled climate chamber (20°C, 65% relative humidity). The material is tested at two N fertilization levels, corresponding to 60 and 120 kg N/ha. The two fertilization levels of each frass are tested against a non-fertilized control (0) and a N fertilization
made with mineral N (urea).
The growing substrate is made by mixing coarse sand (50%), sandy soil (30%) and peat (20%). Rocket (Eruca sativa Mill.) is seed, thinned to 20 plants per pot (3 pots/treatment) then grown for 4 weeks. Total dry matter production is measured.
However, its legal status remains uncertain in many other EU Members States. Therefore this task will also contribute to ongoing discussion on EU level.
WP2 – Chickens feeding trials
1) to perform in vivo poultry feeding trial to determine the optimal supplementation level of live HI larvae for organic chickens production;
2) to assess the gender effect on performances, welfare and health of birds fed live insect larvae;
3) to assess in two different genotypes model (with different growing-rate) the effect on performances, welfare and health of birds fed live insect larvae.
Two in vivo experimental trials are planned in the first and second year of the project, respectively. Bird handling will be in accordance with the guidelines for the Care and Use of Agricultural Animals in Research and Teaching. For each trial, a total of 180 male and 180 female chickens are reared at the Animal Facility Centre of the UNITO partner. An intermediate slow-growing breed (Sasso) chicken is used in the first trial, while a slow-growing Italian (Bianca di Saluzzo, BS, protected by Slow Food trade) breed will be used in the second trial.
Both breeds are suitable for outdoor raising of organic chicken and characterized by different growth rates and nutritional requirements. In both trials the birds are reared separated by sex in order to study the different growth pattern and behavior due to larvae supplementation.
Two in vivo experimental trials are planned in the first and second year of the project, respectively. Bird handling are in accordance with the guidelines for the Care and Use of Agricultural Animals in Research and Teaching. For each trial, a total of 180 male and 180 female chickens are reared at the Animal Facility Centre of the UNITO partner. An intermediate slow-growing breed (Sasso) chicken is used in the first trial, while a slow-growing Italian (Bianca di Saluzzo, BS) breed will be used in the second trial.
Both breeds are suitable for outdoor raising of organic chicken and characterized by different growth rates and nutritional requirements. In both trials birds are reared separated by sex in order to study the different growth pattern and behaviour due to larvae supplementation. The chickens will be allotted according to sex and treatment in 4 groups (15 birds/pen, 90 birds/treatment) and fed with SBM control diet:
1. without HI larvae (control male group);
2. supplemented with live HI larvae (treated male group);
3. without HI larvae (control female group);
4. supplemented with live HI larvae (treated female group).
Sasso birds are individually weighed on day 20, 40, 60, 84 of age while the BS birds on day 20, 50, 80, 110 and 150 of age. The in vivo growth performance (feed intake, body weight gain, feed conversion ratio) and the mortality are recorded.
In both trials, bird welfare is assessed by the measurement of different stress markers in blood and fecal samples. Heterophil/lymphocyte (H/L) ratio, serum lysozyme (LYS), and total plasma antioxidant activity (OXY) will be assayed in blood samples while corticosterone in fecal samples
beacuse in birds corticosterone is the main glucocorticoid; its quantification provides information about adrenocortical activity and is considered to be a reliable indicator of stress levels in birds. To assess the leukocyte formula and then to calculate the H/L ratio, the procedure described by 
is used. LYS (μg/mL) is assessed by the lyso-plate assay. OXY is measured using commercially available kits and is quantified in μM HClO/mL.
Fecal Corticosterone concentration (FCC) will be determined using commercial enzyme immunoassay kits validated for dried fecal extracts.
Blood stress markers are assessed from the beginning to the end of the growing period at regular intervals. The same birds (10 birds/treatment) are sampled at the considered ages. FCC will be assessed at the same age of the birds sampled for blood stress markers but in this case
a pool of faeces for each pen will be utilised. Six pool/treatment will be analysed for each sampling (bird age) time.
Moreover, the bird’s welfare status (in situ behaviour observations) will be evaluated by means of video recording cameras.
 Castellini et al. Italian Journal of Animal Science 15.1 (2016): 37-46.
At the end of both trials, after 8-h feed withdrawal, 12 chickens/group are stunned by electrocution (110 V;350 Hz) before killing. After killing, carcasses are plucked and weighed. Then, non-edible viscera (intestines, proventriculus, gall bladder, spleen, oesophagus and full crop) are removed and the weight of the partial eviscerated carcass (PEC) is recorded. Then the head, neck, legs, edible viscera (heart, liver and gizzard) and fat (perivisceral, perineal and abdominal) are removed to obtain the ready-to-cook carcass (RCC)  and its weight. The proportion of the PEC and RCC on the live body weight (BW) is calculated. The weight of the main viscera (intestines, spleen, heart, liver and gizzard) is recorded. RCC is cooled in a cold tunnel and refrigerated at 4°C for 24 h.
Samples of breast will be collected to perform the evaluations described in Task 3.3 and 3.4.
Samples of small and large intestine are collected and used for the analyses planned in Task 3.5.
 Romboli et al 1996. Metodologie relative alla macellazione del pollame, alla valutazione e dissezione delle carcasse e delle carni avicole. Zoot. Nutr. Anim. 22: 177-180.
WP3 – Laboratory and Sensorial Analyses
1) to perform all the chemical and microbiological analyses needed for the implementation and assessments of WP1;
2) to assess the impact of different feeding regimes on chicken health and meat quality;
3) to deepen the understanding of how feed and animal welfare affect muscle protein composition and fillets nutritional quality.
The health and wellbeing status of the chickens are evaluated by histological and immunological investigations. Chicken breast fillets quality parameters are determined. Sensory attributes including tenderness, juiciness and flavour are evaluated by the sensory panel at CNR. On-line NIR technology will be applied to screen for muscle abnormalities (wooden breast) and estimate macronutrient composition in chicken breast fillets. Macro- and micronutrient composition including fatty acid profiles will be conducted on a subsample. Proteomic techniques are used to assess changes in muscle protein composition and degradation with feed type. Nutritional quality of chicken breast fillet are further assessed by determining their in vitro protein digestibility and amino acid composition.
At the beginning of the first year, all the collected organic sidestream (OS) ingredients are analysed for their nutrient contents to properly formulate the diets to be used in Task 1.1. The analyses are repeated for each batch of OS ingredient collected during the activities performed in Task 1.1. All the diets formulated and used to rear the HI larvae in WP1 are analysed for their proximate composition. The diets used in Task 1.2 are also evaluated for their amino acid profiles (Biasato et al 2016).
In order to guarantee the feed safety, microbiological analyses (total bacteria count, pathogens such as: Salmonella spp., Listeria monocytogenes, prevalence of Campylobacter spp.) are performed on both individual OS ingredients and all the diets used for insect rearing. The microbiological analyses are carried out using ISO published protocols for food and feed. Biasato et al. (2016). Effects of dietary Tenebrio molitor meal inclusion in free‐range chickens. Journal of animal physiology and animal nutrition, 100.6: 1104-1112.
At the end of Tasks 1.1, 1.2 and 1.3, HI larvae are analysed for their proximate and amino acid compositions. For safety purposes, a microbiological assessment, evaluating the total bacteria count, and the presence of pathogens (such as Salmonella spp., Listeria monocytogenes, Campylobacter spp.) is performed on HI larvae, using referenced ISO methods.
The microbiota composition of the larvae is known to affect their health and performance and has to be considered in the effort to optimize larvae biomass yield. The microbiota of the HI larvae produced in Task 1.3 is subjected to total DNA extraction in order to sequence the 16S rRNA gene
by using the Illumina MiSeq platform. 16S rRNA is used as target, and the most discriminant variable regions are selected to get the highest taxonomic definition and assignment.
Bioinformatics tools are used to analyse the sequence data, with taxonomic identification also considering oligotyping. Pathogens detection and links between specific nutrient intakes and structure of the microbiome are established.
At the end of each poultry trial, chicken breasts are sampled and, on their left part, some physical (pH, color, drip loss) and chemical (fatty acid profile) parameters are recorded by the CNR partner staff. The remaining right chicken breast filets will be shipped frozen to NOFIMA, where the thaw loss of each filet will be determined. On-line NIR-technology will be applied to screen for muscle abnormalities (wooden breast) and estimate macronutrient composition in all chicken breast fillets. This composition data will give an overview of within treatment and between treatment variation.
Instrumental tenderness (Warner-Bratzer shear force) will be determined in all chicken filets. A representative subset of 4-8 filets from each treatment group will be picked for further analysis including protein content (combustion), amino acid composition, proteomic and in vitro digestion. Proteomic techniques will be used to assess differences in muscle protein composition including protein oxidation and degradation between groups. As a part of the nutritional quality of the chicken products, protein digestibility will be determined in selected samples by using a human in vitro digestion model combined with customized analytical tools (Size-exclusion chromatography and combustion). Oxidation products (malondialdehyde equivalents) will be determined in chicken filets before and after cooking and in vitro digestion.
Breast meat samples are used to evaluate the consumer perception of meat obtained from animals fed insect meals. As a first step a panel, expert in sensory evaluation, will be specifically trained on breast meat, to define and measure the sensory attributes. Panel test will be performed both through descriptive analysis (DA)  and dynamic tests such as Temporal Dominance of Sensation (TDS) , to provide sensory profiles and predict consumer perception. Once defined the key descriptors of meat product, consumer test will be organized with untrained assessors, selected among regular consumers of poultry products. Consumer test will be conducted through Check-allhat-Apply (CATA)  to assess product acceptability, and drivers of hedonic judgement.
Questionnaire will also include questions about consumers’ expectations and inclination for meat obtained from animals fed with insects, as compared to the traditional product. Test are performed in the sensory laboratory of IBE CNR under controlled conditions, with panellists placed in individual tasting booths, with laptops equipped with a specific software for sensory data acquisition.
 Meilgaard et al. (2016). Descriptive Analysis Techniques. In Sensory Evaluation Techniques, 5th ed. pp. 201–219.
 Labbe et al. (2009). Food Quality and Preference, 20, 216–221.
 Ares & Jaeger (2015). Check-all-that-Apply (CATA) questions with consumers in practice (pp. 227–245). Cambridge, UK: Woodhead Publishing.
Intestinal caeca contents are used for microbiota/microbioma evaluations. The microbiota is studied by high-throughput rRNA gene-targeted amplicon sequencing by using the Illumina MiSeq platform. 16S rRNA are used as target, and the most discriminant variable regions are selected to get the highest taxonomic definition and assignment. Adequate sample coverage is planned to optimally describe the complexity of the cecal microbiota. Alpha-diversity parameters, phylogenetic (UniFrac) and non-phylogenetic beta-diversity, operational taxonomic unit (OTU) network, and OTU co-occurrence and co-exclusion patterns are considered.
In order to functionally describe the observed reassembled gut microbiota resulting from live HI larvae administration, the SCFAs concentrations are investigated. The levels of acetate, propionate, isobutyrate, butyrate, isovalerate and valerate were measured in caeca by gas chromatography. Cecal digesta samples (each about 5 ml) after dilution with oxalic acid (1:1, v/v), were subjected to short chain fatty acids (SCFA) analysis by gas chromatography (Thermo-Electron mod. 8000top, FUSED SILICA Gaschromatograph (ThermoElectron Corporation, Rodano, Milan, Italy) with OMEGAWAX 250 fused silica capillary column 30 m × 0.25 mm × 0.25 mm film thickness; analysis temperature, 125 °C; flame ionisation detector, 185 °C; carrier helium, 1.7 ml/min .
 Taranto et al. (2003). Asian-australasian journal of animal sciences, 16.10: 1540-1544.
Morphological and morphometric investigations are performed on the gut segments. Intestinal segment samples of duodenum, jejunum, ileum and caecum are excised and flushed with 0.9% saline to remove all the content. Samples of liver, spleen, thymus, bursa of Fabricius, kidney, heart and glandular stomach are also collected. The morphometric indices evaluated are villus height (Vh) from the tip of the villus to the crypt, crypt depth (Cd) from the base of the villi to the submucosa and villus height to crypt depth (Vh/Cd) ratio (Laudadio et al 2012).
Morphometric analyses are performed on 10 well-oriented and intact villi and 10 crypts chosen from duodenum, jejunum and ileum. Histopathological alterations are scored using a semiquantitative scoring system. In order to characterize the three types of mucins, formalin-fixed and paraffin-embedded intestinal sections are also submitted to three different histochemical staining. Mucin staining intensity of goblet cells are scored semiquantitatively.
To examine whether the effects of live HI larvae supplementation on the systemic immune response, serum cytokines (IFN-γ, TNF-α, IL-8, and IL-10) and immunoglobulins (IgG and IgA) concentrations are measured using commercially available enzyme-linked immunosorbent assay (ELISA) test kits.
Laudadio et al (2012). Productive performance and histological features of intestinal mucosa of broiler chickens fed different dietary protein levels. Poultry Science, 91.1: 265-270.
WP4 – Sustainability assessments
Quantitative and qualitative comparisons for the possibilities of innovative production concepts of the new types of protein sources for organic poultry feed will be performed. Specific multicriteria assessment frameworks will used to analyze sustainability from integrated perspective.
i) flows of nutrients, energy, degrees of recycling, conversion efficiency;
ii) associated direct and indirect environmental impact;
iii) socio-economic performance of developed solutions and capacity to answer local issues regarding rural development.
These integrated assessment approaches will rely on quantitative approaches such as life cycle assessment (LCA) and qualitative approaches as key performance indicators used for social aspects. LCA will be performed for innovative feed components and formulations to define the comparative sustainability impact criteria applied to common equivalent feed formulations:
i) environmental (global warming, water and energy consumption, soil quality/fertility and biodiversity);
ii) economic (gross margin, including total sales receipts, changes of stocks and direct payments);
iii) social (number of jobs and working hours).
Environmental impacts associated with all the stages of the production, transformation and distribution of the insect larvae will be assessed in a full LCA (Life Cycle Assessment), including a description of the scenarios to be investigated and the indicators applied. This task will quantify and evaluate the environmental impacts of insect production and potential added value of insect meal in feed formulations and in animal feeding. The LCA will offer a structured and quantitative approach to explore the potential environmental impacts throughout the life cycle of the developed applications and products [ISO, 14040, 14044 (2006)].
The LCA analysis of insect production chain will include:
1) acquisition of insect feeding raw materials,
2) insect feeding and harvesting,
3) insect meal manufacturing and processing,
4) storage, materials handling and logistics management,
6) insect based feed application for feeding,
7) end-of-life stage.
A specific focus will be laid on the comparison and reduction of the carbon footprint of different alternatives investigated and thus on the possible potential to mitigate climate change. Then, the most cost-effective option among different competing alternatives to produce, transform and distribute insect larvae will be identified (LCC = Life Cycle Cost Analysis), and the options to reduce energy consumption (carbon foot print) and production costs will be proposed and discussed.
Life Cycle Cost (LCC) analysis will measure and analyze the capital and operational costs resulting from the development and possible implementation of the proposed insect meals in the target applications (Swarr et al., 2011). The LCC will present a similar structure and will be conducted in parallel to the LCA (in terms of functional unit and approach). The analysis will be developed in two basic steps: cost identification and a cost benefits analysis. Therefore, as soon as the process chain for the selected products manufacturing are sketched, cost models can be set up to estimate the product costs as a function of various parameters.
This model will also guide the optimization process e.g. by identifying possible bottlenecks in the process chain. The results of this analysis will make it possible to compare the proposed technology and final products with currently used technologies for the same applications, from an economical point of view. Based on the costs identified, the economic viability of insect production and insect-derived products will be assessed. This task will aim to decide whether the proposed production lines are economically feasible to bring them to the market and what could be the potential economic risks when the real incorporated price is considered.
Swarr TE, Hunkeler D, Klöpffer W, Pesonen H-L, Ciroth A, Brent AC, Pagan R (2011) Environmental life cycle costing: a code of practice. SETAC, Pensacola.
The results from the LCC and LCA as well as results from other WPs will be merged, to make an overall sustainability assessment of the studied systems. Special attention will be devoted to integration of social aspects into overall holistic sustainability assessment. Social aspects of the assessment will be analysed at the last stage of the project, when the complete picture of insect production-consumption chains will be identified. We will also highlight potential trade-offs between different aspects of sustainability. The results will be used to understand how the total cost and environmental impact of insect production systems can be reduced with maximal benefits in economic and social aspects in new innovative ways, and how social attitude towards insect production system and novel bio-based products can be improved. This work will be done by DIL in cooperation with all other involved partners, and with inclusion of selected stakeholders.
WP5 – Communication & Dissemination
For the external communication strategy, several dedicated activities will be employed to engage with different target audiences. The work-plan of Poultrynsect includes dissemination of the concept, vision and results of the project.
A variety of communication activities (targeted press-releases, thematic workshops organized for stakeholders, scientific publications) will be promoted to target groups and to the general public, according to the vision and objectives of this call. Knowledge management and protection will be an integral part of the project. Detailed knowledge management and protection guidelines will define the framework and practical actions for the management and protection of the generated knowledge.
An initial communication and dissemination plan will be developed to outline all the different activities to be carried out throughout the project, to support the project’s objectives and ensure the visibility of the project and its results. The plan will be updated yearly and results from consumer studies will directly inform the plan in order to increase the impact of communication towards the general public.
A visual identity will be created in order to ensure a common graphic line (logo, leaflets, website, presentation templates etc.) for all communications material produced by the consortium. A project website (in English) will be set-up and maintained by CNR for the duration and until 1 year after end of the project. The website will be continuously updated and optimized in line with the project needs.
A six months newsletter will be produced to raise awareness of the project and highlight project findings. Audio-visual: Infographics will be developed explaining key project concepts or results in a visually appealing, easily-understandable way (translated to the partners languages with the help of partners). An animated video will be produced, focused on the value that insect protein sources can bring to consumers. Popular articles, written by partners for non-specialists will also be placed in relevant international and national magazines for relevant audiences (e.g., consumer magazines) where possible. A social Poultrynsect media presence will be ensure outreach to target audiences and enable wider dissemination. A (social) media monitoring strategy (including a Twitter-based sentiment analysis) will be designed to monitor the evolution of online discussions and public attitude around the topic of insect protein sources online and measure the impact of Poultrynsect communications.
A range of tailored materials will be produced for the dissemination of project outcomes and results to policy makers, the feed/food industry and the scientific community:
i) a project leaflet (in English) will be produced to introduce the project and updated yearly with the projects achievements, It will be distributed through project partners’ networks and at relevant conferences and events;
ii) a video will be produced focusing on promoting the main outputs of the project aimed at the industry;
iii) press releases highlighting project results will be sent to the specialist trade media with a focus on relevant topics (e.g., sustainability of insect proteins, organic poultry rearing). Articles will be pitched to specialised magazines targeting animal nutritionist;
iv) open access scientific publications will be produced for relevant scientific journals.
Project partners will attend sector-related events, conferences, workshops, trade fairs to raise awareness of the project objectives and maximise dissemination of results among target audiences. Internet and e-mail information delivery will be used as an information delivery channel. Partners will organize 1 dissemination workshop specifically targeted at PhD and MSc students, contributing to training and knowledge dissemination for building an educated workforce.
A final conference will be organised to present Poultrynsect results to key target audiences (opinion leaders, feed/food industry, consumer organisations, scientific community, and media). Final conference will be held in Bruxelles in order to facilitate the possibility to attend the meeting by the most part of the European stakeholders interested.
WP6 – Project and consortium management
The objectives of this WP are:
1) to coordinate and manage the overall development of the project;
2) to define the project workplan strategies;
3) to coordinate and supervise the project research and innovation activities;
4) to carry out the overall administrative and financial management and reporting of the project;
5) to ensure the compliance with the work programme time schedules.
This WP comprises the activities addressing the management of both the project and the consortium. CNR, as project coordinator, will lead this WP and will be in charge to monitor the overall run and any deviations of the project activities. The coordinator will promote a continuous dialogue between the different partners and guarantee a reliable flow of information between the consortium. All partners will be fully informed on the progression of the project activities, the organisation of meetings, on the main results and on the funding breakdown, as well as receiving a copy of all relevant reports and communications activity. CNR will be in charge maintaining the dialogue between partners and of resolving any conflicts and/or divergences which might arise and interfere with the implementation of the project.
As coordinator, CNR will be responsible of all administrative activities related to the project development such as: day-to-day administrative and logistic issues, gathering all relevant documentation related with contractual activities (agreements, certificates, reports…), assuring the maintenance of the consortium agreement, assist individual partners on administrative issues, update the project website, etc.
CNR will be responsible of co-organizing the project meetings together with the host partner. Three Project Meetings will be organized (M1, M18 and M36) to discuss the follow-up of the project, to present novelties and results of each WP, to decide on possible interactions and collaborative activities between WPs and to decide on future steps to take until the next meeting. Work package leaders will be responsible to organize on line WP meetings every 6 months whenever they consider necessary in order to maintain a smooth running of the WP activities.