The impact of full-fat Hermetia illucens larvae meal on the health and immune system function of broiler chickens

With global warming and environment destruction by the expansion of industry, the land area intended for food production is successively shrinking (11, 43). This situation urges the search for alternative food sources, with the most attention being paid to insects, which are perceived as food of the future (32). This claim is justified because insects have high contents of protein, fat, carbohydrates, fibre, minerals, amino acids and fatty acids, and offer an excellent production efficiency compared to other conventional food groups (21, 51). It is also worth emphasising that edible insects represent a source of many bioactive substances (29). These include chitin, which – as a polysaccharide – accounts for 5–20% of the dry weight of insects and may play an important role as dietary fibre (30). In addition, insects provide multiple peptides exhibiting antibacterial, antifungal and antiprotozoal activity (7, 39); therefore, they can mitigate the growing problem of drug resistance of microorganisms (6, 34). Furthermore, they contain peptides that inhibit the angiotensin-converting enzyme, thereby reducing blood pressure (46, 48), as well as antioxidant enzymes (4) and endopeptidases effective in the treatment of coeliac disease (12). Nevertheless, as van Huis and Oonincx (43) claimed, the nutritional value of insects varies depending on their diet, stage of development, sex, species or the environment they inhabit.

While edible insects are included in the multifaceted strategy of ensuring global food security and environmental sustainability of food production (where their merits are low greenhouse gas emissions, high feed conversion efficiency, low land use, and their ability to transform low-value organic side streams into high-value protein products) (42), their high nutritional value, minimal space requirements, and low environmental impact also make them an attractive option as an animal feedstuff (5, 45). In addition, insect-based animal feeds are particularly attractive in terms of price, especially because the cost of standard feeds accounts for around 70% of livestock production costs today (44). This motivates more and more researchers to address the use of insects as a protein-rich component of feed mixtures for poultry, pigs, and fish (3, 17–19, 27, 33, 35, 37, 45, 47, 49).

Although approximately 2,000 insect species are consumed in at least 113 countries (50), the most thoroughly analysed species that are potentially suitable for mass production for feedstuff purposes are only two, Hermetia illucens (HI) and Tenebrio molitor (11). This is because processed insect protein from HI larvae has been shown to be a practical source of protein in feed mixtures for broiler chickens, replacing up to 4% of fishmeal (3), and been shown not to suppress feed intake, feed conversion efficiency, egg production, hen health or immune status when added to a fodder for laying hens as the only source of protein, replacing soybean meal (27). Research by Ramos-Elorduy et al. (33) has also shown that a 5–10% addition of processed insect protein from Tenebrio molitor larvae could effectively replace 20–31% of soybean meal in a feed mixture for broiler chickens, and a study by Wang et al. (49) has demonstrated that this lifecycle stage of the insect replacing fishmeal in a feed mixture for laying hens increased egg production by 2.4%.

The literature data indicate that further research is necessary to more fully evaluate the possibility of deploying insects in animal nutrition and to assess the impact of such a diet on animal health. Therefore, this study was undertaken feeding broiler chickens a diet containing different proportions of full-fat HI larvae meal as substitutes for the protein of genetically modified soybean meal to assess its impact on their health and selected elements of their humoral and cellular immunity.

Birds and housing. The current study was performed in the poultry house of the Department of Commodity Science and Animal Improvement of the University of Warmia and Mazury (Olsztyn, Poland). A total of 384 male Ross 308 broiler chicks obtained from a commercial hatchery were randomly distributed into four dietary treatment groups (eight pens/treatment and 12 birds/pen) and reared to the 42^nd day of life (dol). Each pen was 1.10 m × 1.25 m (width × length), was equipped with a feeder and an automatic drinker, and provided pellets from straw as litter. During the first 3 weeks, the pens were heated by specialised electric radiators to maintain the temperature recommended for standard breeding practices consistent with the Ross Broiler Management Handbook (2). Fodder and water were provided ad libitum throughout the experiment. At the hatchery, one-day-old chickens were vaccinated against Marek’s disease and coccidiosis. Then, upon delivery to the poultry house, they were vaccinated against Newcastle disease (ND) and infectious bronchitis (IB) and on the 7^th dol the chickens were vaccinated against the diseases caused by avian metapneumoviruses (aMPV) using live-attenuated Nobilis Ma5 + Clone 30 (Merck, Rahway, NJ, USA) and Poulvac TRT (Zoetis, Parsippany, NJ, USA) vaccines applied individually in the dose recommended by the producers with a dropper at one drop (0.05 mL) per bird into one eye.

Diets and feeding programme. The diets were split into three phases for each treatment group: a starter diet (1–14 days), a grower diet (15–35 days) and a finisher diet (36–42 days) with the composition matched to the nutritional demands of broiler chickens. The protein from genetically modified soybean meal in standard diets was replaced with full-fat HI larvae meal in the following amounts: 50% for group HI-50, 75% for group HI-75, and 100% for group HI-100. Broiler chickens from group HI-0 were fed a diet without HI larvae meal and served as the controls. The composition of diets is presented in Table 1.

The dietary formulae in Table 1 were used to produce experimental feed mixture pellets at the Gorzyń Experimental Department of Animal Feeding, Poznań University of Life Sciences (Poznań, Poland). The full-fat HI larvae meal was obtained from a commercial producer (HiProMine S.A., Robakowo, Poland). The composition and energy value of full-fat HI larvae meal are shown in Table 2.

Clinical and anatomopathological examination of chickens. Body weight gains and feed conversion ratio (FCR), the number of culled birds and the mortality rate were registered throughout the experiment. Dead birds were subjected to anatomopathological examinations.

Sample collection. Blood samples were collected from the wing vein into sterile tubes without anticoagulant for serological (n = 23) and biochemical (n = 10) analyses as well as into sterile tubes containing ethylenediaminetetraacetic acid K2 anticoagulant for haematological (n = 5) and flow cytometry analyses (n = 8). For haematological, biochemical, serological and cytometric analyses, blood was sampled on the 21^st and 42^nd dol. The birds were sacrificed on the 42^nd dol, and spleens were collected from eight birds (n = 8) from each group. These were used together with the blood samples for isolation of mononuclear cells and determination of the sizes of the CD3⁺CD4⁺, CD3⁺CD8a⁺ and CD4⁺CD8a⁺ T cell subpopulations and the CD3⁻Bu-1⁺ B cell subpopulation by flow cytometry.

Haematological analysis. The blood samples were analysed for red blood cell count, haemoglobin concentration, packed cell volume, white blood cell count, and percentage composition of leukocytes (by leukogram) – including percentages of lymphocytes, monocytes and granulocytes (heterophils, eosinophils and basophils). Haematological analyses were performed in accordance with the methodology described by Krasnodębska-Depta and Koncicki (23).

Biochemical analysis. Blood serum samples were evaluated for levels of total protein and activity of selected enzymes, including alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, lactate dehydrogenase (LDH) and creatine kinase (CK) as well as for total concentrations of calcium (CALC), phosphorus (PHOS) and uric acid (UA). The tests were performed using a VetTest Chemistry Analyzer (IDEXX Laboratories, Westbrook, MN, USA) according to the manufacturer’s instructions.

Serological analysis. Vaccine-induced immunoglobulin Y titres of anti-ND, anti-IB and anti-aMPV antibodies were evaluated with the use of a commercial ELISA kit (IDEXX Laboratories, Westbrook, MN, USA) according to the manufacturer’s recommendations. The ELISA assay was performed with an Eppendorf epMotion 5075 LH automated pipetting system (Eppendorf, Hamburg, Germany), a BioTek Elx405 washer and Elx800 absorbance microplate reader (Agilent, Santa Clara, CA, USA) and a KBF 115 constant climate chamber (Binder, Tuttlingen, Germany).

Isolation of mononuclear cells and flow cytometry. Mononuclear cells from the blood and spleens were isolated according to a previously described procedure (22). The cells were counted, and their viability was evaluated using the Vi–Cell XR cell counter (Beckman Coulter, Brea, CA, USA). Viable mononuclear cells (1 × 10⁶) were stained with Pacific Blue–conjugated Mouse Anti–Chicken CD3-PACBLU clone CT-3, fluorescein–conjugated Mouse Anti–Chicken CD4-FITC clone CT-4, Alexa Fluor 647–conjugated Mouse Anti–Chicken CD8a-AF647 clone CT-8, and phycoerythrin–conjugated Mouse Anti–Chicken Bu-1–PE clone AV-20 (Southern Biotech, Birmingham, AL, USA). Data were acquired using a FACSAria II digital flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) in the FACSDiva 8.0 environment (Becton Dickinson). A total of 100,000 events were recorded for each sample. The immunophenotype and sizes of subpopulations of T CD3⁺CD4⁺, CD3⁺CD8a⁺ lymphocytes, double-positive cells (CD4⁺CD8a⁺), and B cells (CD3⁻Bu-1⁺) were analysed using FlowJo V10 software (Becton Dickinson). Fluorescence minus one controls for Pacific Blue, FITC, Alexa Fluor 647 and PE fluorochromes were used to determine the cut–off point between background fluorescence and positive populations.

Statistical analysis. All calculations were made using Statistica 13.1 software (StatSoft, Kraków, Poland). The significance of differences in values of the measured parameters between the investigated groups was analysed with Student’s t-test for independent variables and post-hoc Tukey’s test. Differences were considered statistically significant at P ≤ 0.05.

Health of broiler chickens. The production parameters for the broiler chickens are presented in Table 3.

On the 42 dol, the birds differed significantly by treatment group in terms of body weights, especially in the group fed the diet with the highest content of insect larvae meal. As observed at this time point (experiment end), the average group body weights of broiler chickens were significantly lower in correlation with the increasing content of full-fat HI larvae meal in the diet group by group, and only reached 2,375 g in group HI-100 compared to 3,046 g in group HI-0. The body weights of birds from groups HI-50 and HI-75 were significantly lower than those from group HI-0 and significantly higher than those from group HI-100. Also the FCR evaluated for birds from group HI-100 was significantly higher than the FCR of birds from the other groups and was 1.77 vs 1.63 in HI-0. In groups HI-50 and HI-75 FCR, it was 1.59 and was lower than in group HI-0 despite the statistically significant body weight differences between the HI meal-fed birds and the control group birds. The culling and mortality rate reached 28.2% in group HI-100 but was only 3.15% in group HI-0. The high number of culled and dead birds in group HI-100 was due to growth inhibition and gizzards becoming filled with pellets of the litter on which the birds were raised.

Haematological parameters. The haematological parameters are summarised in Table 4 (for 21-day-old chickens) and in Table 5 (for 42-day-old chickens).

The substitution of soybean meal with the full-fat HI larvae meal had no effect on the values of the haematological markers evaluated in either 21-day-old or 42-day-old chickens, regardless of the dietary inclusion level of larvae meal.

Biochemical parameters. The results of the biochemical analyses are set out in Table 6 (for 21-day-old chickens) and Table 7 (for 42-day-old chickens).

The levels of soybean meal substitution with full-fat HI larvae meal had no significant effect on the majority of the analysed parameters in 21-day-old chickens. In all groups that received full-fat HI larvae meal, there was a significant decrease in CK activity and PHOS levels compared to the control group. In addition, the HI-100 group chickens showed a statistically significant increase in serum CALC and UA levels compared to the control group (HI-0).

A statistically significant increase in AST, CK and LDH activity and UA level was found in 42-day-old chicks that had received full-fat HI larvae meal compared to control birds. In addition, there was a statistically significant increase in CALC and decrease in PHOS levels in the HI-75 and HI-100 groups compared to the control chickens from group HI-0.

Serological parameters. The results of serological analyses are provided in Table 8.

On the 42^nd of life, the broiler chickens fed a diet with the addition of full-fat HI larvae meal had highly significantly lower titres of anti-infectious bronchitis virus (IBV) antibodies compared to the control birds (HI-0).

Immunological parameters. Tables 9 and 10 present the T and B cell subpopulations in blood and spleen samples of broiler chickens.

On the 21^st dol, the percentage of T cells which were CD3⁺CD4⁺ was significantly higher in the blood samples of group HI-75 than in those of HI-0 and HI-50. The size of the CD3⁺CD8a⁺ T cell subpopulations was significantly lower in group HI-100 in comparison with HI-50. The full-fat HI larvae meal had no significant effect on the size of the T and B cell subpopulations in blood samples collected from birds on day 42 of life.

No significant differences between treatment groups were found in the percentages of immune cells which were CD4⁺CD8a⁺ T cells and CD3⁻Bu-1⁺ B cells in blood and spleen samples. On the 42^nd dol, the percentage of T cells which were CD3⁺CD4⁺ was significantly lower in the spleen samples of group HI-100 than in those of HI-0 and HI-50. In general, the increasing proportion of full-fat HI larvae meal in the diet increased the percentage of CD3⁺CD8a⁺ T cells and decreased that of CD3⁺CD4⁺ T cells in both blood and spleen samples in 42-day-old chickens.

Insects are receiving much attention these days as a proposition for a food of the future (32). Interest in edible insects increased sharply when the Food and Agriculture Organization (FAO) began promoting them as a viable dietary option for humans (44). They have also drawn growing interest as an attractive source of animal feedstuff (5, 45), especially in response to Commission Regulation (EU) 2017/893 of 24 May 2017 on the possibility of using insect protein in feeding strategies for aquaculture and to EU Regulation No. 2021/1372 allowing the use of processed animal proteins derived from farmed insects in fodders for poultry and pigs (13).

In response to the greater awareness of the exploitability of insect protein, numerous studies have been conducted for many years on the economic effects of feeding poultry with diets with different inclusion levels of this protein as fishmeal or soybean meal replacement (3, 27, 33, 49). Józefiak et al. (18) showed that a 0.2% addition of full-fat Hermetia illucens and Tenebrio molitor meal to fodders for chickens increased feed intake and improved the microbiota of their gastrointestinal tract. However, on the aspects of poultry health and immune system function, to the best of the authors’ knowledge there were no studies prior to the present research on the impact of feed mixtures with insect meal.

The analysis of the production results from the present study indicates a progressive negative impact of the increasing content of insect protein in the feed mixtures on the body weight gains, FCR values, and bird mortality. This seems to be in contradiction to the results of previous insect meal feed studies conducted on various species of birds including broiler chickens, in which the meal yielded a beneficial effect on the production performance of birds (9, 20). Earlier studies with laying hens demonstrated better body weight gains and better FCR in the groups receiving feed mixtures with the addition of partly de-fatted HI larvae meal (28, 31). In the study by Marono et al. (27) with laying hens, although a more favourable feed conversion rate was noted in the experimental group than in the control group, other production parameters such as laying percentage, feed intake and average egg weight were significantly lower. Interestingly, in the case of studies by Schiavone et al. (37), replacing 50% and 100% of soybean oil with fat from HI larvae had no effect on the growth or body weight of the birds. Similarly, Sypniewski et al. (41) found no effect of soybean meal substitution with insect protein on the production performance of turkeys. It seems that the influence of the insect component on the growth and development of birds is not clear or obvious. These differences in findings may depend on the species and utility type of birds, the period of feeding them with mixtures containing the insect component, the content of chitin in the diet and its potential adverse impact on protein digestibility, the content of insect protein in the diet, the process of preparation of feed components, in particular larvae peeling, and even its form and colours, as confirmed by previous studies (9, 36). Some authors suggested that daily feed intake could be affected by fodder colour, because HI meal is dark brown in colour and darker than soybean meal, and therefore is less willingly consumed by birds. Perhaps only purified protein and insect fat should be used for poultry feed production.

The results of haematological analyses show that the diet with insect protein addition had no significant influence on evaluated parameters, which is consistent with the findings reported by Schiavone et al. (38) and Li et al. (25). However, a decrease in the number of peripheral white blood cells was observed in 21-day-old chicks, which corresponds to the results obtained by de Souza Vilela et al. (10). The percentages of lymphocytes, granulocytes and monocytes remained normal both in the control group and in the experimental groups, which leads to the conclusion that full-fat HI larvae meal had no effect on the white blood cell profile. The leukogram results obtained correspond with the findings of Marono et al. (27).

Based on the selected biochemical parameters, it is possible to determine the impact of the diet on liver, kidney, bone and muscle functions in birds (15). Although there were statistically significant differences between the groups in the levels of CALC, PHOS, and UA and the activities of AST, CK, and LDH, the values of these parameters were still within the reference ranges for this poultry species (23). The presented results correspond to the observations of other authors. Marono et al. (27) noted a significant increase in calcium and a decrease in serum phosphorus levels in laying hens fed fodders with HI meal. It is not easy to explain the significantly higher levels of CALC in blood of broiler chickens from the HI-75 and HI-100 groups compared to the HI-0 group, because the fodders used in the experiment contained similar levels of calcium. Uric acid is the major poultry nitrogenous waste product and its level in serum reflects protein catabolism (15). Our study showed that increasing levels of HI larvae meal increased serum UA levels in chickens. These results are inconsistent with those obtained by Marono et al. (27): those authors did not show any effect of HI larvae meal on UA levels in the serum of laying hens.

When assessing the post-vaccination response, it should be noted that out of the three vaccinations given to the broiler chickens as part of the experiment, the one against infectious bronchitis should elicit the strongest immune response, because IBV antigens are characterised by the highest immunogenicity. This is reflected in the results of serological tests: the six-week-old birds showed more clearly marked IBV seroconversion than the three-week-old birds. As our observations show, feeding birds a diet in which the standard protein component was replaced by HI larvae protein significantly impaired the effectiveness and efficacy of vaccination against IBV, as manifested by the statistically significantly lower titres of post-vaccination antibodies in six-week-old birds from these groups. This situation is most likely due to lower daily feed intake, which resulted in poorer body weight gains and deterioration of the general condition of birds in these groups, which translated into low production results and debilitated immune systems. Unfortunately, so far there has been no similar research on the development of post-vaccination immunity in birds fed with insect protein supplemented diets. The only available reports on the protein obtained from HI larvae and the efficacy of vaccination concern fish and indicate a complete lack of its influence on the level of anti-infectious pancreatic necrosis virus specific antibodies (26). On the other hand, the results of the present study correspond with findings of other authors concerning the impact of deficient feeds on post-vaccination immunity in poultry, e.g. with the research by Chen et al. (8), in which birds fed a diet low in lysine had lower titres of anti-NDV antibodies, which confirms the effect of the feed provided to birds on the functioning of their immune systems.

The cytometric analysis of blood and spleen samples conducted in the present study showed no effect of the protein component on the size of the B cell subpopulation, but indicated an increase in the percentage of T cells which were CD3⁺CD8a⁺ and a decrease in the percentage of them which were CD3⁺CD4⁺. These results contradict the research of Lee et al. (24), where the CD3⁺CD4⁺ subpopulation of spleen T lymphocytes significantly increased with the addition of increasing levels (1, 2 or 3%) of HI larvae protein in fodder. On the other hand, de Souza Vilela et al. (10) observed not only a general decrease in the percentage of white blood cells in haematological analyses, but also a decrease in the percentage of CD3⁺CD8⁺ jejunal intraepithelial T lymphocytes in chickens fed fodder supplemented with 20% full-fat HI larvae meal. According to the observations of Huo et al. (16), the fatty acid profile in feed materials has a substantial impact on the formation of immune mechanisms and the ratio of CD4⁺ to CD8⁺ T cells. Hermetia illucens larvae have a high content of lauric acid, which, in addition to its antibacterial properties, elicits an immunomodulatory effect (14).

One conclusion of the present study results is that full-fat HI larvae meal used as a protein and substitute for genetically-modified soybean meal has a negative effect on the health of broiler chickens. The low production results and high mortality in experimental birds may indicate the need to use feeds with a lower insect component than those tested in this study or to use only purified insect proteins.