Abstract
The use of black soldier fly larvae (BSFL) as an alternative protein source in broiler nutrition has attracted increasing interest; however, information on the effects of defrosted full-fat BSFL supplementation remains limited. This study evaluated the dose-response effects of top-dressed defrosted full-fat BSFL (10 and 20% of the previous day’s feed intake) on growth performance, gut histology, and blood metabolic markers of Ross 308 broilers. A total of 150 one-day-old male broilers were randomly allocated to three groups. The control group (CON) received a commercial corn-soybean meal-based basal diet ad libitum, while the BSFL10 and BSFL20 groups received the same diet supplemented with 10% or 20% BSFL. Growth performance and blood samples were collected on days 21 and 35. The BSFL20 group exhibited a significant increase in average daily gain (ADG) during days 22–35 and 1–35 (
1 Introduction
The search for sustainable and high-quality protein alternatives in poultry nutrition has led to increased interest in insect-based feed sources. Among these, black soldier fly larvae (BSFL; Hermetia illucens) offer a promising solution due to their ability to convert organic waste into a nutrient-rich protein source while simultaneously reducing environmental impact (Rehman et al., 2023). Unlike conventional feed components that require intensive agricultural inputs, BSFL production can be integrated into local farming systems, enhancing circular agriculture and reducing dependence on imported feed ingredients (Jagtap et al., 2021; Siva Raman et al., 2022). In regions such as Southeast Asia, where organic waste generation is substantial, local BSFL farming presents an opportunity to contribute to sustainable poultry production and lower transport-related emissions (Kim et al., 2021).
BSFL are nutritionally rich, offering crude protein levels of approximately 40% in dry matter (Gasco et al., 2021; Lu et al., 2022; Shumo et al., 2019; Wong et al., 2019) along with bioactive compounds such as lauric acid, chitin, and antimicrobial peptides (Furtado et al., 2024; Mousavi et al., 2020). These components may influence intestinal health, gut-associated immune function, nutrient efficiency, and systemic metabolism in poultry (Abd El-Hack et al., 2020; De Smet et al., 2018; de Souza Vilela et al., 2021; Li et al., 2025). Studies on Ross 308 broilers have shown that dietary inclusion of BSFL meal enhances weight gain and improves feed conversion ratio (FCR) (de Souza Vilela et al., 2021; Ipema et al., 2020; Seyedalmoosavi et al., 2022). These effects have been suggested to be associated with enhanced intestinal immune response linked to chitin, as well as improvements in intestinal morphology and digestive and absorptive function linked with alterations in the gut microbiome, contributing to better gastrointestinal health (Chen et al., 2022; Dabbou et al., 2018; Kim et al., 2020). While most poultry studies involving insect-based diets have focused on dried BSFL meal incorporated into formulated feed (Abd El-Hack et al., 2020), the direct feeding of defrosted full-fat BSFL remains underexplored. Fresh larvae differ significantly from processed meal in moisture content and nutrient profile (Pornsuwan et al., 2023; Silvan et al., 2025), particularly in free fatty acid yield and composition. Fresh BSFL may contain higher levels of free fatty acids, including lauric acid (C12:0), potentially due to maintained endogenous lipase activity. These characteristics have been associated with antibacterial activity in vitro (Silvan et al., 2025), although their in vivo relevance in poultry remains to be fully elucidated. These differences may influence feed intake, digestion, and microbiota adaptation, potentially impacting intestinal health and nutrient absorption. However, for smallholder farms with limited processing facilities, top-dressing defrosted full-fat larvae is a practical, low-cost strategy. Research on the dose-response effects of top-dressed defrosted full-fat BSFL at different levels remains unexplored. This study addresses this gap by investigating the dose-response effects of top-dressed defrosted full-fat BSFL at 10% and 20% inclusion rates on Ross 308 broilers, evaluating growth performance, feed efficiency, gut histology, blood lipid profiles, metabolic parameters and oxidative stress markers. These findings support the potential of BSFL as a sustainable and functional feed source for Southeast Asian smallholder poultry production.
2 Materials and methods
Birds and husbandry
The experimental protocol was approved by the Institutional Animal Care and Use Committee of Mahanakorn University of Technology (ACUC-MUT-2024/005). The experiment was conducted in a closed, concrete-floor pen house equipped with an evaporative cooling system at the Student Training Center, School of Agricultural Technology at King Mongkut’s Institute of Technology Ladkrabang, Thailand. Birds were raised following standard practices for lighting and temperature (the light-dark cycle was 22:2 h during the first 14 days, followed by 12:12 h from day 15 until the end of the experiment), with room temperature recorded and analysed twice daily. The indoor temperature was 25.8 ± 2.2 °C, with afternoon temperatures reaching 32.5 ± 2.0 °C; the relative humidity averaged 84.5 ± 5.7% (mean ± SD), which are typical of an evaporative cooling system used in tropical poultry production.
A total of 150 one-day-old male Ross 308 broilers were obtained from a commercial hatchery (Panus hatchery, Chon Buri, Thailand) and used in the trial, with an average initial body weight of 40.17 ± 0.05 g (mean ± SD). Birds were randomly assigned to pens in a completely randomised design, allocated into three groups (5 replicates of 10 birds each). Each pen measured 150 × 200 × 55 cm and consisted of metal-mesh pallet cages bedded with rice husk as litter material. They were equipped with a feeding tray and an automatic drinker, ensuring consistent access to ad libitum feed and water. Chicks were vaccinated against Newcastle disease and infectious bronchitis by spraying in the hatchery, and all broilers were intraocularly immunised against Newcastle disease and infectious bronchitis on day 7, followed by infectious bursal disease vaccination at 14 days.
Ingredients and nutrient composition of the experimental diets and defrosted full-fat black soldier fly larvae (BSFL)
Citation: Journal of Insects as Food and Feed 12, 9 (2026) ; 10.1163/23524588-bja10360
Diets
The trial evaluated the effects of two levels of defrosted full-fat black soldier fly larvae provided as a top-dressed supplement to a basal diet. The larvae were not incorporated into the basal diet but were provided separately as a top-dressed supplement. A commercial corn-soybean meal basal diet, formulated to meet or exceed Ross 308 nutrient requirements (Aviagen, 2022), served as the basal diet (Table 1). A commercial vitamin and mineral premix (purchased from Betagro Public Co., Ltd., Bangkok, Thailand) was incorporated into the basal diet at an inclusion rate of 0.20%. The detailed composition of the premix is presented in the footnote of Table 1. A crumbled starter diet was used from day 1 to day 21, and a pelleted grower diet was used from days 22 to 35 of the trial. Kasetsart University, Thailand, provided the full-fat BSFL used in this study. Upon harvesting, larvae were frozen and stored at −20 °C. For daily supplementation, larvae were transferred to a 4 °C refrigerator approximately 12 h before distribution for thawing and then maintained at room temperature for portioning. The two treatment groups received BSFL as a top-dressed supplement at levels of 10% (BSFL10) and 20% (BSFL20), calculated based on the previous day’s actual feed intake of the basal diet. The larvae were scattered daily on top of the basal diet to ensure immediate consumption. The analysed nutrient composition of defrosted full-fat BSFL is presented in Table 1. Proximate analyses of the diets and full-fat BSFL were performed according to the Association of Official Analytical Chemists (AOAC) International methods (AOAC International, 2005).
Birds were initially reared in pens until three days of age, after which they were transitioned to their respective dietary treatments, which were maintained until day 35. The CON group received only the basal commercial diet, while the BSFL10 and BSFL20 groups were supplemented with defrosted full-fat BSFL as a top-dressed supplement at 10% and 20% calculated based on the previous day’s feed intake. The daily larvae portion was divided into equal portions and provided at 09:00 and 16:00 h.
The larvae were top-dressed directly onto the feeding trays without homogenisation into the basal diet. This method allowed the birds to interact naturally with the larvae, which were highly palatable and consumed entirely and rapidly (within approximately 5 minutes) after each supplementation, with no larval residues observed in any pen throughout the experimental period. The basal diet was provided ad libitum, and no selective refusal of the basal feed was observed following larval consumption. To ensure precise inclusion levels (10% and 20% relative to the basal diet intake), any remaining basal diet was collected and weighed daily at 09:00 h to determine actual feed consumption. The amount of larvae provided for the following day was then adjusted based on the actual feed consumption over the preceding 24 hours. Both the basal diet and clean drinking water were provided ad libitum throughout the experimental period.
Growth performance
Growth performance was measured separately for days 1 to 21, and days 22 to 35, and also collectively for days 1 to 35. The mortality rate was recorded and calculated at the end of the experiment. Body weight (BW) and feed intake (FI) were recorded at 21 and 35 days of age, and these data were used to calculate daily feed intake (DFI) and average daily gain (ADG). The feed conversion ratio (FCR) was calculated by dividing DFI by ADG.
3 Sample Collection and Analysis
Blood biochemical and haematological parameters
Blood samples were obtained on day 35 from 10 birds per treatment (2 randomly selected chickens per pen). The blood was collected from the wing vein, and blood samples from each bird were divided into two tubes for further examination. The first blood sample was collected in a serum tube, and the blood samples were centrifuged at 3,000 g for 15 minutes. The serum samples were stored frozen at –80 °C, and measurements included clinical biochemistry parameters, malondialdehyde (MDA), d-lactate, alkaline phosphatase (ALP), lipopolysaccharide binding protein (LBP), alpha 1-acid glycoprotein (AGP) concentrations, and diamine oxidase (DAO) activity. d-lactate and ALP concentrations were measured via colourimetric assays (D-Lactate Colourimetric Assay Kit, and Alkaline Phosphatase Assay Kit, Sigma-Aldrich, St. Louis, MO, USA). DAO activity was quantified using a competitive ELISA kit (Chicken Diamine Oxidase ELISA Kit, Mybiosource, San Diego, CA, USA). LBP levels were determined using an ELISA kit (Chicken Lipopolysaccharide Binding Protein ELISA Kit, Mybiosource). AGP concentrations were measured using an immunoperoxidase assay (chicken alpha 1-acid glycoprotein Immunoperoxidase Assay Kit, Mybiosource). The absorbance of ALP was measured at 405 nm, while the absorbances of d-lactate, DAO, LBP and AGP were measured at 450 nm using a SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA, USA). MDA concentration was measured by the thiobarbituric acid reactive substances method (Zeb and Ullah, 2016). Protein was determined using bovine serum albumin as a standard. The absorbance of MDA was measured at 535 nm using a UV-Vis spectrophotometer. The second blood sample was collected in tubes containing EDTA as an anticoagulant. Whole blood was used for white blood cell counts and differentials (heterophils, lymphocytes, monocytes, eosinophils, and basophils), analysed by the Hemavet Multi-Species Haematology System (Drew Scientific, Oxford, CT, USA). Heterophil:lymphocyte (H:L) ratios were calculated using a previously described method (Gross, 1989). The remaining EDTA blood samples were centrifuged for 10 min at 3000
Feather corticosterone concentration
Feathers were cut from the attachment point on the right wing and processed for the assay as described previously (Lattin et al., 2011). Four to five feathers were collected per bird and stored in a dark, dry environment at room temperature after the calamus was removed. Surface contaminants were eliminated by washing the feathers in 2.5 ml of isopropanol with continuous shaking for 3 min at room temperature, followed by air-drying for 12 hours. A methanol-based extraction method was used to isolate corticosterone (CORT) (Ataallahi et al., 2020; Bı́lková et al., 2019; Bortolotti et al., 2008). Following washing and drying, the barb was separated from the rachis, and 50 mg of dried feather barb was homogenised into a fine powder under continuous cooling with liquid nitrogen. A tissue homogeniser (Precellys Evolution Touch Homogeniser, Bertin Technologies) was used at 6800 rpm for five cycles of 20 s to ensure complete pulverisation. The pulverised feather samples were suspended in 1.8 ml methanol and sonicated at room temperature for 30 min to enhance CORT solubilisation. Samples were subsequently incubated overnight at room temperature. The methanol supernatant was dried using a vacuum concentrator at 40 °C overnight, and the dried extract residues were reconstituted in 400 μl ELISA Buffer. Particulate material was eliminated through centrifugation of the buffer-reconstituted samples. The CORT quantification of the final extracts was performed using a corticosterone ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA) with measuring the absorbance at 420 nm using a SpectraMax iD3 plate reader (Molecular Device).
Intestinal histomorphology
On day 35 of age, the segment of mid-jejunum was taken using two birds per replicate pen. The intestinal tissue was flushed and immediately fixed in 10% formaldehyde solution. Afterwards, the samples were placed on a glass slide and then stained using hematoxylin and eosin. Villus height (VH) and crypt depth (CD) were measured using a light microscope at 100× magnification (Primostar3, Zeiss, Jena, Germany). The villus height-to-crypt depth ratio (VH:CD) was calculated.
Statistical analysis
All data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene’s test. Data are presented as means ± standard error of the mean (SEM). Differences in growth performance, intestinal histology, and blood biomarkers among experimental groups were assessed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparisons. In addition, orthogonal polynomial contrasts (linear and quadratic) were performed to evaluate the dose-response relationship of increasing BSFL supplementation levels. For growth performance, the pen was the experimental unit; for blood and histological parameters, the individual bird was the experimental unit. Statistical significance was defined as
Effect of defrosted full-fat black soldier fly larvae supplementation on the growth performance of broiler chickens
Citation: Journal of Insects as Food and Feed 12, 9 (2026) ; 10.1163/23524588-bja10360
Blood profiles and intestinal histomorphology of broiler chickens supplemented with defrosted full-fat black soldier fly larvae at day 35
Citation: Journal of Insects as Food and Feed 12, 9 (2026) ; 10.1163/23524588-bja10360
4 Results
Growth performance
The effect of defrosted full-fat BSFL supplementation on the growth performance of broiler chickens is shown in Table 2. During the starter phase (days 1-21), no significant differences were observed among treatments for ADG (
Haematological and chemistry profiles
Effects of defrosted full-fat BSFL supplementation on haematological parameters and plasma/serum chemistry profiles of broiler chickens on day 35 are shown in Table 3. The haematological parameters were not significantly different across treatments. Regarding plasma chemistry, circulating triglyceride concentration decreased linearly (
Intestinal histomorphology
Jejunal morphological measurements are presented in Table 3. BSFL supplementation significantly influenced gut architecture; villus height (VH) and the VH:CD ratio increased linearly (
Feather corticosterone concentration
Feather CORT concentrations were numerically decreased in the defrosted full-fat BSFL inclusion groups, with mean values of 4.48 pg/mg in the control group, 3.70 pg/mg in BSFL10, and 3.97 pg/mg in BSFL20. However, these differences were not statistically significant (
5 Discussion
Growth performance and intestinal morphology
The present study investigated the effects of top-dressed defrosted full-fat BSFL at 10 or 20% on broiler performance and gut health. The rationale was based on BSFL’s distinctive fatty acid profile, particularly their reported high lauric acid content, which has been linked to antimicrobial activity and improved gut health, and on evidence showing that whole larvae act as a behavioural enrichment, stimulating natural foraging and improving welfare (Borrelli et al., 2017; Ipema et al., 2020; Moula et al., 2018; Pichova et al., 2016). Providing defrosted full-fat BSFL at 20% significantly improved ADG and final body weight. These enhancements are primarily attributed to the increased total nutrient density provided by the top-dressed feeding method. However, the concurrent improvements in gut morphology and reduction in d-lactate suggest that the observed performance benefits cannot be explained solely by increased nutrient intake. Since BSFL was provided in addition to the basal diet, the BSFL20 group consumed a more nutrient-dense diet without a significant reduction in daily feed intake (DFI). This provided additional substrates for protein deposition and accelerated growth, especially during the grower phase. It is important to acknowledge a primary limitation of the current study: the experimental diets were not formulated to be isocaloric or isonitrogenic. Since the defrosted full-fat BSFL were provided as a top-dressed supplement based on the previous day’s feed intake, the total nutrient density in the BSFL10 and BSFL20 groups was effectively higher than that of the control group. Consequently, the enhanced growth performance observed, especially in the BSFL20 group, likely reflects the additional nutrient supply provided by the larvae. Future studies using isocaloric or isonitrogenic formulations are needed to further isolate the functional bioactive effects of defrosted full-fat BSFL from its primary nutrient contribution.
Although these improvements may be partly attributed to the numerically higher daily feed intake (DFI) observed in the BSFL groups, this increase in DFI was not statistically significant. Previous studies have shown that full-fat BSFL has a highly nutritious profile, with 42% crude protein and 30% lipid (Salahuddin et al., 2024). Furthermore, we observed that chickens rapidly approached and consumed the larvae immediately upon supplementation. These behaviours are consistent with findings highlighting that insects are an evolutionarily familiar part of the avian diet (Bovera et al., 2016). This active interaction may enhance bird vitality and feed efficiency. However, several studies report no performance effects of whole larvae under similar inclusion levels (Dabbou et al., 2018; de Souza Vilela et al., 2021; Khan et al., 2018; Moula et al., 2018), while others observed improved body weight gain or FCR with different inclusion rates and feeding durations (Cullere et al., 2016; Onsongo et al., 2018). The discrepancies among studies may be attributed to differences in experimental conditions, such as bird age, breed, management conditions, diet formulation, and the rearing substrate of black soldier fly larvae, which could influence the outcomes (Danieli et al., 2019; Kawasaki et al., 2019). One potential contributor to this improved performance is the modification of intestinal architecture. In the present study, the significant linear increase in the VH:CD ratio in the BSFL20 group indicates an expanded absorptive surface area (Garagna et al., 2020; Wang et al., 2025) and an efficient epithelial cell renewal rate in the intestine (de Verdal et al., 2010). Conversely, villus atrophy and increased crypt depth can impair nutrient absorption, thereby reducing growth performance (Xu et al., 2003). These findings align with previous studies investigating insect-based functional ingredients (Anas et al., 2024; Chen et al., 2022; Kim et al., 2021) and suggest that BSFL components, such as chitin and lauric acid, may contribute to a healthy gut environment. Although lauric acid was not directly analysed in this study, its well-documented presence in Hermetia illucens larvae has been proposed as a possible mechanism underlying the improved gut morphology and barrier function observed (Kim et al., 2021; Zeitz et al., 2015). The improvements in intestinal architecture observed in this study were further corroborated by the assessment of serum biomarkers for gut permeability. Specifically, birds supplemented with 20% defrosted full-fat BSFL exhibited a significant reduction in D-lactate levels. d-Lactate is a microbial metabolite produced in the gastrointestinal tract and is typically maintained at low concentrations in the bloodstream. However, when intestinal integrity is compromised and permeability increases, d-lactate can leak into the circulatory system (Levitt and Levitt, 2020). Therefore, the lower d-lactate levels in the BSFL20 group suggest enhanced intestinal barrier integrity and reduced epithelial disruption or inflammation, as previously documented in broilers under various dietary interventions (Simon et al., 2016; Zou et al., 2016). This outcome may be associated with the presence of BSFL bioactive lipids, such as lauric acid, and structural polysaccharides, such as chitin. These components have been shown to support the upregulation of tight junction proteins (Anas et al., 2024) and promote a beneficial cecal microbiota (Zhao et al., 2023), thereby collectively fortifying the gut barrier. While a reduction in D-lactate strongly points toward improved epithelial barrier function, it is also possible that BSFL supplementation modified the gut environment to favour a decrease in lactate-producing bacterial communities. Consequently, while these findings are promising, the specific molecular mechanisms and microbial shifts involved warrant further investigation to elucidate how defrosted full-fat BSFL influence gut health.
Physiological stress and metabolic status
The absence of significant differences in MDA, a marker of lipid peroxidation, and the heterophil to lymphocyte (H:L) ratio suggests that the levels of defrosted full-fat BSFL up to 20% did not induce oxidative or physiological stress in broilers. The uniform and low mortality rates across all groups further support this. Although we hypothesised that environmental enrichment associated with defrosted full-fat BSFL supplementation would reduce stress as measured by feather corticosterone, this hypothesis was not supported by the data. This indicates that while BSFL supplementation is safe and maintains physiological homeostasis, it may not further alleviate stress under the already optimal management conditions of this study.
Regarding lipid metabolism, the significant reduction in plasma triglycerides in the BSFL20 group on day 35 (
In contrast to triglycerides, plasma total cholesterol levels remained unaffected by BSFL supplementation, indicating that the defrosted full-fat BSFL does not perturb cholesterol homeostasis under these conditions. While previous studies have reported cholesterol-lowering effects often attributed to chitin’s ability to bind bile acids in the gut (Hossain and Blair, 2007; Marono et al., 2017), the current study suggests that the metabolic influence of BSFL was more targeted toward triglyceride clearance. This improved lipid metabolism appears to be synchronised with enhanced liver efficiency and gut integrity, as evidenced by the significant reduction in ALP and d-lactate levels in the BSFL20 group. This selective lipid modulation improved the metabolic efficiency and gut health of broiler chickens without compromising their physiological status. Further studies are warranted to clarify the effects on specific lipid fractions and metabolic pathways.
Liver function and intestinal health
Beyond systemic metabolism, the health status of the liver and intestinal barrier was further evaluated. In this study, serum AST levels remained unaffected across all groups, indicating that supplementation with up to 20% defrosted full-fat BSFL did not induce hepatic stress or tissue damage (Senanayake et al., 2015). Interestingly, serum ALP levels were significantly lower in the BSFL20 group than in the control group (
Notably, the concurrent decrease in both triglycerides and ALP in the BSFL20 group reflects more efficient lipid clearance and overall enhanced liver metabolism (Gariglio et al., 2019). This hepatic efficiency is likely a secondary benefit of the improved intestinal integrity discussed previously. When the gut barrier is robust, as indicated by the lower d-lactate levels in this study, the translocation of pro-inflammatory metabolites to the liver is minimised, thereby supporting optimal hepatobiliary function. Regarding other intestinal health markers, although we intended to use DAO, LBP, and AGP as indicators of dysfunction, no significant differences were observed among the treatments. Since these markers typically rise in response to acute intestinal barrier failure or systemic inflammation (Alhotan et al., 2021; Chen et al., 2015), their stable and low levels in this study confirm that BSFL supplementation is safe and does not trigger inflammatory pathways. Collectively, these findings reinforce the conclusion that 20% defrosted full-fat BSFL supports a healthy gut-liver axis, promoting both structural integrity and metabolic efficiency in broiler chickens. Although lauric acid, chitin, and other bioactive components have been proposed as potential contributors to the observed effects, their specific roles were not directly evaluated in this study. Therefore, future research incorporating fatty acid profiling and mechanistic analysis is warranted.
6 Conclusions
Supplementation of defrosted full-fat BSFL at 20% of daily feed intake effectively enhances growth performance and final body weight in broiler chickens. These improvements appear to result from a synergistic effect, whereby the larvae provide a high-quality nutrient top-up while simultaneously fortifying gut-liver health. The significant increase in the VH:CD ratio and the reduction in D-lactate and ALP levels indicate that BSFL supplementation is associated with improvements in the intestinal barrier and with more efficient lipid metabolism without inducing physiological or oxidative stress. While the lack of isocaloric or isonitrogenic diet formulation means the performance gains are partly due to increased nutrient intake, the observed improvements in gut histology and metabolic markers suggest functional benefits. As a sustainable and evolutionarily familiar feed ingredient, defrosted full-fat BSFL supports circular food systems by recycling local waste into high-value protein. These findings support the potential of BSFL as not only a viable alternative protein source but also a functional feed that optimises the metabolic status of poultry. Future research should focus on trials using isocaloric or isonitrogenic formulations to isolate the specific molecular mechanisms underlying these metabolic shifts and to explore further the long-term impacts of supplementation levels exceeding 20% to maximise production efficiency and improve bird welfare.
Corresponding author; e-mail: sucheera@mut.ac.th
Acknowledgements
S. Chotikatum and C.M. Li contributed equally to this work. The authors acknowledge the valuable assistance of the staff members and students at the farm.
Conflict of interest
All authors declare no conflict of interest.
Funding
The work described in this paper was partially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. CityU 11104124).
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