The BSF larva meal, obtained by processing larvae reared on inedible food substrates, consisting of biowaste, including fruit and vegetable peels, discarded crop residues, and food processing by-products, was provided by NextProtein Tunisia. The composition in terms of nutrients, essential amino acids, and minerals is presented in Table 1 .

Three hundred unsexed one-day-old Arbor Acres broiler chicks having an average initial weight of 75 ± 2.5 g were acquired from a Tunisian commercial hatchery and delivered by an authorized truck to the pedagogical farm of the High School of Agriculture of Mateur, where the trial was held for 35 days, from 24 April to 28 May 2023. The chicks were transported in well-ventilated cardboard boxes, each with a capacity of 50 chicks. The transport duration was relatively short, approximately 2 hours, and the ambient temperature was around 23°C. The chicks were randomly split into two experimental treatment groups, the control group (n = 150) and the experimental group (n = 150), in a completely randomized block design. The two experimental treatments comprised six replicates, with twenty-five chicks per replicate, kept on a 5 cm-deep litter of wood shavings in an area of 2.5 × 1.5 m/ 25 chicks/pen. Each pen was equipped with one feeder and one drinker. The light/dark applied cycle was 23L/1D. Exhaust fans ensured air renewal and temperature control was managed by the pad cooling system. The chicks were raised according to the husbandry guidelines provided by Aviagen, Inc. A three-stage feeding regimen was implemented: starter: 1–14 days of age; grower: 15–28 days of age; and finisher: 29–35 days of age. Five diets were formulated for the starter (control and experimental), grower (control and experimental), and finisher (BSF-free) feeding regimen. The inclusion rates of full-fat BSF larvae meal were set at 5% for the starter diet and 10% for the grower diet, based on existing literature (Dabbou et al., 2018; Biasato et al., 2020) and a strategy to balance the chicks’ nutritional requirements with sustainability. The 5% inclusion in the starter phase minimizes potential disruptions to gut health, while positively influencing the cecal microbiota and gut mucin dynamics by selecting potentially beneficial bacteria and increasing villi mucins (Biasato et al., 2020). In contrast, the 10% inclusion in the grower phase takes advantage of their improved digestive capacity, allowing for better utilization of insect meal without compromising growth performance (Dabbou et al., 2018). These rates were chosen to optimize growth while reducing reliance on traditional protein sources like soybean, aligning with both nutritional requirements and sustainable feeding practices.

Experimental and control diets were formulated to be isocaloric/isonitrogenous. Table 2 presents the ingredients and the chemical composition of the control and experimental diets during the starter and grower phases, as well as the finisher’s BSF-free diet. Chemical analyses of the BSF larvae meal, control, and experimental diets were performed in triplicate according to the AOAC International (2004) procedures: Dry matter (method 934.01), ether extract (method 920.39), ash (method 942.05), crude protein (method 954.01), and crude fiber (method 945.18). Digestible lysine, digestible methionine, methionine + cysteine, and metabolizable energy were calculated according to Cerrate and Corzo (2019).

Health status and mortality were monitored daily throughout the whole experimental period. The live weight (LW) was recorded individually at the beginning of the trial and weekly thereafter (days 7, 14, 21, 28, and 35). Feed intake was measured daily for each pen. The daily weight gain (DWG; Equation 1) in grams and the feed conversion ratio (FCR; Equation 2) were calculated weekly (weeks 1, 2, 3, 4, and 5) at individual and pen levels, respectively.

All the measurements were made on a pen basis using high-precision electronic scales (Accurex DSK, Gram Group, Barcelona, Spain).

At 35 days of age, 24 animals (two birds per replicate pens) from each feeding group (chosen based on the average final LW in each pen) were individually identified with a shank ring and weighed. The chickens were slaughtered at a commercial abattoir. Plucked and eviscerated carcasses were obtained, and the head, neck, feet, liver, heart, spleen, bursa of Fabricius, and abdominal fat were removed to obtain the chilled carcass. Then, eviscerated carcass, thigh and breast weights were immediately recorded. The hot eviscerated carcass yield were expressed as percentages of the LW (Equation 3) and, the breast and thigh were expressed as percentage of eviscerated carcass weight (Equations 4, 5).

For the measurements of pH, color, myoglobin concentration, cooking loss, and water-holding capacity (WHC), the meat was vacuum sealed and refrigerated (4 ± 1°C) to maintain its freshness. For the microbiological analyses, a sample of meat was placed in sterile containers and stored at -20°C to prevent bacterial proliferation during the storage period.

The pH of the breast was recorded immediately after slaughter and 24 hours postmortem (pHu) using a calibrated pH meter (Hanna HI-99163, Italy). The values of 2 replicates were considered, and their mean was subsequently utilized. The meat color was measured at room temperature (20°C) after slaughter and at 24 h postmortem on the inner surface of the Pectoralis major muscle on the breast and on the Biceps femoris muscle on the thigh and using a portable colorimeter Chroma Meter CR-410 Konica Minolta Sensing device (Minolta Sensing Inc., Osaka, Japan). The color measurements were reported in terms of lightness (L*), redness (a*), and yellowness (b*) in the CIELAB color space model (Commission Internationale de l’Éclairage, 1976). The values were recorded for the CIE standard illuminant D65 and the CIE 2° standard observer. The color values were obtained considering the average of three readings per sample.

Myoglobin concentration in the breast was determined using a spectrophotometric method as outlined by Viriyarattanasak et al. (2011). Water-holding capacity (WHC) was determined according to Bowker et al. (2014). Briefly, 10 g of minced muscle (initial weight) was placed in 15 mL of 0.6 M NaCl solution in a 50 mL centrifuge tube. The sample was mixed for 1 minute, stored at 4°C for 15 minutes, and then centrifuged at 7000 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was removed, and the meat was collected and weighed. WHC was calculated as the percentage of retained water using the formula provided in Equation 6.

Cooking loss was measured following the procedure of Petracci and Baéza (2011). The process involved placing the breast meat into hermetically plastic food bags in a water bath operating at 80°C until reaching a final internal temperature at the thickest part of the breast of 75°C.

The temperature was monitored using a hand-held digital thermometer (Testo 104, Germany). Meat samples were then cooled under tap water, dried, weighed, and calculated according to Equation 7:

Meat chemical composition was performed by determining the dry matter (method 950.46), ash (method 920.153), and total fat contents (method 960.39) of the breast meat according to the AOAC’s official methods of analysis AOAC International (2000).

Meat microbiological analysis was based on the counts of total aerobic mesophilic flora, the Enterobacteriaceae family, and E. coli in 1 gram of minced meat. To determine colony-forming units (cfu/g), 1 ml of all decimal dilutions, ranging from 10^-1 to 10^-4 for each bacterial species, was inoculated in specialized bacterial growth media and incubated according to the times and temperatures specific for each bacterial class (Downes and Ito, 2001). To count total aerobic mesophilic flora, samples were inoculated on Plate count agar (PCA) and incubated at 30°C for 24 hours. Incubation of samples at 37°C for 48 hours was performed using HEKTOEN agar to isolate the Enterobacteriaceae family. Finally, for the determination of the total number of E. coli strains, samples were inoculated in Violet Red Bile Agar with Lactose (VRBL) agar and incubated at 44°C for 24 hours.

Cecal contents were carefully extracted during the dissection of broilers sampled from the control and experimental groups, using sterile gloves and a sterile spatula to collect the cecal material directly from the cecum, ensuring that no external surfaces came into contact with the sample. The samples were transferred into sterile containers and stored at -20°C until processing. Enterobacteriaceae, Echerichia coli, Clostridium spp., and Lactobacillus spp. were enumerated following the same protocol used for the microbiological quality of meat. For Clostridium spp. and Lactobacillus spp., samples were inoculated in Meat-Liver agar and De Man, Rogosa, and Sharpe (MRS) agar and incubated at 37°C for 24 to 48 hours and at 40°C for 24 to 48 hours, respectively. Two counts were conducted after 24 and 48 hours.

Data obtained from this study were analyzed using the SAS software (SAS Institute Inc. SAS^® 9.4, 2014). Data were tested for normality and homogeneity of variance. All values were grouped, and the mean and standard error were calculated. A one-way analysis of variance (ANOVA) in a fully randomized design using the general linear model (GLM) was performed for all parameters to examine differences between groups. P <0.05 was considered a significant value.

The overall mortality rate during the trial was identical for both groups (equal to 0.66%), and it was recorded during the two first weeks (starter period) as shown in Table 3 . Statistical analysis revealed no significant differences in all measured parameters between the control and experimental groups (P> 0.05), except the average weight of broilers during the second week which was higher in the experimental group than the control group (545.7 ± 24.1 vs. 541.0 ± 43.0, P= 0.016).

The effect of BSF larvae meal on carcass characteristics, meat quality, and cecal analysis is shown in Table 4 . The results indicated that BSF larvae meal had a positive impact (P=0.001) on hot eviscerated carcass yield. The experimental group exhibited a significant increase in carcass yield (80.2%) compared to the control group (76.8%; P=0.001). However, no significant differences were observed in breast and thigh yields (P>0.05). No significant differences were found in post-mortem or ultimate pH values, myoglobin content, water-holding capacity, cooking losses, or meat chemical composition between the control and experimental groups. Enterobacteriaceae counts in the cecal content showed a significant difference between the control and experimental groups (P=0.006), while no significant differences were observed in total aerobic mesophiles and E. coli counts between the groups (P>0.05).

There were no significant differences in color parameters in broiler breast and thigh meat 24 hours after slaughter ( Table 5 ). However, 1 hour post-mortem, the breast meat from the experimental group was redder (a*= 13.0 ± 2.68) than that from the control group (a*= 10.2 ± 1.70; P=0.026). For thigh meat, the experimental group had significantly lower yellowness (b*= 8.09 ± 1.68) compared to the control group (b*= 9.89 ± 1.06; P=0.023).

Throughout the trial, broilers exhibited normal vitality, with no pathological symptoms observed. The mortality rate was extremely low, at 0.66% in both the control and experimental groups. One chick in the experimental group was stunted from the beginning of the trial, while another in the control group developed joint inflammation in its leg, which prevented it from eating and drinking.

Our results are consistent with those of Dabbou et al. (2018), who reported a zero-mortality rate in all groups of broiler chickens fed diets containing 5%, 10%, and 15% BSF larvae meal. Similarly, Cullere et al. (2016) observed mortality rates ranging from 0% to 0.2% in broiler quails fed diets incorporating 15% and 10% BSF larvae meal, respectively. However, Hartinger et al. (2022) reported a higher mortality rate of 16.66% in chickens fed a diet containing 30% BSF larvae meal, whereas the group fed a 15% BSF larvae meal diet had a mortality rate of 1.38%. In our study, no significant differences were observed in average weight, daily weight gain, or feed conversion ratio between the control and experimental groups, except for the average weight during the second week. It should be noted that throughout the 5 weeks of broiler chicken rearing, the experimental group numerically showed higher weights during the second, third, fourth, and fifth weeks. However, the difference was only statistically significant during the second week. This suggests that while the experimental group exhibited higher weights across the later weeks, the effect was most pronounced in the second week. The lack of statistical significance in the subsequent weeks may indicate that the initial advantage diminished over time, possibly due to adaptation to the diet. Overall, these findings suggest that the inclusion of BSF larvae meal as a protein source in broiler chicken diets did not negatively affect growth performance. Similar conclusions were drawn by Hartinger et al. (2022), who reported no adverse effects with a 15% inclusion level of BSF larvae meal in broiler diets, and by Al-Qazzaz et al. (2016) in laying hens supplemented with 1% or 5% BSF larvae meal. Other studies have reported a positive impact of insect meal inclusion on growth performance. For instance, De Souza Vilela et al. (2021) found that a diet containing 20% BSF larvae meal reduced the FCR by 10% in broilers. Similarly, Hwangbo et al. (2009) demonstrated that diets supplemented with 10% to 15% maggots improved the growth performance of broilers. Dabbou et al. (2018) found that BSF larvae meal positively influenced broiler growth performance at inclusion levels up to 10%, although performance declined with a 15% inclusion level. Meanwhile, Cullere et al. (2016) observed no significant differences in body weight gain, feed intake, FCR, or mortality rate between control and experimental groups of broiler quails fed 10% or 15% BSF larvae meal.

The variability in findings among these studies may be attributed to several factors, including differences in the nutrient profiles of the insect meals used, which are influenced by the substrates used for larvae production and directly affect their chemical composition (Tschirner and Simon, 2015). Additionally, factors such as insect processing methods, inclusion rates, experimental protocols, and the specific bird species studied play a critical role. These results highlight the potential of BSF meal as a sustainable protein source for the broiler feed industry. Moreover, this study provides valuable insights into how BSF meal influences broiler performance, and overall feed efficiency, contributing to efforts aimed at reducing variability in its composition. However, further research is needed to establish consistent and universal standards that ensure the reliability of insect-based feed ingredients.

The inclusion of BSF larvae meal in poultry diets did not affect carcass traits in this study. Research on BSF larvae meal as a protein source for poultry has shown mixed effects on carcass characteristics. Some studies align with our findings, reporting no significant impact on carcass traits in Cobb 500 broilers fed diets containing 5%, 10%, and 15% BSF larvae meal (Pieterse et al., 2019), in broiler quails (Coturnix coturnix japonica) fed diets with 10% and 15% BSF larvae meal (Cullere et al., 2016), and in Gramasree hens receiving BSF larvae as a protein replacer at varying levels (25%, 50%, 75%, and 100%) (Avinash et al., 2022). In contrast, other studies have reported improvements in carcass traits following BSF larvae meal inclusion (Hoffman, 2019; Chia et al., 2021). Popova et al. (2020) found that while BSF larvae meal enhanced carcass weight and breast yield, it reduced thigh yield. These discrepancies may be attributed to differences in bird species, dietary formulations, or environmental factors influencing growth.

Colorimetric analysis showed that chicken thighs from birds fed BSF larvae meal exhibited significantly lower yellowness one hour post-slaughter and slightly reduced yellowness after 24 hours. Meat color is influenced by multiple factors, including heme pigments, genetics, and diet composition (Fletcher, 1999). The lower yellowness observed in this study could be due to the reduced carotenoid content in BSF larvae, which is highly dependent on the rearing substrate (Borel et al., 2021; Leni et al., 2022). Similarly, Schiavone et al. (2019) reported a decrease in breast meat yellowness with increasing levels of BSF larvae meal. In contrast, redness (a*) values increased in the breast meat of broilers fed BSF larvae meal. This finding aligns with previous studies showing that BSF larvae meal or oil increases breast meat redness (Schiavone et al., 2019; Aprianto et al., 2023; Kierończyk et al., 2023). The increase in a* values may be attributed to xanthophyll pigments accumulated in BSF larvae, which originate from vegetables and fruits in their rearing substrate (Aprianto et al., 2023).

In this study, dietary treatments did not significantly affect (P>0.05) the physico-chemical, nutritional, technological, or microbiological quality of breast and thigh meat. This consistency in meat quality can be attributed to the formulation of diets as isocaloric and isonitrogenous, ensuring similar levels of essential nutrients across all groups. Additionally, our dietary strategy, starting with a lower inclusion rate during the starter phase to avoid digestibility issues and excluding BSF larvae meal during the finisher phase, may have limited the duration of exposure, preventing significant differences from manifesting. Similar findings have been reported by Schiavone et al. (2019), who observed no changes in meat quality parameters, such as cooking loss, with BSF larvae meal inclusion up to 10%. Pieterse et al. (2019) also found no significant effect of diets containing up to 15% BSF larvae meal on broiler meat pH, color, and cooking loss. However, Popova et al. (2020) reported that BSF larvae meal reduced pH and myoglobin concentration in broiler meat, attributing this effect to the lauric acid content of the larvae. Differences in inclusion levels, basal diet composition, bird breed, or BSF larvae processing and storage methods may explain these conflicting results, as such factors could influence the bioavailability of lauric acid and its impact on meat quality.

To our knowledge, no prior studies have assessed the effect of BSF larvae meal on the microbiological quality of broiler meat. In this study, its inclusion did not affect total aerobic mesophilic flora, Enterobacteriaceae, or Escherichia coli counts. This outcome is likely due to identical handling conditions during slaughter, storage, and analysis for all groups. However, further research is needed to explore the impact of different BSF larvae meal inclusion levels on meat microbiological quality.

BSF larvae are recognized as a source of antimicrobial compounds that can modulate fecal microbiota, potentially reducing harmful bacteria (Borrelli et al., 2017). This effect is attributed to their bioactive molecules, including lauric acid (Borrelli et al., 2021), chitin (Lagat et al., 2021; Kemboi et al., 2022), and various antimicrobial peptides (Moretta et al., 2020; Xu et al., 2020). Lauric acid, which makes up to 60% of BSF larvae fat (Spranghers et al., 2017), exhibits strong antimicrobial activity against various bacterial species. It achieves this through a membrane-lytic mechanism, destabilizing bacterial cell membranes, increasing permeability, and ultimately leading to cell lysis (Yoon et al., 2018). Additionally, lauric acid inhibits the MurA enzyme, which is essential for bacterial cell wall biosynthesis. Chitin, the primary component of the insect exoskeleton and crustacean shells (Joseph et al., 2021), is a natural polysaccharide and the second most abundant polymer after cellulose. It holds significant value in the pharmaceutical, food, and agricultural industries due to its diverse biological activities, including antimicrobial properties (Kumar et al., 2020; Lagat et al., 2021). Insects, including BSF larvae, are also rich sources of antimicrobial peptides that can target both Gram-positive and Gram-negative bacteria. These include Staphylococcus aureus, Micrococcus gluteus, Bacillus subtilis, Bacillus thuringiensis, Aerococcus viridans, and Bacillus megaterium, as well as Escherichia coli (Moretta et al., 2020). These peptides exert antimicrobial effects either through membranolytic mechanisms or by interacting with intracellular targets such as DNA, RNA, and proteins. This is consistent with our findings, which showed a significant decrease (P=0.006) in Enterobacteriacae counts, a group of gram-negative bacteria.

Building on the well-documented antimicrobial properties of chitin and insect-derived peptides, the inclusion of BSF larvae meal in animal diets may enhance gut microbiota composition by leveraging these bioactive compounds. The insect-based diet led to an increase in Lactobacillus spp. and a decrease in Clostridium spp. and Enterobacteriaceae, while Escherichia coli strains abundance was not significantly affected. Lagat et al. (2021) reported that chitin and chitosan extracted from BSF pupal exuviae exhibit broad-spectrum antimicrobial activity, inhibiting the growth of both Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria. Similarly, Borrelli et al. (2021) demonstrated that lauric acid inhibits Clostridium difficile by disrupting cell membranes. Borrelli et al. (2017) suggested that BSF larvae may act as a prebiotic, with chitin playing a key role in this effect. The inclusion of BSF larvae in poultry diets has been associated with an increase in Oscillospira, a genus considered one of the next-generation probiotics, known to prevent Clostridium difficile infections (Yang et al., 2021). Bonomini et al. (2025) further confirmed that BSF larvae and their lipid fractions exhibit both antibacterial and prebiotic effects, supporting their potential role in gut health optimization. Lauric acid, a major component of BSF larvae, exhibits strong antimicrobial activity against pathogenic Clostridium species, such as C. perfringens, a causative agent of necrotic enteritis in poultry (Matsue et al., 2019). Importantly, lauric acid selectively targets pathogens while sparing beneficial bacteria like Lactobacillus spp., potentially modulating intestinal health. This could explain the increase in Lactobacillus spp. and decrease in Clostridium spp. observed in our study. Furthermore, chitin, a major structural component of BSF larvae, has been reported to act as a functional fiber, promoting the growth of beneficial bacteria and enhancing gut barrier function (Edo et al., 2024). The combined effects of chitin and lauric acid may synergistically inhibit pathogens while fostering a microbiota composition favorable for gut health (Borrelli et al., 2021). Future research should explore ways to enhance the functional benefits of BSF meal, including adjusting inclusion levels, processing methods (e.g., defatting, enzymatic hydrolysis), or combining BSF-derived bioactives with other functional feed additives.

Additionally, Lactobacillus spp. exhibit antibacterial activity, making them valuable for poultry feed formulation. Their probiotic properties help inhibit pathogen growth through competitive exclusion in the gastrointestinal tract (Ahmed et al., 2019) and contribute to improved feed efficiency (Yan et al., 2017). Based on these findings, we hypothesize that an insect-based diet could positively influence cecal microbiota, creating a gut environment that supports poultry health and performance. Given the antimicrobial properties of BSF-derived compounds, future research should investigate BSF meal as a microbial modulator, particularly its impact on gut microbiota composition and its potential role in improving meat safety and quality.