The use of insect-derived by-products as litter amendments may represent a sustainable strategy in broiler production. The present study evaluated the effects of supplementing wood shavings with different inclusion levels of yellow mealworm (Tenebrio molitor) frass on litter quality, growth performance, slaughter traits, and the meat quality of broiler chickens.
Methods
A total of 352 one-day old Ross 308 male broilers were allocated to 16 pens (4 replicates per treatment; 22 chicks per replicate) with four bedding treatments: 100% wood shavings (FO, control), or wood shavings supplemented with 10% (F10), 20% (F20), or 30% (F30) of frass. Individual live weight (LW) and daily weight gain (DWG), and pen-level daily feed intake (DFI) and feed conversion ratio (FCR) were recorded during the trial. At 42 days of age, chickens were slaughtered to assess carcass yield, cut proportions, and the occurrence of breast myopathies, while meat quality parameters were measured on breast (pectoralis major) and thigh (iliotibialis lateralis) muscles.
Results and Discussion
The DFI was significantly reduced at all frass inclusion levels compared to the control group. Final LW (P = 0.012), and overall DWG (P = 0.004) were significantly lower in F10 and F30 compared with FO, while overall FCR was not affected. Carcass and breast yields did not differ among treatments, while thigh yield was significantly reduced in FO group (P = 0.023). Breast myopathies and meat quality parameters remained unaffected by the bedding treatments. These findings indicate that yellow mealworm frass can be supplemented in conventional bedding materials in broiler production without compromising meat quality traits. However, in F30 group growth performance and breast yield were impaired, suggesting that frass inclusion should not exceed 20%.
bedding materialinsect frassmeat qualitypoultry litter qualityproductive performanceUniversità degli Studi di Padova10.13039/501100003500The author(s) declared that financial support was received for work and/or its publication. This research was supported by the University of Padova (Italy) funds (2023-prot. BIRD234733/23).section-at-acceptanceAnimal Physiology and ManagementIntroduction
With the global population projected to reach nearly 10 billion by 2050, food demand is expected to rise by about 70%, intensifying the global need for animal proteins and sustainable feed resources (FAO, 2018). As a result, there is growing interest in alternative protein sources that simultaneously meet nutritional requirements and align with principles of environmental sustainability (Amorim et al., 2024). Among the potential alternative protein sources, insects are widely regarded as one of the most promising options for use in animal feed and human food applications (Dalle Zotte, 2021).
Insect farming has shown significant growth in recent years and is expected to continue expanding. European production was estimated at 11,000 tons in 2023 and is predicted to increase by 10–60 times by 2030 (De Volder et al., 2025). Currently, eight insect species are authorized in the EU as ingredients in feeds for aquaculture and non-ruminant livestock species (European Commission, 2021). The most widely farmed insect species in Europe are yellow mealworm (Tenebrio molitor) and black soldier fly (Hermetia illucens), which dominate current industrial insect production in the feed and food (Thrastardottir et al., 2021). Insect-based ingredients have been evaluated in the diets for poultry (Dabbou et al., 2018; Biasato et al., 2025), fish (Rawski et al., 2020; Ido et al., 2024), rabbits (Martins et al., 2018; Dalle Zotte et al., 2018) and pigs (Yoo et al., 2019; Meyer et al., 2020) and have shown a favorable nutritional potential.
Insect farming primarily focuses on protein and fat production; however, it also generates a considerable amount of secondary output, known as frass. Frass is defined by the Commission Regulation (EU) 2021/1925 as: “a mixture of excrements derived from farmed insects, the feeding substrate, parts of farmed insects, dead eggs and with a content of dead farmed insects of not more than 5% in volume and not more than 3% in weight” (European Commission, 2021). The insect-rearing process yields a substantial quantity of frass relative to the produced insect biomass. In black soldier fly and yellow mealworm farming, it is typically produced at 2–4 times the harvested larval biomass (He et al., 2021).
Currently, frass is mainly used as an organic fertilizer, where it contributes to reducing dependence on synthetic agrochemicals and supports sustainable crop production (Poveda, 2021). Indeed, it is rich in nutrients and microorganisms, and has shown potential as a soil amendment, bio-fertilizer, and biostimulant (Barragán-Fonseca et al., 2022). Several studies have reported its beneficial effects on plant growth and soil health (Menino et al., 2021; Houben et al., 2020; Ferruzca-Campos et al., 2023). However, the growing volume of frass calls for alternative applications beyond agronomic use. Beyond its physical properties, frass also provides several functional benefits arising from its complex composition, which includes not only insect excreta but also dead insects or larvae, unhatched eggs, and residual biomass (Adams and Koutsos, 2024). In addition, consequent to the molting process of insects, frass may contain exoskeletons (exuviae), rich in chitin and chitin-derived components, which have been shown to exert a prebiotic effect on the gut microbiome of broiler chickens, thereby supporting healthy growth and development (Subbarayudu et al., 2020). Notably, certain insect species exhibit varying quantities of bioactive compounds. For instance, lauric acid—a medium-chain fatty acid known for its antimicrobial properties- can be present in different concentrations depending upon the insect species (Borrelli et al., 2021; Wu et al., 2021). Insects also produce antimicrobial peptides as part of their innate immune system, which have been shown to exhibit bactericidal, antifungal, and antiviral activity (Xia et al., 2021). It is hypothesized that these bioactive compounds may remain active in frass and contribute to modulating the litter microbiota, reducing pathogenic loads, and ultimately improving animal health and welfare.
Bedding materials commonly used in broiler production vary globally and include wood shavings, rice hulls, chopped straw, shredded paper, sand, and peat (Grimes et al., 2002; Diarra et al., 2021; Brink et al., 2022). In Europe, wood shavings and chopped wheat straw are among the most prevalent options (Brink et al., 2022). Bedding material and litter play a critical role in poultry production, influencing bird behavior, welfare, performance, carcass quality, health, and environmental outcomes (Dunlop et al., 2016; Munir et al., 2019; Wilcox et al., 2024). Selecting an appropriate bedding material requires consideration of several factors, such as cost-effectiveness, local availability, moisture absorption, water-holding ability, drying property, biosecurity concerns, and the potential to support natural bird behaviors without compromising health (Brink et al., 2022). In addition, poultry express various natural behaviors such as litter pecking, scratching, and dust bathing through interaction with litter, which may reduce stress and promote better health and immune status (Shields et al., 2005; Scholz et al., 2010; Regmi et al., 2018). Furthermore, bedding materials must be either free from potential pathogens, contaminants, and other harmful substances, or contain them only at regulated, safe levels. This requirement is crucial not only for the protection of animal health and welfare, but also for minimizing risks related to food safety and ensuring the safe agronomic utilization of spent litter (Gerber et al., 2020). However, frass does not necessarily meet this requirement. The use of untreated insect frass may pose microbiological risks, as high counts of Enterobacteriaceae, lactic acid bacteria, and bacterial endospores have been reported in various studies (Gold et al., 2020; Osimani et al., 2018, 2021; Lopes et al., 2022; Verardi et al., 2025). Several species within these microbial groups are considered potential pathogens (e.g., Salmonella spp., Escherichia coli, Enterococcaceae, Bacillus cereus, Clostridium perfringens), although their loads and species composition vary with insect species and feeding substrate used (De Volder et al., 2025).
To mitigate these risks, Regulation (EU) 2021/1925 establishes harmonized standards for the production and commercialization of insect frass when used as an organic fertilizer or soil improver. These standards are aligned with those applied to processed animal manure. Specifically, frass must undergo a heat treatment of at least 70 °C for a minimum of 60 minutes and must demonstrate a significant reduction in spore-forming bacteria and toxin production where such hazards are identified.
Given the physical characteristics, absorptive properties, and chemical composition, frass may be a viable alternative bedding material in broiler production. Therefore, the present study aimed to investigate the effects of incorporating yellow mealworm (Tenebrio molitor) frass at 0%, 10%, 20%, and 30% into wood shavings bedding on litter quality, growth performance, mortality, carcass and meat quality traits in broiler chickens.
Materials and methodsExperimental design and bird management
The study was performed at the poultry facility of the experimental farm of University of Padova (Legnaro, Padova, Italy). A total of 352 one-day-old male, fast growing broiler chicks (Ross 308; Aviagen Group, UK) were obtained from a local commercial hatchery (Avizoo/Euroagricola s.s., Longiano (FC), Italy). All chicks were vaccinated against Marek’s disease (bivalent HVT + RISPENS), infectious bronchitis and avian pseudo plague, and they were transported by an authorized truck from a commercial hatchery to the experimental farm.
Wood shavings were used as basal bedding material and were supplemented with Tenebrio molitor frass at varying inclusion levels. Before use, frass underwent thermal treatment at 70°C for 24 h to reduce microbial contamination and ensure biosecurity. Three days before the beginning of the trial, all litter materials were placed in the pens to allow acclimatization to room temperature. Wood shavings were evenly distributed, and the designated proportion of frass was uniformly layered on top.
Four treatment groups were formed based on the proportion of frass mixed with wood shavings: Control (F0; 100% wood shavings), 10% frass (F10; 90% wood shavings + 10% frass), 20% frass (F20; 80% wood shavings + 20% frass), and 30% frass (F30; 70% wood shavings + 30% frass). In all pens, an equal amount of initial bedding material was provided (50 kg/pen; 16.7 kg/m²). No additional bedding was added during the trial.
Upon arrival, birds were individually weighed, identified with wing tags, and randomly assigned to one of four bedding treatment groups, each consisting of four replicates (16 pens; 22 chicks per replicate. Each pen measured 2.60 m in length, 1.25 m in width, and 1.20 m in height, providing a total surface area of 3.25 m².
Continuous lighting (24 h/day) was provided on the first 3 days post-placement using a combination of natural and artificial light sources (Osram L 36W/640 cool white; OSRAM Licht AG, Munich, Germany). From day 3 to day 12, the dark period was gradually increased until reaching six consecutive hours, which was then maintained until the end of the trial. The experimental house was environmentally controlled, and temperature and humidity were maintained according to Ross 308 management guidelines (Aviagen, 2018).
Birds had free access to feed and water throughout the 42 days. Each pen had 5 automatic nipple drinkers with drip cups and one 37-cm diameter manual circular feeder. Diet formulation followed Aviagen Ross 308—Broiler Nutrition Specifications (Aviagen, 2022). All groups received the same commercial diet (Consorzio Agrario di Treviso e Belluno, Treviso, Italy) in a three-phase program: starter (0–14 day, mash; CP 21.0%; EE 5.7%; CF 3.5%; ash 6.6%; Ca 1.10%; P 0.81%; Lys 1.25%; Met 0.33%; Na 0.15%), grower (14–28 day, pellet; CP 18.6%; EE 5.0%; CF 3.7%; ash 6.2%; Ca 1.10%; P 0.70%; Lys 1.03%; Met 0.28%; Na 0.15%), and finisher (28–42 day, pellet; CP 17.3%; EE 5.0%; CF 3.7%; ash 6.0%; Ca 1.10%; P 0.65%; Lys 0.93%; Met 0.27%; Na 0.14%).
Microbiological analysis, moisture content, and oocysts count of bedding material, litter and droppings
Bedding materials (wood shavings and frass) were sampled for microbiological analysis before the trial initiation: frass (n = 6 batches; after thermal treatment) and wood shavings (n = 3 batches; untreated). At the end of the trial, at 42 days of age of birds, litter samples were taken from five different locations within each pen (at the four corners, and at the center), and from both the surface and the bottom layers of the litter. Afterwards, samples were homogenized, obtaining a pool for each pen to be subjected to microbiological analysis, which was performed in an accredited analytical laboratory using methods established by specific ISO standards or, in the absence of a specific ISO standard, by internal laboratory test methods. For each sample, 10 g were added to 90 mL of Buffered Peptone Water and homogenized in Stomacher Colworth 400. Each sample has been analyzed for salmonellae (ISO 6579) and Listeria monocytogenes (ISO 11290; both parameters in 25 g). Furthermore, on each sample several bacterial counts were also determined: total aerobic mesophilic count (TBC; UNI EN ISO 4833-1:2022), Enterobacteriaceae (ISO 21528-2:2017), beta-glucuronidase-positive Escherichia coli (with internal laboratory test method, validated), coagulase-positive Staphylococci (i.e. Staphylococcus aureus and similar species) (UNI EN ISO 6888-2:2023 part 2), sulphite-reducing clostridia (ISO 15213-1:2023). Microbiological results were expressed as log CFU/g.
At day 42, another litter sampling (n = 16; 1/pen; 4/treatment) was performed for moisture content determination and oocysts count. Litter samples were taken as indicated above. After retaining an aliquot (preserved at +4°C) for oocysts count, samples were oven-dried at 70°C until reaching a constant weight to determine dry matter content and calculate moisture percentage. Additionally, at day 42 freshly voided droppings samples (n=16, 1/pen, 4/treatment) were collected from each pen as above described, homogenized in a pool/pen and preserved at +4°C. Both droppings and litter samples were analyzed by using the Miniflotac technique [sensitivity 5 oocysts per gram-OPG; (Barda et al., 2013)] within 2 days from sampling.
Growth performance
Individual live weight (LW) was recorded weekly until commercial slaughter age (42 days). Daily feed intake (DFI) was recorded daily on pen basis throughout the experimental period. Daily weight gain (DWG), and feed conversion ratio (FCR) were calculated on a weekly basis. Mortality and health status were monitored daily during the entire experimental period.
Slaughtering, carcass and meat quality traits
At 42 days of age, all birds were individually weighed immediately before to transportation to the slaughterhouse to determine the final LW, following a 9-hour feed withdrawal period. Broilers were slaughtered according to standard commercial procedures, including electrical stunning, exsanguination, scalding, plucking, and evisceration, with removal of the heads, necks, and shanks. After 2 hours of chilling at 2°C, all carcasses were individually weighted and 120 carcasses (30 per group) were selected as representative of their respective experimental groups, based on average LW and standard deviation. The selected carcasses were transported to the laboratories (LaChi laboratory of the DAFNAE Department, and LabCNX laboratory of the MAPS Department), where they were stored under chilled conditions (2°C) for 24 h. Subsequently, the carcasses were individually weighed both with and without feet to determine the cold carcass yield. Afterward, these carcasses were visually assessed to determine the presence of breast muscle myopathies. The pectoralis major muscles were subjected to gross examination to assess the occurrence and severity of white striping myopathy according to the classification systems described by Kuttappan et al. (2012), wooden breast myopathy by Sihvo et al. (2014), and spaghetti meat myopathy by Baldi et al. (2018).
Then, 80 birds (20 per treatment, 5 per pen) were used to evaluate carcass yield parameters. Carcasses were subsequently dissected into major cuts, including breast, wings, drumsticks, and thighs (Petracci and Baéza, 2011). Breast-related parameters were further evaluated: breast yield, defined as whole chicken breast with bone and skin, and breast meat yield: boneless, skinless edible breast meat (pectoralis major and pectoralis minor muscles). All examined parameters were expressed as a percentage of the cold carcass (CC) weight.
Meat quality and femur fracture toughness
After dissection and skin removal, right breast fillets (pectoralis major) and right thighs (iliotibialis lateralis) were used for the main meat quality analyses, following the harmonized criteria of Petracci and Baéza (2011). The ultimate pH (pHu) was measured with a portable pH meter (FG2-Five GoTM; Mettler Toledo, Greifensee, Switzerland) calibrated at pH 4.0 and 7.0. Colour measurements (Lightness—L*, redness—a* value, yellowness—b* value; CIE, 1976) were performed with a portable colorimeter Chroma Meter CR-400 Minolta (Minolta Sensing Inc., Osaka, Japan). The pHu and color measurements were performed in duplicate.
After measuring pH and color, the right thighs were deboned. Subsequently, femurs were measured for fracture toughness (FFT) using a three-point flexure test with a TA-HDi Texture Analyzer dynamometer (Stable Macro System, London, UK) at a loading rate of 5 mm/min. Measurements were performed at the mid-diaphysis, positioning each femur with its natural convex side facing downwards on the flexure fixture, with the distance between the two supporting fulcra set at 60 mm (Dalle Zotte et al., 2014).
Breast meat was then used to assess thawing and cooking losses, to evaluate the meat water-holding capacity (WHC). From each fillet, a standardized meat sample (8 cm × 4 cm × 3 cm) was excised from the cranial portion of the p. major muscle, aligned parallel to the muscle fibers. Samples were weighed, vacuum-sealed in plastic bags, and stored at −18°C until analysis of thawing and cooking losses. For thawing losses determination, samples were thawed overnight at room temperature, removed from the packaging, gently dried with paper towels, and weighed. Subsequently, each sample was vacuum sealed in a new plastic bag and cooked in a water bath at 80°C for 45 min. After cooling at room temperature for 40 min, the samples were dried again and reweighed to calculate cooking losses (Petracci and Baéza, 2011). For meat shear force analysis, a breast subsample (4 cm × 2 cm × 1 cm) was cut from the cooked portion, and the maximum shear force was determined using a single-column LS5 texture analyzer (Lloyd Instruments Ltd., Bognor Regis, UK) equipped with an Allo-Kramer shear cell (10 blades; 500 kg load cell; blade spacing: 5 mm; blade thickness: 2 mm; crosshead speed: 250 mm/min) (Mudalal et al., 2015).
Statistical analysis
The pen was considered the experimental unit, as treatments were applied at pen level, while individual birds measured within each pen were considered subsampling units and were individually identified and measured. LW and DWG were analyzed using linear mixed models (PROC MIXED, SAS 9.4; SAS Institute Inc., Cary, NC, USA), including the experimental group as a fixed effect and the pen nested within treatment as a random effect. Weekly LW and DWG were analyzed separately for each time point, while overall DWG was analyzed over the whole experimental period. Pen-level data on DFI, FCR, litter moisture, oocysts count, and microbiological count were analyzed using the GLM procedure of SAS, setting the experimental group as a fixed effect. The occurrence of myopathies and mortality were analyzed using the GENMOD procedure of SAS. Carcass traits and meat quality parameters were analyzed using linear mixed models with the experimental group as a fixed effect and the pen nested within treatment as a random effect. Individual carcasses measured within each pen were considered subsamples. Differences were considered statistically significant at P ≤ 0.05, and the Bonferroni post-hoc comparison test was used to compare means.
ResultsMicrobiological analysis, moisture content, and oocysts count of bedding material, litter, and droppings
The initial (before use) TBC did not differ between wood shavings and frass (5.00 vs 5.36 log CFU/g, respectively) (data not shown). As expected, at day 42 the TBC in the litter increased across all treatments (range: 8.03–8.08 log CFU/g), but no statistically significant differences were observed (Table 1). Similarly, Enterobacteriaceae, β-glucuronidase-positive Escherichia coli, and sulphite-reducing clostridia did not differ among groups. Counts of coagulase-positive Staphylococci remained below the detection threshold (<1.0 log CFU/g) across all experimental treatments. Furthermore, Salmonella spp. was absent (NR/25 g) from all samples analysed.
Microbiological counts (log CFU/g) of broiler litter containing increasing levels of insect frass (F0, 0%; F10, 10%; F20, 20%; F30, 30%) at day 42.
Item
Treatments
Pooled SE
P-value
F0
F10
F20
F30
Litter, n
4
4
4
4
TBC1
8.03
8.07
8.08
8.08
0.12
0.896
Enterobacteriaceae
7.73
7.53
7.78
7.69
0.17
0.251
Beta-glucuronidase-positive E. coli
7.36
7.11
7.32
7.12
0.21
0.260
Sulphite-reducing Clostridia
6.74
6.67
6.71
6.74
0.13
0.858
Coagulase-positive Staphylococci
<1.0
<1.0
<1.0
<1.0
Salmonella enterica (NR/25 g)2
ND
ND
ND
ND
1TBC: total aerobic mesophilic count
2ND: not detected in 25 grams
At day 42, litter moisture content did not differ among the experimental groups (F0 = 28.9%; F10 = 26.3%; F20 = 27.2%; F30 = 26.4%; data not shown). At day 42, means OPG values detected in dropping pool samples showed high variability among groups, with higher oocysts count found in F10 (458 OPG) and F30 (210 OPG), while F0, and F20 remained near zero (4 and 3 OPG, respectively). No (F0; F20, F30) or very few oocysts (F10: 10 OPG) were detected in litter pool samples (data not shown).
Growth performance
At the end of the trial, overall mortality was 2.3% (8 birds: F0 = 1; F10 = 2; F20 = 4; F30 = 1), with no significant differences among groups (data not shown). The effects of frass incorporation in bedding on growth performance traits are presented in Table 2. All birds had comparable initial LW at day 1. However, by day 7, F0 birds exhibited significantly higher LW compared to those in frass-supplemented bedding groups (P = 0.004). This pattern was observed also on day 14 (P = 0.005) and day 28 (P = 0.004). At day 35, the treatment effect was not significant. At the end of the trial, final LW was higher in F0 (3171 g) compared to F10 (3044 g) and F30 (3021 g) groups (P = 0.012), while F20 (3119 g) did not differ from other treatments.
Effect of frass incorporation in the bedding at 0% (F0), 10% (F10), 20% (F20), and 30% (F30) on live weight (LW) and daily weight gain (DWG) of broiler chickens.
Item
Treatments
Pooled SE
P-value
F0
F10
F20
F30
Pens, n
4
4
4
4
LW, g
Day 1
44.6
44.0
44.1
44.1
0.46
0.531
Day 7
154a
140b
141b
139b
2.52
0.004
Day 14
430a
390b
398b
384b
7.72
0.005
Day 21
888a
828ab
832ab
808b
15.6
0.020
Day 28
1557a
1478b
1498ab
1454b
20.8
0.004
Day 35
2341
2248
2299
2243
42.1
0.120
Day 42
3171a
3044b
3119ab
3021b
35.6
0.012
DWG, g/d
Week 1
18.2a
15.9b
16.2b
15.8b
0.58
0.004
Week 2
39.5a
35.8b
36.7ab
35.0b
0.83
0.011
Week 3
65.4
62.6
61.9
60.7
1.39
0.156
Week 4
95.5
92.8
95.2
92.2
1.26
0.153
Week 5
112
110
114
113
1.81
0.440
Week 6
119
114
117
111
1.93
0.068
Week 1-6
77.1a
73.2b
75.0ab
72.6b
0.97
0.004
Values are least-squares means. The pen was the experimental unit, while all birds were individually identified and measured within pens (subsampling).
a,bValues with different superscript letters within the same row differ significantly (P < 0.05).
During week 1, F0 chickens had higher DWG compared to all frass-supplemented groups (P = 0.004). This trend continued in week 2 (P = 0.011). No differences in DWG were observed during weeks 3, 4, 5 and 6. Overall, DWG from day 1 to 42 was significantly greater in the F0 group compared to F10 and F30 (77.1 vs 73.2 and 72.6 g/d, respectively; P = 0.004), while birds in the F20 group showed intermediate DWG (75.0 g/d).
DFI differed among groups from the early phases of the trial, with birds in the F0 group showing higher intake levels than those in the frass-treated groups (Figure 1). In week 1, DFI was higher in F0 compared to frass-supplemented groups, with F30 showing the lowest value (P < 0.001; Table 3). At week 2 and from week 4 onwards the F20 group consistently showed DFI comparable to that of F0 group. In contrast, F10 and F30 groups generally resulted in lower DFI compared to F0 group, with some variations across weeks. No differences were observed in FCR among treatment groups at any time point. Overall, the DFI resulted higher in F0 and F20 groups compared to F10 and F30 ones. No treatment effect was detected for FCR over the experimental period (Table 3).
Effect of frass incorporation in the bedding at 0% (F0), 10% (F10), 20% (F20), and 30% (F30) on daily feed intake of broiler chickens.
Line graph showing feed intake in grams per day (g/d) of four groups(F0, F10, F20, F30) evaluated over 41 days. Daily feed intake increases steadily with a peak aroundday 39. Each group is represented by a different colored line.
Effect of frass incorporation in the bedding at 0% (F0), 10% (F10), 20% (F20), and 30% (F30) on daily feed intake (DFI), and feed conversion ratio (FCR) of broiler chickens.
Item
Treatments
Pooled SE
P-value
F0
F10
F20
F30
Pens, n
4
4
4
4
DFI, g/d/bird
Week 1
19.0a
16.7b
16.9b
15.2c
0.33
<0.001
Week 2
49.8a
45.2b
45.9ab
44.1b
0.97
0.007
Week 3
91.6a
86.4ab
85.7b
84.1b
1.38
0.013
Week 4
143a
136b
137ab
135b
1.41
0.006
Week 5
192a
184b
190ab
184b
1.52
0.008
Week 6
221a
209b
217ab
210ab
2.92
0.033
Week 1-6
122a
115b
118ab
114b
1.01
0.001
FCR, g/g
Week 1
1.05
1.05
1.04
0.97
0.02
0.056
Week 2
1.26
1.26
1.25
1.26
0.02
0.978
Week 3
1.40
1.38
1.38
1.39
0.02
0.893
Week 4
1.50
1.46
1.44
1.46
0.02
0.257
Week 5
1.71
1.67
1.66
1.63
0.02
0.192
Week 6
1.87
1.84
1.85
1.89
0.03
0.644
Week 1-6
1.60
1.57
1.57
1.58
0.01
0.296
a,bValues with different superscript letters within the same row differ significantly (P < 0.05).
Breast myopathies
The occurrence of breast muscle myopathies is shown in Table 4. The occurrence of breast muscle myopathies is shown in Table 4. No statistically significant treatment effect was detected for any of the evaluated myopathies. Overall, white striping was the most prevalent condition, accounting for 66.8% of the observed cases, followed by wooden breast (20.0%). Spaghetti meat was detected at relatively low frequencies across the experimental groups.
Effect of frass incorporation in the bedding at 0% (F0), 10% (F10), 20% (F20), and 30% (F30) on occurrence (mean ± standard error) of white striping, wooden breast, and spaghetti meat in the pectoralis major muscles of broiler chickens.
Item
Treatments
P-value
F0
F10
F20
F30
Pens, n
4
4
4
4
Without myopathy, %
20.0 ± 7.4
33.3 ± 8.8
23.3 ± 7.9
46.6 + 9.3
0.112
White striping, %
76.7 ± 7.9
66.7 ± 8.8
70.0 ± 8.5
53.3 ± 9.3
0.276
Wooden breast, %
23.3 ± 7.9
30.0 ± 8.5
20.0 ± 7.4
6.7 ± 4.6
0.106
Spaghetti meat, %
10.0 ± 5.6
13.3 ± 6.3
20.0 ± 7.4
6.7 ± 4.6
0.451
The pen was the experimental unit, while carcasses were individually identified and measured within pens (subsampling).
Slaughter traits and cut yields
Carcass traits and cut yields are depicted in Table 5. No significant differences were observed in CC weight or carcass yield among groups. No treatment effect was detected for breast, drumstick, and wing yields. In contrast, thigh yield was affected by treatment (P = 0.005), being higher in the F10 and F30 groups (17.1 and 17.2%, respectively) compared with F0 (15.7%).
Effect of frass incorporation in the bedding at 0% (F0), 10% (F10), 20% (F20), and 30% (F30) on carcass traits of broiler chickens.
Item
Treatments
Pooled SE
P-value
F0
F10
F20
F30
Pens, n
4
4
4
4
Cold carcass (CC) weight, g
2295
2181
2253
2186
39.2
0.167
Carcass yield, % slaughter weight
71.9
71.9
71.7
71.7
0.56
0.996
Breast yield, % CC
40.2
39.2
39.9
38.6
0.54
0.050
Breast meat yield, % CC
31.5
30.7
31.5
30.0
0.59
0.068
Thighs, % CC
15.7b
17.1a
16.7ab
17.2a
0.47
0.023
Drumsticks, % CC
13.2
13.0
13.1
13.3
0.22
0.563
Wings, % CC
9.51
9.57
9.52
10.1
0.41
0.374
Values are least-squares means. The pen was the experimental unit, while carcasses were individually identified and measured within pens (subsampling).a,b Values with different superscript letters within the same row differ significantly (P < 0.05).
Meat quality and femur fracture toughness
Table 6 presents the meat quality traits of breast and thigh muscles, and FFT as influenced by the litter treatments. No statistically significant differences were observed among groups in terms of breast meat L*a*b* color values, pHu, thawing and cooking losses, or meat toughness. No treatment effect was detected for thigh iliotibialis lateralis muscle traits including L*a*b* color values, pHu, and FFT.
Effect of frass incorporation in the bedding at 0% (F0), 10% (F10), 20% (F20), and 30% (F30) on breast (pectoralis major), thigh (iliotibialis lateralis) meat quality parameters and femur fracture toughness (FFT) of broiler chickens.
Item
Treatments
Pooled SE
P-value
F0
F10
F20
F30
Pens, n
4
4
4
4
Breast
Lightness (L*)
54.0
53.3
53.4
54.2
0.77
0.604
Redness (a*)
4.84
4.63
4.96
5.08
0.29
0.479
Yellowness (b*)
4.36
3.97
4.13
4.36
0.19
0.171
pHu
5.95
5.96
5.90
5.86
0.05
0.203
Thawing losses, %
11.0
11.0
12.5
13.3
0.01
0.153
Cooking losses, %
24.0
24.3
24.0
24.6
0.76
0.832
Shear force, kg/g
3.20
3.36
3.31
3.58
0.24
0.475
Thigh
Lightness (L*)
47.4
47.7
46.9
47.6
0.56
0.787
Redness (a*)
5.53
5.45
5.50
5.96
0.35
0.475
Yellowness (b*)
2.98
3.14
2.78
3.11
0.25
0.485
pHu
6.37
6.38
6.35
6.35
0.05
0.951
FFT, N
390
369
341
369
27.7
0.408
Values are least-squares means. The pen was the experimental unit, while carcasses were individually identified and measured within pens (subsampling).a,b Values with different superscript letters within the same row differ significantly (P < 0.05).
Discussion
The expansion of insect farming requires the exploration of sustainable applications for its by-products, particularly frass. Given that the quality of bedding material and litter quality directly affect poultry live performance, health, carcass traits, and welfare (de Jong et al., 2014; Diarra et al., 2021; Wilcox et al., 2024), it is of paramount importance to identify sustainable, but effective, bedding alternatives that can mitigate negative outcomes while supporting circular economy principles in agriculture.
To the best of our knowledge, this is the first study to investigate the use of insect frass as a bedding material in poultry farming, bridging a significant gap in the literature and providing valuable insights into the practical applications of insect farming by-products in poultry husbandry.
Frass supplementation up to 30% did not significantly alter litter moisture content at the end of the rearing period, with all experimental groups maintaining moisture levels below the critical 30% threshold associated with the development of FPD and other pododermatitis in poultry (Martland, 1984; 1985; Shepherd and Fairchild, 2010).
Counts of coagulase-positive Staphylococci remained below the detection limit and Salmonella spp. were absent (NR/25 g) from all samples analysed, confirming the microbiological safety of both bedding substrates. These results support the efficacy of the thermal treatment (70°C, 24 h) applied to frass before its utilisation. These findings indicate that the incorporation of frass up to the 30% level did not adversely affect the litter microbiological composition. Contrary to initial hypotheses, no reduction in litter microbial load was observed, suggesting that the antimicrobial peptides and lauric acid naturally present in insects (Borrelli et al., 2021; Xia et al., 2021) were either insufficient in quantity or inactivated by heat treatment.
It is well known that different litter types and amendments (e.g. superphosphate, meta-bisulfide, charcoal) can significantly affect Eimeria infection in poultry, providing a bed environment with more or less suitable conditions (e.g. humidity, Temperature, pH) for oocysts sporulation and survival (Soliman et al., 2018). However, no information is currently available concerning the effects of insect farming by-product (frass) on this concern. In this study, Eimeria infections were confirmed in the pool faecal samples from each treatment group, but with very high variability probably due to sampling effect, in the context of an uneven or aggregated distribution of parasites in the host population (McVinish and Lester, 2020). Results of oocyst counts in litter pool samples seem to indicate that no effect on litter contamination was related to different proportions of frass. This result may have been influenced by the fact that our analysis on pooled litter samples was performed only at 42 days, showing null or very few OPG values. On this concern, it has been proven that oocyst counts in litter of commercial poultry could be very low during the first or last weeks of broiler growth, while very high during the period from week 3 to 6 (Reyna et al., 1983). These results involve only preliminary observations, and further study is needed to better evaluate the effect of insect frass on Eimeria sporulation and survival.
Consistent with previous studies on alternative bedding materials (Toghyani et al., 2010; Kuleile et al., 2019; Durmuş et al., 2023; Duman et al., 2024), no significant differences in mortality were observed among groups. Despite the fine, sand-like texture of frass, no respiratory alterations or clinical symptoms indicative of respiratory distress were noted, suggesting that frass inclusion up to 30% did not compromise litter quality or chicken’s health.
The incorporation of insect frass into the bedding influenced growth performance primarily through its effect on DFI. Across the experimental period, broilers reared on frass-supplemented bedding consistently showed lower DFI compared with birds housed on conventional litter, indicating that the bedding composition itself can modulate feeding behavior. The effect on growth-related traits was more pronounced during the early phases of rearing, when differences in LW and DWG were detected, whereas during the later growing stages treatment-related effects were less evident. Overall, despite the attenuation of weekly differences over time, DWG over the entire production cycle remained affected by treatment, reflecting the cumulative effects associated with treatment-related differences in DFI. This outcome may be attributed to a behavioral mechanism. Poultry may consume litter, accounting for up to 4% of DFI Malone et al. (1983) and engage in foraging behaviors directly from the litter (Diarra et al., 2021). The presence of residual insect biomass in frass may have stimulated innate foraging behaviors (scratching, pecking, digging) typically elicited by nutrient-rich substrates like worms and insects (Belhadj Slimen et al., 2023). Earlier studies have reported increased feeding activity in poultry provided with live yellow mealworm larvae (Dalle Zotte et al., 2024). Although the majority of the frass had a sand-like texture, residual larval fragments were still present. While the larvae in the present study were non-viable due to heat treatment, their structural integrity may have been preserved, potentially triggering natural foraging responses and promoting greater activity. Additionally, poultry generally exhibit a preference for insect-containing diets due to their palatability and nutritional value (Cullere et al., 2016; Belhadj Slimen et al., 2023), which may have further reinforced these behaviors. These mechanisms could explain the reduced DFI and subsequent impacts on LW. In contrast, FCR was not influenced by the bedding treatments, suggesting that the observed changes in growth performance were mainly associated with differences in feed consumption rather than with alterations in feed utilization efficiency.
Beyond the specific effects of frass, previous studies have also demonstrated that bedding type itself can influence bird performance and feed intake. Previous studies have shown that birds perform differently depending on the type of bedding material used. Toghyani et al. (2010) reported lower feed intake in broilers reared on rice hulls, whereas higher intake was recorded in birds kept on other bedding types such as wood shavings, paper rolls, and sand, with the highest feed intake observed in birds raised directly on the floor without litter. In that study, the reduced DFI on rice hulls was also associated with lower LW. Durmuş et al. (2023), on the other hand, reported higher feed intake in broilers reared on rice hulls compared to those kept on sand, although no significant differences in LW were observed between the groups. In contrast, other authors found no significant differences in DFI or LW among birds raised on different bedding materials (Kuleile et al., 2019; Şen et al., 2023).
The incorporation of frass into the bedding material was initially hypothesized to reduce growth rate, potentially exerting a protective effect against the development of breast myopathies. Indeed, factors that limit growth rate or breast muscle accretion have been associated to a decreased myopathy occurrence (Lilburn et al., 2019; Caldas-Cueva and Owens, 2020). In the present study, although frass incorporation was associated with reduced birds growth performance, no treatment effect was detected for the occurrence of breast muscle myopathies. Consequently, no conclusions can be drawn regarding the influence of frass incorporation on the development of these conditions. Further studies, involving with larger sample sizes are needed to clarify whether frass-based bedding may play a role in modulating myopathy occurrence.
Although carcass yield, breast yield and breast meat yield did not differ significantly among treatments, thigh yield was higher in F10 and F30 compared with F0 group. This pattern may reflect growth-related trade-offs between muscle groups, and warrants further investigation into the underlying behavioral and physiological mechanisms. Rapid growth rates are genetically linked with increased breast muscle development (Zuidhof et al., 2014), often at the expense of other muscles, including the thigh (Santos et al., 2021).
Conclusions
The results of the present study indicate that yellow mealworm frass can be included in broiler litter at levels up to 30% without compromising litter quality or meat characteristics. However, a 30% inclusion rate appeared excessive, as significant reductions were observed in feed intake, final live weight, and daily weight gain. In contrast, 20% inclusion did not result in any significant differences compared with the control group.
These findings suggest that insect frass represents a promising alternative bedding material for broilers, although its optimal inclusion level requires further investigation. Future research should focus on evaluations of bird behavior, particularly litter foraging, litter consumption, and changes in daily activity patterns, to better understand the mechanisms underlying the performance responses observed in this study.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Organismo Preposto al Benessere degli Animali, OPBA, University of Padova, Italy. The study was conducted in accordance with the local legislation and institutional requirements.
The authors are grateful to James Caon, the owner of the INEF-Insect Novel Ecologic Food (Piombino Dese, Padova, Italy) for providing the frass used in this study. We thank Barbara Contiero for her assistance with the statistical analysis of the data.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Edited by: John Michael Gonzalez, University of Georgia, United States
Reviewed by: Servet Yalcin, Ege University, Türkiye
Rogers Ruyu Azabo, Sokoine University of Agriculture, Tanzania