Effects of Tenebrio molitor meal supplemented with Saccharomyces cerevisiae and/or Bacillus velezensis on production performance and egg quality in laying hens

Abstract

This study investigated whether probiotic supplementation (Bacillus velezensis and Saccharomyces cerevisiae) can enhance the nutritional value of mealworm (Tenebrio molitor) and improve performance and eggshell quality in 68-week-old Lohmann Selected Leghorn-Lite hens. A total of 180 hens were randomly assigned to five dietary treatments in a completely randomized design with six replicates per treatment: (1) basal diet (control); (2) basal diet + 2% mealworm powder (MW); (3) MW + 500 mL S. cerevisiae suspension (1 × 10⁸ CFU/mL; MW + SC); (4) MW + 500 mL B. velezensis suspension (1 × 10⁹ CFU/mL; MW + BV); and (5) MW + a mixture of 250 mL B. velezensis and 250 mL S. cerevisiae suspensions at the same concentrations (MW + MIX). Egg production, egg mass, feed conversion ratio, and internal egg quality (yolk index, egg shape index, Haugh unit, albumen weight, and yolk weight) were not significantly affected (P > 0.05). Egg weight increased in MW and MW + BV groups, while MW + SC slightly reduced it (P = 0.04). Eggshell weight and thickness were highest in the control and MW + SC groups and lowest in MW + MIX (P = 0.002 and P = 0.01, respectively). Overall, these results indicate that inclusion of 2% MW powder in the diets of aged laying hens did not adversely affect performance. The effects of probiotic supplementation were strain-dependent, and combined probiotic treatments did not demonstrate synergistic benefits, highlighting the need for careful selection and formulation of probiotic strains.

Introduction

The global poultry industry is increasingly challenged to adopt feed strategies that are both environmentally sustainable and supportive of long-term productivity and animal health (Pexas et al., 2023). Dependence on conventional protein sources, particularly soybean meal, has raised concerns related to environmental impact, competition with human food systems, and supply instability (Andretta et al., 2021; Song et al., 2021). In parallel, regulatory restrictions and consumer pressure to reduce antibiotic use have intensified interest in natural dietary alternatives capable of supporting gut health and performance without contributing to antimicrobial resistance (Abou-Jaoudeh et al., 2024; Acosta et al., 2025). These pressures underscore the need for innovative feed ingredients that simultaneously address sustainability, efficiency, and bird health.

Insect-derived proteins, especially those from yellow mealworm (Tenebrio molitor) larvae, have gained attention as a viable alternative protein source in poultry nutrition (Hong et al., 2020). T. molitor larvae are rich in high-quality protein (47–60%), lipids, and essential amino acids, while requiring fewer land and water resources than conventional protein crops (Hong et al., 2020; Sajid et al., 2023). In addition to their nutritional value, insect meals contain bioactive components such as chitin and antimicrobial peptides, which have been associated with immunomodulatory and gut health–related effects (Syahrulawal et al., 2023). However, the chitinous exoskeleton of insects can also act as an anti-nutritional factor, potentially reducing nutrient digestibility and lipid utilization when included at higher dietary levels (Selaledi et al., 2020). This limitation has constrained the broader application of insect meals in poultry diets.

One approach to improving the nutritional utilization of insect-derived feeds is the inclusion of probiotics with enzymatic capabilities. Probiotic species such as Saccharomyces cerevisiae and Bacillus velezensis are known to produce enzymes that can degrade complex polysaccharides, including chitin, while also supporting intestinal health, microbial balance, and nutrient absorption (Kang et al., 2025). In laying hens, S. cerevisiae supplementation has been associated with improved feed efficiency and eggshell quality, whereas B. velezensis has been shown to enhance intestinal morphology and calcium metabolism, both of which are critical for maintaining eggshell strength (Ye et al., 2020; Mazur-Kuśnirek et al., 2025). These effects are particularly relevant during the late laying phase, when hens experience age-related declines in nutrient utilization efficiency and eggshell quality due to reduced calcium absorption and compromised intestinal function (Gu et al., 2021).

Although insect meals and probiotics have been individually studied in poultry nutrition, their combined application, particularly in late-phase laying hens, remains poorly characterized. The extent to which probiotics may enhance the utilization of insect-derived nutrients, including alleviating chitin-related limitations, is not well understood, and evidence for additive or complementary effects on laying performance and egg quality is limited.

Therefore, the present study aimed to evaluate the effects of dietary inclusion of T. molitor larvae powder (MW), either alone or supplemented with S. cerevisiae and/or B. velezensis, on performance and egg quality parameters in late-phase laying hens, thereby addressing a specific knowledge gap regarding the combined application of insect-derived proteins and probiotics in this production phase.

Materials and methods

Animals, experimental design, and housing

All experimental protocols were conducted in compliance with animal welfare guidelines and were approved by the Animal Ethics Committee of Razi University (Kermanshah, Iran).

A total of 180 68-week-old Lohmann Selected Leghorn-Lite (LSL-Lite) laying hens were randomly distributed into five dietary treatments with six replicates per treatment in a completely randomized design (CRD). Each replicate consisted of two adjacent cages, with three birds housed per cage (45 × 45 × 45 cm), resulting in six birds per replicate. The experimental period lasted 56 days, from 68 to 76 weeks of age.

Hens were provided 110 g/bird/day of a mash diet and had ad libitum access to water. The photoperiod was maintained at 16 hours of light and 8 hours of darkness. Ambient temperature and relative humidity were consistently regulated within ranges of 18–24°C and 40–45%, respectively.

Rearing and processing of yellow mealworm larvae for poultry diet

Yellow mealworm larvae were cultivated in 100 plastic trays (11 × 27 × 40 cm) containing a substrate of wheat bran and barley flour. They were maintained in a climate-controlled room at 25 ± 1 °C and 50 ± 5% relative humidity. A photoperiod of 12-h light/12-h dark was maintained. Moisture requirements were met by providing vegetables such as cabbage, lettuce, and carrots.

Beetles, larvae, and pupae were reared separately. Pupae were collected daily and placed in isolated trays to mature into adults (8–9 days). Dead beetles were regularly removed. The rearing substrate was replaced monthly to remove waste.

Larvae were reared for 16–18 weeks until reaching maturity. Prior to pupation, they were separated from the substrate and other life stages using a sieve. To empty the gut content, larvae were subjected to a 48-hour fasting period (gut-cleaning). They were then frozen at −21 °C for 48 hours to euthanize them.

Larvae were dried either using sunlight or in an oven at 60 °C for 24 hours. The dried larvae were ground into a powder. This powder was subsequently analyzed for proximate composition (crude protein, ether extract, crude fiber, ash, chitin, calcium, and phosphorus) according to AOAC (2005) methods. The analyzed components included dry matter (Method 934.01), crude protein (nitrogen × 6.25; Method 984.13), ash (Method 942.05), crude fiber (Method 978.10), and ether extract (Method 920.39). Besides, by the following formula (ash-free acid etergent fibre [%] − acid detergent insoluble protein [%]), the chitin content was calculated (Marono et al., 2015; Table 1).

Table 1.

Chemical analysis of Tenebrio molitor larvae meal (% Dry matter basis).

Component	% Dry matter
Dry matter	97.02
Crude protein	53.81
Ether extract	28.03
Crude ash	6.99
Crude fiber	7.53
Chitin	5.60
Calcium	3.50
Phosphorus	6.80

Preparation of bacterial and yeast suspensions

To investigate the effects of dietary supplementation with S. cerevisiae and B. velezensis in a MW-based diet, the microbial strains were procured from the culture collection of the Department of Plant Pathology (Razi University, Kermanshah, Iran).

A full inoculating loop of a 48-hour pure culture of B. velezensis, grown on Nutrient Agar (Merck, Darmstadt, Germany), was aseptically transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterile Nutrient Broth (Oxoid, Basingstoke, UK). The culture was incubated for 48 hours at 21 °C in an incubator shaker (New Brunswick Scientific Innova® 44, Edison, NJ) set at 120 rpm. Following incubation, bacterial cells were harvested by centrifugation at 6000 × g for 10 min at 4°C using a refrigerated centrifuge (Eppendorf 5804 R, Hamburg, Germany). The pellet was washed twice with 0.14 molar sterile physiological saline (NaCl, Merck, Darmstadt, Germany) to remove residual medium constituents. The final pellet was resuspended in saline, and the optical density was measured at 600 nm using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). Based on a previously established OD₆₀₀–CFU calibration curve, the bacterial suspension was adjusted to a final concentration of 1 × 10⁹ CFU/mL, as specified in the experimental design.

For the yeast suspension, S. cerevisiae was cultured on Potato Dextrose Agar (PDA; Difco, Detroit, MI) plates for 7 days at 25 °C to induce sporulation. Yeast cells were harvested by flooding the agar surface with 10 mL of sterile distilled water and gently scraping with a sterile loop. The resulting suspension was filtered through sterile cheesecloth to remove hyphal debris. The yeast cell concentration was determined using an improved Neubauer hemocytometer (Marienfeld, Lauda-Königshofen, Germany) and subsequently adjusted with sterile distilled water to obtain a final concentration of 1 × 10⁸ CFU/mL.

Enumeration of microbial load in feed

To confirm the viable microbial counts in the inoculated feeds, the number of B. velezensis and S. cerevisiae cells was determined using the serial dilution and spread-plate method. Briefly, 1 g of feed was aseptically transferred into 9 mL of sterile 0.1% peptone water (Oxoid, Basingstoke, UK) and homogenized for 5 min using a vortex mixer (IKA® MS 3 digital, Staufen, Germany). Ten-fold serial dilutions were prepared up to 10⁻⁷.Aliquots (100 µL) from appropriate dilutions were spread-plated in duplicate onto Nutrient Agar for bacterial enumeration and PDA for yeast enumeration. Plates were incubated at 30°C for 24–48 h, after which colonies were counted manually. Only plates containing 30–300 colonies were considered for analysis, and results were expressed as CFU/g of feed.

Dietary treatments and formulation

The isoenergetic and isonitrogenous experimental diets were: (1) basal diet (control); (2) basal diet + 2% MW; (3) MW + 500 mL of S. cerevisiae suspension (1 × 10⁸ CFU/mL, MW + SC); (4) MW + 500 mL of B. velezensis suspension (1 × 10⁹ CFU/mL, MW + BV); and (5) MW + 250 mL B. velezensis + 250 mL S. cerevisiae suspension (MW + MIX).

The microbial suspensions were sprayed evenly onto the feed and mixed thoroughly to ensure uniform distribution. All inoculated feeds were prepared fresh and air-dried at room temperature prior to feeding to avoid excessive moisture accumulation.

The diets were formulated to meet the nutritional specifications for laying hens as outlined by the LSL-Lite management guide and the NRC 1994. The complete formulation and chemical composition of the basal diet are presented in Table 2. The proximate composition of the experimental diets was analyzed in accordance with the official methods of AOAC (2005).

Table 2.

Ingredients and chemical composition of the experimental diets.

Ingredients (%)	Control	Mealworms
Corn	61.65	60.18
Soybean meal	24.15	21.23
Mealworm	-	2.00
Bacillus velezensis	-	0.05
Saccharomyces cerevisiae	-	0.05
Wheat bran	-	2.29
Oil	0.36	0.36
Calcium carbonate	4.00	4.00
Dicalcium phosphate	1.76	1.71
Oyster	6.34	6.37
Salt	0.38	0.37
Vitamin mixture	0.3	0.3
Mineral mixture	0.3	0.3
Sodium bicarbonate	0.1	0.1
Toxin binder	0.1	0.1
Acidifier	0.1	0.1
DL-Methionine	0.28	0.3
L-Lysine HCl	0.18	0.19
Nutrient analysis (g/kg)
Metabolisable energy (kcal/kg)	2650	2650
Crude protein	16	16
Sodium	0.17	0.17
Threonine	0.03	0.03
Methionine	0.11	0.15
Calcium	4.4	4.4
Available phosphorus	0.45	0.45

^a Vitamin premix provided per kilogram of diet: vitamin A, 7700 IU; vitamin D₃, 3300 IU; vitamin E, 6.6 mg; vitamin K₃, 0.55 mg; thiamine, 2.2 mg; riboflavin, 4.4 mg; vitamin B₆, 4.4 mg; calcium pantothenate, 0.55 mg; nicotinic acid, 0.2 mg; folic acid, 0.11 mg; choline chloride, 275 mg; biotin, 0.055 mg; vitamin B₁₂, 0.0088 mg. ^b Mineral premix provided per kilogram of diet: Mn, 66 000 mg; Zn, 66 000 mg; Fe, 33 000 mg; Cu, 8800 mg; Se, 300 mg.

Measurements

Productive performance

Feed intake (FI) was recorded weekly on a per-replicate basis, while the number of eggs produced and individual egg weight (EW) were recorded daily. From these data, egg mass (EM) was calculated as the product of mean EW and the hen-day egg production percentage (EP). The feed conversion ratio (FCR), expressed as g of feed per g of egg, was derived by dividing the total FI by the total EM produced per replicate.

Egg quality traits

To evaluate egg quality traits at the end of the experimental period, a total of 24 eggs per treatment were randomly selected, corresponding to two eggs collected from each replicate over two consecutive days. Each egg was individually numbered and transported to the laboratory for analysis. The weight of each egg was determined to an accuracy of 0.01 g using an analytical balance (Model AX324, Ohaus Corp., Parsippany, NJ). A comprehensive set of internal and external quality parameters was assessed.

Albumen quality was characterized by measuring the Haugh Unit and albumen weight. The Haugh Unit was calculated using the established formula: HU = 100 × log (H + 7.6 − 1.7W^0.37) (Eisen et al., 1962), where H represents the height of the thick albumen, measured in millimeters with a tripod micrometer (Mitutoyo, 0.01 mm, Kawasaki, Japan), and W is the weight of the egg in g. The albumen weight was recorded after its careful separation from the yolk and shell.

Yolk quality was evaluated based on the yolk index and yolk weight. The yolk index, defined as the ratio of yolk height to its diameter, was determined using a digital caliper (Model 500-196-20, Mitutoyo Corp., Kawasaki, Japan). The yolk was then separated and weighed.

Shell quality was determined by measuring shell percentage, and shell thickness. The shell percentage was calculated as (shell weight / whole EW) × 100; prior to weighing, shells were washed and air-dried for 48 hours at ambient temperature. Shell thickness was measured to the nearest 0.01 mm using a dial and pipe gauge (Ozaki MFG. Co., Tokyo. Japan) at three distinct points on the egg: the sharp end, the blunt end, and the equator. The average of these three measurements was used for subsequent analysis.

Following measurement of the egg's length and width with a compass (Swordfish, 0.02 mm, Zhejiang, China), the egg shape index was calculated using the formula (width/length) × 100.

Statistical analysis

The data were initially processed using Microsoft Excel 2013 (Microsoft Corp., Redmond, WA). All statistical analyses were performed using the GLM procedure of SAS software, version 9.4 (SAS Institute, 2015). Treatment means were compared using Duncan's multiple range test at a significance level of P ≤ 0.05. The following statistical model was used:

Y_ij = µ + T_i+ e_ij

Where:

• •Y_ij is the individual observation,
• •µ is the overall population mean,
• •T_i is the fixed effect of the ith dietary treatment,
• •eij is the random residual error, assumed to be normally and independently distributed with a mean of zero and constant variance.

Results

Production performance

The effects of dietary treatments on laying hen performance are summarized in Table 3. Overall, dietary interventions had little effect on EP, EM, or FCR (P > 0.05). However, a clear trend was observed for EW. Hens fed MW or MW + BV diets from 68 to 76 weeks of age tended to produce heavier eggs, whereas the control and MW + SC groups showed slightly lighter eggs (P = 0.04), suggesting a potential positive influence of MW and B. velezensis on EW during the late production phase.

Table 3.

Effects of Tenebrio molitor meal and probiotic supplementation on productive performance in laying hens.

Period	Control	MW	MW + SC	MW + BV	MW + MIX	P value	SEM	CV (%)
			Egg production (%)
68-72wk	71.92 ± 7.59	69.04 ± 6.16	72.71 ± 9.09	74.79 ± 7.95	69.34 ± 9.35	0.71	3.31	11.33
72-76wk	79.56 ± 7.68	79.95 ± 5.64	81.64 ± 7.19	79.36 ± 20.91	79.45 ± 11.27	0.99	4.91	15.04
68-76wk	75.00 ± 7.38	72.99 ± 7.81	77.17 ± 8.10	77.21 ± 12.12	74.40 ± 10.02	0.91	3.78	12.28
				Egg weight (g)
68-72wk	60.73 ± 1.36	65.38 ± 6.57	60.02 ± 1.94	61.89 ± 1.38	60.96 ± 2.07	0.07	1.35	5.37
72-76wk	60.91 ± 1.07	62.86 ± 1.69	61.48 ± 1.56	63.03 ± 1.19	62.81 ± 2.22	0.10	0.65	2.58
68-76wk	60.83 ± 1.09b	64.65 ± 4.03a	60.79 ± 1.74b	62.52 ± 1.14a	61.82 ± 2.21ab	0.04	0.94	3.72
			Egg mass (g/hen/day)
68-72wk	43.60 ± 3.75	42.74 ± 2.84	43.51 ± 4.20	46.35 ± 5.50	42.19 ± 5.10	0.53	1.79	10.04
72-76wk	46.42 ± 5.40	51.52 ± 4.99	50.12 ± 3.52	50.14 ± 13.5	49.67 ± 6.66	0.82	3.12	15.45
68-76wk	45.58 ± 3.98	46.47 ± 2.44	46.82 ± 3.83	49.61 ± 7.91	45.93 ± 5.76	0.68	2.10	10.97
			Feed conversion ratio
68-72wk	2.53 ± 0.21	2.57 ± 0.17	2.54 ± 0.24	2.30 ± 0.15	2.65 ± 0.34	0.14	0.09	9.36
72-76wk	2.39 ± 0.26	2.14 ± 0.19	2.19 ± 0.15	2.44 ± 1.15	2.25 ± 0.34	0.86	0.22	24.59
68-76wk	2.43 ± 0.19	2.36 ± 0.12	2.35 ± 0.19	2.27 ± 0.46	2.42 ± 0.35	0.88	0.12	12.47

Diets: Control (basal diet); MW (basal diet + 2% Tenebrio molitor meal); MW + SC (MW + 500 mL Saccharomyces cerevisiae); MW + BV (MW + 500 mL Bacillus velezensis); MW + MIX (MW + 250 mL S. cerevisiae + 250 mL B. velezensis). Performance: 6 pens/treatment (6 hens/pen). Values are presented as mean ± SD. Different superscript letters within a row indicate significant differences (P ≤ 0.05).

Egg quality traits

The effects of the dietary supplements on internal and external egg quality parameters are presented in Table 4. Internal egg quality parameters, such as egg shape index, yolk index, albumen weight, yolk weight, and Haugh unit, were generally consistent across all dietary treatments (P > 0.05). In contrast, external quality traits showed more noticeable trends. Shell weight and shell thickness appeared to decline in hens receiving the combined MW + MIX supplementation, while the control and MW + SC groups maintained stronger shells (P = 0.002 and P = 0.01, respectively).

Table 4.

Effects of Tenebrio molitor meal and probiotic supplementation on egg quality characteristics of laying hens.

	Control	MW	MW + SC	MW + BV	MW + MIX	P value	SEM	CV (%)
Haugh unit	78.44 ± 6.39	79.69 ± 5.27	80.38 ± 7.58	79.38 ± 5.82	81.66 ± 4.86	0.44	1.23	7.58
Egg shape index (%)	74.82 ± 2.77	74.56 ± 2.34	73.99 ± 2.13	74.52 ± 1.62	74.82 ± 3.12	0.76	0.50	3.29
Yolk index (%)	30.93 ± 2.10	32.79 ± 2.53	31.53 ± 3.51	30.97 ± 2.15	31.83 ± 2.40	0.08	0.52	8.20
Shell weight (g)	10.58 ± 0.51a	10.26 ± 0.64abc	10.43 ± 0.76ab	10.08 ± 0.69abc	9.91 ± 0.46c	0.002	0.12	6.11
Shell thickness (10-2 mm)	0.41 ± 0.017a	0.40 ± 0.018ab	0.41 ± 0.026a	0.40 ± 0.023ab	0.39 ± 0.018b	0.01	0.004	5.23
Albumen weight (g)	34.53 ± 3.36	34.65 ± 3.18	35.22 ± 4.08	35.46 ± 3.67	37.12 ± 2.96	0.08	0.70	9.82
Yolk weight (g)	19.59 ± 3.33	18.92 ± 1.87	18.63 ± 1.86	20.00 ± 1.67	18.75 ± 1.96	0.15	0.45	11.60

Diets: Control (basal diet); MW (basal diet + 2% Tenebrio molitor meal); MW + SC (MW + 500 mL Saccharomyces cerevisiae); MW + BV (MW + 500 mL Bacillus velezensis); MW + MIX (MW + 250 mL S. cerevisiae + 250 mL B. velezensis). Egg quality: 2 eggs/replicate over 2 days (n = 24). Values are presented as mean ± SD. Different superscript letters within a row indicate significant differences (P ≤ 0.05).+.

Discussion

Productive performance

The present study evaluated the effects of dietary supplementation with MW powder, either alone or in combination with B. velezensis and S. cerevisiae, on production performance and egg quality in 68-week-old LSL-Lite laying hens. Overall, inclusion of 2% MW, with or without probiotic supplementation, did not significantly influence EP, EM, or FCR, indicating that MW powder can be incorporated into late-lay diets without compromising production performance. The limited response in feed efficiency may, in part, reflect the influence of feed physical characteristics and processing conditions, such as texture and conditioning parameters, which are known to modulate nutrient availability and digestive efficiency and may attenuate the measurable effects of dietary additives (Iravani et al., 2024).

The generally modest effects of probiotic supplementation observed in this study are consistent with reports in post-peak laying hens and suggest that advanced hen age may constrain the responsiveness to nutritional interventions. Several studies have reported no significant effects of S. cerevisiae supplementation on EP, EM, or FCR in older hens (Liu et al., 2021; Gong et al., 2025), whereas beneficial responses have been observed more frequently in younger or mid-lay birds (Qiu et al., 2024). Although Zhang et al. (2020) reported improved laying performance in 67-week-old hens, such outcomes appear to be context-dependent and influenced by factors including strain characteristics, dosage, and diet composition. Meta-analytical evidence further highlights substantial variability in probiotic efficacy, even within the same species, reflecting differences in fermentation processes, metabolite profiles, and host physiological status (Sjofjan et al., 2021).

Similarly, the absence of performance responses to B. velezensis supplementation contrasts with modest improvements reported in younger laying hens (Ye et al., 2020) and may be attributed to age-related alterations in gut microbiota composition and reduced intestinal plasticity in older layers (Li et al., 2023; Yu et al., 2025). Although B. velezensis is known to support gut health through mechanisms such as competitive exclusion, enzyme secretion, and immune modulation, these pathways may be less effective under conditions of reduced intestinal adaptability. Importantly, the absence of additive or synergistic effects in the MW + MIX treatment does not support the original hypothesis that combined MW and probiotic supplementation would enhance performance beyond individual interventions. Instead, this outcome suggests overlapping modes of action between B. velezensis and S. cerevisiae, potentially leading to competition for intestinal niches or convergence on similar physiological pathways related to gut function and nutrient utilization (Suarez et al., 2012). Such interactions are likely exacerbated in late-lay hens, where nutrient bioavailability and intestinal adaptability are already constrained (Attia et al., 2020).

The effects of insect-based ingredients on laying performance remain inconsistent across studies, largely due to differences in processing methods, inclusion levels, basal diet composition, and bird age. While Sedgh-Gooya et al. (2021) reported improvements in EM, EP, and FCR with T. molitor supplementation, other studies observed no significant effects on laying rate at comparable or lower inclusion levels (Ko et al., 2020; Ait-Kaki et al., 2022). These discrepancies underscore the importance of standardized processing techniques and age-specific evaluations when assessing the nutritional value of insect meals in layer diets.

In contrast to performance traits, EW was positively influenced by MW supplementation. In the present study, MW alone and MW combined with B. velezensis significantly increased EW compared with both the control and MW + SV treatments. This response may be explained by the high crude protein content of MW and its balanced essential amino acid profile, which can support yolk and albumen deposition and thereby increase EM (Stastnik et al., 2021). Reported effects of probiotics and insect meals on EW remain variable. Ye et al. (2020) observed increased EW following B. velezensis supplementation in mid-lay hens, potentially due to improvements in intestinal morphology and protein digestion. In contrast, responses to yeast supplementation are inconsistent, with studies reporting positive, neutral, or negative effects depending on yeast form, dosage, and hen age (Özsoy et al., 2018; Liu et al., 2021; Gong et al., 2025). The reduced EW observed in the MW + SV group in the present study aligns with these variable findings and suggests that yeast supplementation may not uniformly enhance egg formation in older hens. Similarly, insect meal inclusion has produced mixed outcomes, with several studies reporting no significant effects on EW at inclusion levels comparable to those used here (Ko et al., 2020; Sedgh-Gooya et al., 2021). Collectively, these inconsistencies indicate that responses to MW and probiotic supplementation are highly context-dependent and influenced by additive type, dosage, hen age, and dietary composition, reinforcing the need for targeted strategies rather than assuming synergistic benefits of combined interventions (Jensen et al., 2008).

Egg quality traits

In the present study, internal egg quality parameters, including yolk index, egg shape index, Haugh unit, and albumen and yolk weights, were not significantly influenced by dietary supplementation with MW, probiotics, or their combination. This stability is noteworthy given that albumen height and Haugh unit typically decline with advancing hen age (Anene et al., 2021). The absence of treatment-related deterioration indicates that neither MW inclusion nor probiotic supplementation compromised egg compositional integrity in post-peak laying hens. However, these findings should be interpreted cautiously, as they reflect maintenance rather than enhancement of egg quality.

The lack of treatment effects contrasts with studies reporting positive impacts of yeast or bacterial probiotics on albumen-related traits. For example, Gong et al. (2025) observed that 60-week-old hens fed S. cerevisiae fermentation products had significantly higher Haugh units and albumen height compared with controls, while Liu et al. (2021) similarly reported improvements with yeast culture supplementation. Other investigations also highlighted beneficial effects of yeast or yeast extracts on internal egg quality (Zhong et al., 2016; Özsoy et al., 2018; Gaboardi et al., 2019). These improvements are generally attributed to bioactive compounds in yeast, including enzymes, vitamins, amino acids, polypeptides, and oligosaccharides, which may enhance protein metabolism and albumen deposition (Jensen et al., 2008). Conversely, several studies corroborate the present results by showing no significant effects of yeast supplementation on albumen height or Haugh unit (Yalçin et al., 2008; Zhang et al., 2020). Such inconsistencies likely reflect differences in yeast strain, dosage, or hen age, with evidence suggesting that the efficacy of yeast-based supplements diminishes in post-peak layers.

Similarly, variable outcomes have been reported for Bacillus-based probiotics. Positive effects on Haugh unit, yolk weight, and albumen height have been documented in younger or mid-lay hens (Forte et al., 2016; Mazanko et al., 2018; Yang et al., 2020; Ye et al., 2020). These responses are often linked to enhanced gut microbial balance and improved digestive enzyme activity, which can facilitate nutrient absorption. The lack of comparable effects in the current study suggests that probiotic-mediated mechanisms may be less effective in older hens or that the applied strains and dosages were insufficient to induce detectable changes in egg quality traits.

In contrast to probiotics, existing evidence indicates that insect meal supplementation generally exerts minimal influence on internal egg quality. Studies by Ko et al. (2020), Shariat Zadeh et al. (2020), and Sedgh-Gooya et al. (2021) reported no significant effects of T. molitor meal on albumen height or Haugh unit, consistent with the present findings. These results suggest that MW can be used as an alternative protein source in layer diets without negatively affecting internal egg quality, though its capacity to enhance such traits appears limited.

Eggshell weight and thickness were significantly affected by dietary treatments, with the control group showing superior shell quality compared with the MW plus MIX group. The reduction in shell quality observed with combined supplementation may be associated with altered mineral bioavailability. In late-phase laying hens, age-related declines in calcium absorption and trace mineral utilization increase sensitivity to dietary factors that affect mineral solubility and uptake (Gu et al., 2021; Alfonso-Carrillo et al., 2021). Trace minerals such as zinc, manganese, and copper are essential for eggshell matrix formation and calcium carbonate deposition, and reduced availability can impair shell integrity (Gu et al., 2021; Sinclair-Black et al., 2023). Dietary components with mineral-binding or chelating properties have been shown to compromise mineral absorption and eggshell quality in older hens (Nejad et al., 2023). Accordingly, the combined inclusion of MW-derived components and multiple probiotics may have modified mineral interactions in the gut, limiting mineral availability for shell formation.

Evidence from the literature suggests that probiotics may both support and impair shell traits. Some studies reported positive effects of Bacillus-based probiotics, such as improved shell weight and thickness through enhanced calcium absorption and retention (Li et al., 2013; Forte et al., 2016; Mazanko et al., 2018; Ye et al., 2020). However, Khogali et al. (2022) observed reduced shell thickness with Clostridium butyricum and B. subtilis, highlighting the potential for negative outcomes depending on probiotic species, dosage, and interactions.

In the present study, shell thickness was maintained in the S. cerevisiae-only group at levels comparable to the control, suggesting that single probiotic supplementation may be more effective than combined approaches in supporting eggshell quality in older laying hens. This effect may be related to the functional role of S. cerevisiae in promoting gastrointestinal health and intestinal integrity, thereby facilitating more efficient mineral utilization (Gong et al., 2025). Yeast-derived components, particularly cell wall polysaccharides such as beta-glucans and mannan oligosaccharides, have been shown to support gut barrier function, modulate microbial balance, and enhance the absorptive capacity of the intestine (Gong et al., 2025; Vakili et al., 2025), which are critical factors for calcium uptake and eggshell formation. Similar findings have been reported by Liu et al. (2021), Gong et al. (2025), and Hassanabadi et al. (2025), who attributed improvements in shell strength, thickness, and shell percentage to enhanced nutrient digestibility and calcium absorption following yeast supplementation. Beyond mineral absorption, S. cerevisiae may contribute to broader digestive and detoxification-support processes by binding undesirable luminal compounds and reducing intestinal stress, thereby indirectly preserving calcium availability for shell deposition (Vakili et al., 2025).

The combination of MW and probiotics did not yield improvements in shell traits, despite the mineral contribution of MW. This lack of additive effect may reflect overlapping modes of action between B. velezensis and S. cerevisiae, potentially resulting in competition for intestinal niches or convergence on similar physiological pathways affecting gut function and nutrient utilization, as discussed earlier (Suarez et al., 2012). Additionally, the chitin present in insect exoskeletons may bind minerals and reduce their bioavailability (Selaledi et al., 2020), and probiotic supplementation at the tested levels may have been insufficient to counteract this effect. Findings for insect meal inclusion remain similarly inconsistent. Ko et al. (2020), Shariat Zadeh et al. (2020), and Sedgh-Gooya et al. (2021) reported no significant effects of MW on shell thickness or hardness, whereas full-fat Hermetia illucens larvae meal has been associated with decreased shell thickness when used to completely replace soybean meal (Mancini et al., 2025). These results underscore the importance of processing method, nutrient composition, and inclusion level in shaping shell quality outcomes.

Conclusions

In conclusion, dietary inclusion of 2% MW powder, alone or combined with B. velezensis and S. cerevisiae, did not adversely affect production performance or internal egg quality in 68-week-old LSL-Lite hens. However, the anticipated synergistic effects of combining MW with probiotics were not observed, likely due to overlapping physiological functions of the additives and age-related limitations in intestinal responsiveness and mineral utilization, which may constrain calcium absorption and eggshell formation in older hens. Egg weight increased with MW alone and MW + BV, while MW + SC reduced egg weight; the control group maintained superior eggshell thickness and shell weight compared with the MW + MIX treatment. Future studies should explore optimized MW inclusion levels, nutritional strategies to support shell quality, and the mechanistic basis of probiotic interactions through longer-term trials.

CRediT authorship contribution statement

Bahareh Yalveh: Writing – original draft, Investigation, Formal analysis, Data curation. Mehran Torki: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. Maryam Darbemamieh: Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Kianoosh Cheghamirza: Validation, Software, Methodology, Formal analysis, Data curation. Rouhallah Sharifi: Writing – review & editing, Validation, Resources, Data curation.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.