Combined Bifidobacterium bifidum and Bacillus toyonensis supplementation in drinking water improves productive efficiency, physiological status, and meat quality in broiler chickens

Abstract

Developing combined, multistrain probiotic strategies in drinking water represents a promising approach to improve broiler productivity, health, and economic returns under intensive farming. This study evaluated the effects of supplementing broiler drinking water with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination on growth performance, physiological status, meat quality, and economic efficiency. A total of 320 one-day-old, unsexed IR broiler chicks were randomly allocated to four treatments with eight replicates of 10 birds each: an unsupplemented control, BB (0.5 g/L; 5 × 10⁸ cfu/L), BT (0.5 g/L; 5 × 10⁸ cfu/L), and BB+BT (1.0 g/L; 5 × 10⁸ cfu/L of each probiotic). Birds receiving probiotics exhibited improved growth performance compared with the control. The combined BB+BT treatment produced the greatest advancements in final body weight, feed conversion ratio, and European Production Efficiency Factor. Carcass yield was unaffected by treatments. However, probiotic supplementation improved meat proximate composition, oxidative stability, color characteristics, tenderness, and juiciness, without affecting muscle pH or water-holding capacity. Hematological indices, including hemoglobin concentration, red and white blood cell counts, and packed cell volume, were optimized by probiotic supplementation, whereas erythrocyte indices remained unchanged. The combined treatment also increased serum total protein, reduced circulating transaminases, lowered total cholesterol and low-density lipoprotein cholesterol, and enhanced immune and antioxidant status through higher immunoglobulins (G and M) and superoxide dismutase activity, together with lower malondialdehyde concentration. Despite the additional supplementation cost, the combined probiotic treatment generated the greatest economic return. Overall, combined supplementation with B. bifidum and B. toyonensis via drinking water was associated with improved productive performance, physiological status, and meat quality compared with individual supplementation or the control.

Graphical abstract

Introduction

Broiler production is a major livestock sector that provides affordable, high-quality animal protein. Growing demand for poultry meat has increased the need to improve feed utilization, flock health, meat quality, and profitability (Abd El-Hack et al., 2022; Mulyono, 2025). However, the widespread use of antibiotic growth promoters (AGP) has been curtailed due to concerns about antimicrobial resistance and antibiotic residues in poultry products. (Mulyono, 2025; Yu, 2026). Consequently, diverse sustainable and non-antibiotic approaches are being explored in poultry production, including carbon-based feed additives (Zahed et al., 2025), plant-derived feed additives (Niknia et al., 2024), and microbial feed additives such as probiotics (Wang et al., 2025). Probiotics are now recognized as suitable alternatives because they can enhance productivity, support gut integrity, promote production sustainability, and reduce reliance on antibiotics (Abd El-Hack et al., 2022; Mulyono, 2025). The effectiveness of probiotics depends on the strain and management conditions; however, they generally help maintain gut microbiota, enhance immunity, strengthen intestinal barrier integrity, support intestinal architecture, and increase nutrient digestibility (Jha et al., 2020; Yosi and Metzler-Zebeli, 2023).

The significance of evaluating targeted microbial candidates relies on their distinct biological properties. B. bifidum has received considerable attention because it is a natural member of the intestinal microbiota. It can ferment indigestible carbohydrates, produce organic acids, and inhibit pathogen colonization (Zhang et al., 2025). In broilers, supplementation with BB has been associated with beneficial effects on growth and feed utilization, antioxidant status, immunoglobulin synthesis, intestinal morphology, and beneficial microbial populations (Abdel-Moneim et al., 2020; Wang et al., 2025). Its postbiotic compounds strengthen gut epithelial barrier function, regulate inflammatory responses, and restore gut microbial balance under disease conditions (Chen et al., 2025b). Concurrently, B. toyonensis is another promising probiotic, and Bacillus-based products are widely recognized as potential substitutes for AGP in poultry (Qiu et al., 2021; Yu, 2026). Bacillus spp. stabilize the gut microbial community, suppress pathogenic bacteria, improve nutrient utilization, and support immune homeostasis (Naeem and Bourassa, 2025; Liu et al., 2026). Fortification of poultry feeds with Bacillus has been associated with improved growth performance, feed efficiency, meat quality, and economic returns, as well as enhanced gut integrity and immunity (Qiu et al., 2021; Alqahtani et al., 2024). It may also improve antioxidant and digestive functions, increase short-chain fatty acid production (SCFA), and reduce lipid peroxidation and inflammatory responses (Zhang et al., 2021; Liu et al., 2024). Combining BB with BT is highly significant because the two probiotics perform complementary functions in different compartments of the gastrointestinal tract. As an obligate anaerobe, BB supports intestinal homeostasis through carbohydrate fermentation, gut microbiota modulation, and immune regulation (Gavzy et al., 2023; Chen et al., 2025b). In contrast, B. toyonensis, a spore-forming aerobic/facultatively anaerobic bacterium, improves enzymatic digestion, digestibility, intestinal morphology, and epithelial barrier integrity (Wang et al., 2021; Yosi and Metzler-Zebeli, 2023). This dual-action framework is hypothesized to generate enhanced physiological benefits compared with single-strain formulations (Mirsalami and Mirsalami, 2024).

Despite these documented benefits, a distinct knowledge gap remains regarding the separate and combined impacts of BB and BT administered specifically via drinking water in IR broiler chicks. Consequently, the aim of the present study was to evaluate the effects of BB, BT, and their combination in drinking water on broiler growth performance, blood biochemical profiles, meat quality characteristics, and overall economic efficiency. The underlying hypothesis was that the combined drinking water administration would yield enhanced productive and physiological advantages due to the complementary live microbial traits.

Materials and methods

The experimental work was carried out at the Poultry Research Farm, Department of Animal and Poultry Production, Faculty of Technology and Development, Zagazig University, Zagazig, Egypt, during September and October 2024. All experimental procedures involving birds were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC-ZU-2026) of Zagazig University.

Probiotic preparation and administration

Pure cultures of Bifidobacterium bifidum and Bacillus toyonensis were prepared, propagated, harvested, freeze-dried, and standardized for viable counts under laboratory conditions at the Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, Zagazig, Egypt. The preparations were standardized to 1 × 10⁹ cfu/g, using maltodextrin as the carrier for B. bifidum and calcium carbonate for B. toyonensis. Each preparation was administered in drinking water at 0.5 g/L, corresponding to an initial viable dose of 5 × 10⁸ cfu/L. Fresh probiotic solutions were prepared daily before administration using chlorine- and disinfectant-free drinking water, and provided through drinking equipment protected from direct sunlight to ensure maximum bacterial viability. The drinking water was provided to the birds at ambient house temperature.

Experimental design, birds, and management

A total of 320 healthy, newly hatched, unsexed IR broilers (Tiba Poultry Grandparents, Al-Salheya Al-Gadida, Sharkia, Egypt) with similar initial live weights (approximately 42 g) were employed in this trial. Upon arrival, birds were individually weighed and randomly allocated to 32 wire cages (100 × 60 × 60 cm), ensuring similar average initial weights among cages. The cages (replicates) were then allocated at random to 4 drinking water supplementation groups, with 8 replicates for each treatment group. The treatments were as follows: (1) control group receiving probiotic-free drinking water; (2) drinking water supplied by Bifidobacterium bifidum (BB) at 0.5 g/L (5 × 10⁸ cfu/L); (3) drinking water supplied by Bacillus toyonensis (BT) at 0.5 g/L (5 × 10⁸ cfu/L); and (4) drinking water supplied by their equal combination (BB+BT) at 1.0 g/L (5 × 10⁸ cfu/L of each probiotic)

The experiment was conducted in an open-sided broiler house, where standard cleaning, disinfection, and general biosecurity measures were implemented before and throughout the experimental period to maintain hygienic conditions and minimize the risk of pathogen introduction. Prior to chick placement, the facility was thoroughly cleaned by removing accumulated manure and debris, followed by high-pressure washing with a 2% detergent solution and room disinfection using 4% formaldehyde mist. General measures were also implemented to limit the entry of rodents and insects and to maintain appropriate farm hygiene. During the 35-day trial, chicks were housed under similar environmental and husbandry conditions. House temperature was initially maintained at 32 ± 2°C on day 1 and progressively reduced by approximately 2–3°C weekly to reach 22–24°C by week 4, after which it remained constant until the end of the experiment. Relative humidity was maintained at 55–65%. Lighting was provided using monochromatic warm-white LED bulbs at an intensity of 25 lux during the first 3 days and 20 lux thereafter, with a daily lighting program of 22 h light and 2 h darkness. All birds were fed the same basal diets that were prepared to fulfill or exceed the nutritional needs (NRC, 1994) for broilers over the starter phase extending from hatch to day 21, followed by the finisher phase from day 21 to day 35. The basal diets’ ingredients and calculated nutrimental value are detailed in Table 1. Drinking water was provided using an individual opaque water bottle connected to a nipple drinker for each cage. Water was obtained from a deep local groundwater well, with a measured pH of 7.3 and total dissolved solids of 320 mg/L. Fresh drinking water and feed were provided ad libitum throughout the experimental period. The routine vaccination program included infectious bronchitis vaccination on days 0 and 18, Newcastle disease vaccination on days 7, 18, and 28, and infectious bursal disease vaccination on days 7 and 14. No antibiotics or antibiotic growth promoters were administered throughout the experimental period. Birds were inspected daily to monitor health status and record mortality

Table 1.

The basal diets’ ingredients and calculated nutrimental value.

Diet ingredients (kg/ton)	Starter phase (0–21 days of age)	Finisher phase (21–35 days of age)
Ground yellow corn	554.50	587.00
Soybean meal (44% CP)	335.00	287.50
Corn gluten meal (60% CP)	55.00	55.00
Dicalcium phosphate	19.30	20.00
Ground limestone	10.80	9.50
Vitamin-mineral premix a	3.00	3.00
L-lysine	1.40	0.00
Sodium chloride	3.00	3.00
Soy oil	18.00	35.00
Toal (kg)	1000	1000
Calculated nutrimental valueb
CP (%)	23.00	21.10
ME (Kcal/kg)	2968.70	3109.70
Ca (%)	0.98	0.94
Available P (%)	0.45	0.45
TSAA (%)	0.79	0.74
Lysine (%)	1.30	1.04

^aThe vitamin-mineral premix (Multi Vita Animal Nutrition®, Tenth of Ramadan City, Sharkia Governorate, Egypt) supplied the following nutrients per kilogram of diet: vitamin A, 12,000 IU (retinyl acetate); vitamin D₃, 2,500 IU; vitamin E, 20,000 IU (dl-α-tocopherol acetate); vitamin K₃, 2 mg; vitamin B₁, 2 mg; vitamin B₂, 5 mg; vitamin B₆, 2 mg; vitamin B₁₂, 0.05 μg; niacin, 30 mg; biotin, 0.05 μg; folic acid, 1 mg; pantothenic acid, 10 mg; manganese, 60 mg; zinc, 50 mg; iron, 40 mg; copper, 10 mg; iodine, 0.6 mg; and selenium, 0.3 mg. DL-methionine (Evonik Industries, Essen, Germany) contained 99% methionine, whereas L-lysine hydrochloride (Evonik Industries, Essen, Germany) contained 70% lysine.

^bIn accordance with NRC (1994).

Growth performance measurements

Broiler performance was evaluated on a replicate basis throughout the 35-day experimental period. Live body weight (LBW) was recorded at the initial measurement and then monitored weekly (weeks 1, 2, 3, 4, and 5 of age) employing a precision electronic weighing scale (Model USS-DBS51, U.S. Solid, Cleveland, OH) with a sensitivity of 0.1 g. The mean value of body weight gain (BWG; g/chick/day) was computed for each replicate during the intervals of 0–1, 1–2, 2–3, 3–4, and 4–5 weeks, as well as for the entire experimental period (0–5 weeks). Average feed intake (FI) was determined on a replicate basis during the same intervals and expressed as g/chick/day. The feed conversion ratio (FCR; g:g) was obtained by relating FI to BWG. Mortality was documented throughout the experiment, and the percentage of surviving birds was estimated for each replicate. The European Production Efficiency Factor (EPEF) was calculated at the end of the experiment (day 35 of age) according to the formula described by Kryeziu et al. (2018) as follows:

[(LBW (kg) × Survival rate (%)) / (Age (35 days) × FCR)] × 100

Slaughter and carcass evaluation

At the end of the 35-day trial, one representative chick with a body weight close to the corresponding replicate mean was randomly selected from each replicate (eight birds per treatment) for slaughter, carcass evaluation, blood sampling, and subsequent meat quality assessment. Selected birds were subjected to an 8–10 h fasting period, with drinking water provided continuously, before being individually weighed and humanely slaughtered following the Islamic method for subsequent carcass evaluation and sample collection (Mohamed et al., 2025). After exsanguination, defeathering, and evisceration, the weights of carcass, liver, gizzard, and heart were recorded and expressed as percentages of LBW before slaughter. The giblet percentage was determined by summing the percentages of the liver, gizzard, and heart, while the dressing percentage was computed by summing carcass and giblet percentages (Sweed et al., 2026).

Meat quality

Breast meat samples were collected from chilled carcasses for physicochemical and sensory-related measurements. Proximate composition was determined for moisture, ether extract (lipid), crude protein, and ash content following the analytical procedures of AOAC, which are widely adopted in poultry meat-quality research (Petracci and Baéza, 2011; Challioui et al., 2025). All proximate nutrients were calculated and expressed as percentages strictly on a fresh tissue (as-is) basis. Breast meat pH was measured directly with a digital pH meter (Model HI98161, Hanna Instruments, Romania) fitted with a penetration glass electrode (Stavropoulos et al., 2026). Lipid oxidation was assessed as thiobarbituric acid (TBA) and reported in units of mg malondialdehyde (MDA) per kilogram breast meat following the TBA method, with absorbance read at 532 nm against 1,1,3,3-tetraethoxypropane standards (Stavropoulos et al., 2026). Breast muscle samples for TBARS analysis were stored at −80°C for 14 d and analyzed in duplicate. For extraction, 5 g of meat was homogenized with 25 mL of 7.5% trichloroacetic acid (TCA), filtered, and subsequently incubated with thiobarbituric acid reagent. Meat color of the pectoralis major was determined on the meat surface using a Minolta colorimeter (Chroma Meter CR-400, Konica Minolta Inc., Tokyo, Japan) in the CIELAB system, and color coordinates (L, a, and b*) were recorded at three locations per sample (Stavropoulos et al., 2026). Water-holding capacity (WHC; %) was evaluated using the drip-loss technique, in which samples were suspended in bags under refrigeration and percentage fluid loss was calculated relative to initial weight (Sarkar et al., 2024). Tenderness was measured instrumentally as shear force by a texture analyzer equipped with a Warner-Bratzler blade on standardized meat cores aligned parallel to the muscle fibers (Stavropoulos et al., 2026). Juiciness was evaluated using a trained sensory panel on cooked breast meat using standardized sample preparation and descriptive scoring procedures for poultry meat texture and palatability attributes (Murata, 2025). The sensory panel included 10 trained members (5 males and 5 females, aged 25–40 years). Samples were cooked to an internal temperature of 75°C, cut into 2-cm cubes, and served warm. A randomized complete block design with blinded panelists was used, and attributes were scored on an anchored 9-point scale (1 = extremely dry/tough; 9 = extremely juicy/tender).

Blood sampling, hematology, and biochemistry

During slaughter, two tubes of blood were obtained from each selected bird. The first tube was an EDTA-coated tube for hematological analyses, gently mixed by inverting the tube several times, and processed at once for hematological evaluation. Following dilution of whole blood with Natt and Herrick solution (Natt and Herrick, 1952), red blood cells (RBC; ×10⁶ cells/mm³) and white blood cells (WBC; ×10³ cells/mm³) were manually counted with an improved Neubauer counting chamber. The packed cell volume (PCV, %) was assessed by the capillary microhematocrit method, whereas hemoglobin concentration (Hb, g/dL) was measured by spectrophotometric analysis. Erythrocyte indices were calculated using the following standard hematological formulas:

Mean corpuscular hemoglobin (MCH, pg) = (Hb × 10)/RBC count

Mean corpuscular volume (MCV, fL) = (PCV × 10)/RBC count

Mean corpuscular hemoglobin concentration (MCHC, g/dL) = (Hb × 100)/PCV.

The second blood sample was obtained in an anticoagulant-free tube, kept at room temperature until clot formation, and subjected to centrifugation to isolate serum, which was stored at −20°C for further biochemical assays. Commercial reagents (Biodiagnostic, Giza, Egypt) were employed following the supplier's recommendations to assess protein and lipid fractions and liver function enzymes. Endpoint colorimetric assays were used to determine serum total protein (g/dL), albumin (g/dL), total cholesterol (TC; mg/dL), triglycerides (TG; mg/dL), and high-density lipoprotein (HDL; mg/dL). Serum globulin (g/dL) was derived by subtracting albumin from total protein, and the corresponding albumin-to-globulin (A/G) ratio was subsequently derived. The serum concentrations of very low-density lipoprotein (VLDL; mg/dL) were determined as one-fifth of the serum triglyceride concentration; subsequently, serum low-density lipoprotein (LDL; mg/dL) concentrations were derived by subtracting the HDL and VLDL fractions from total cholesterol. The serum liver enzyme activities of alanine aminotransferase (ALT; U/L) and aspartate aminotransferase (AST; U/L) were assayed by the continuous-monitoring kinetic method, whereas serum creatinine (mg/dL) concentration was measured using the fixed-time kinetic Jaffé method. Serum antioxidant status and humoral immune function were assessed using commercial ELISA kits according to the manufacturer’s instructions (MyBioSource, San Diego, CA). The serum oxidative and antioxidant status was characterized by quantifying MDA (nmol/mL) concentration and superoxide dismutase (SOD; U/mL) activity (respectively), while humoral immune response was determined by measuring serum levels (mg/dL) of immunoglobulin A, G, and M.

Economic evaluation

An economic evaluation was performed per bird on a replicate basis to evaluate the economic benefits of the different probiotic treatments. Feed cost was calculated by multiplying the total feed consumption per bird by the diet price (US$ 0.47/kg). Probiotic cost was calculated by multiplying the total probiotic consumption per bird by the corresponding unit cost of each probiotic preparation (US$ 8.00/kg for Bifidobacterium bifidum, US$ 6.00/kg for Bacillus toyonensis, and US$ 7.00/kg for the combined probiotic preparation). All laboratory preparation and carrier costs for each probiotic were fully incorporated into the total probiotic cost. Total feed cost was calculated as the sum of feed cost and probiotic cost. Other production costs (e.g., chicks, housing, labor, management, and veterinary expenses) were not included in the economic analysis because they were identical across all experimental treatments and therefore did not influence the comparative economic evaluation. The total return per bird was estimated by multiplying the final LBW (kg) and the commercial selling price (US$ 2/kg), with the resulting value adjusted for the corresponding survival rate. The feed and commercial live-weight selling prices were based on prevailing local Egyptian poultry market averages during the experimental period. Net return was calculated by subtracting total feed cost from total return. Economic efficiency (EE) was estimated as the net return expressed as a percentage of total feed cost. Relative economic efficiency (REE) was estimated as the EE of each replicate relative to the mean EE of the control treatment, multiplied by 100.

Statistical methods

IBM SPSS Statistics software (Version 25.0, IBM Corp., Armonk, NY) was used to analyze all experimental data (IBM Corp., 2017). The statistical procedures followed the principles of a completely randomized design. The cage was considered the experimental unit for all response variables. For destructive measurements, including carcass traits, meat quality, and blood parameters, one representative bird was randomly selected from each cage (n = 8 birds per treatment). Thus, individual birds within the same cage were not treated as independent replicates, avoiding pseudoreplication. Before statistical analyses were performed, normality and homogeneity of variances were verified using the Shapiro-Wilk and Levene's tests, respectively. Treatment effects were assessed using one-way ANOVA through the GLM procedure. Percentages of survival rate were arcsine square-root transformed before conducting the ANOVA. When a significant treatment effect was detected (P ≤ 0.05), treatment means were separated using Tukey's HSD test. The statistical model used to analyze the experimental data was as follows:

Y_ij = μ + T_i + e_ij

Where: Y_ij represents the observed response variable value for the j^th experimental unit receiving the i^th probiotic drinking water treatment; μ is the overall population mean; T_i is the fixed effect of the i^th probiotic drinking water treatment (i = 1, 2, 3, 4); and e_ij represents the random experimental error.

Results

Growth performance

Adding probiotics in drinking water of broilers exerted significant effects on growth performance, particularly when BB and BT were administered in combination (Table 2, Table 3). Initial LBW did not differ significantly (P = 0.673) among the experimental groups, confirming the uniformity of chicks at the beginning of the experiment. The BB+BT treatment produced significantly higher LBW at weeks 1, 3, 4, and 5 (P-values: < 0.001, 0.025, 0.003, and < 0.001, respectively). Birds receiving BB+BT had higher LBW relative to the control and BT treatment groups, while the BB group showed mid-range values. Likewise, the BB+BT treatment significantly increased BWG during weeks 0–1, 3–4, 4–5, and 0–5 (P < 0.001, P = 0.003, P < 0.001, and P < 0.001, respectively), followed by the BB, whereas the control and BT treatments generally recorded the lowest values of BWG. During weeks 1–2 and 2–3, no significant differences were detected (P = 0.882 and P = 0.087, respectively) in BWG. By contrast, FI was not significantly affected during weeks 0–1 (P = 0.205), 1–2 (P = 0.554), 2–3 (P = 0.403), 3–4 (P = 0.823), 4–5 (P = 0.726), or over the overall experimental period (0–5 weeks; P = 0.441), although birds receiving BB+BT consistently showed numerically greater FI than the other treatments. In contrast, FCR was significantly improved during weeks 0–1, 4–5, and 0–5 (P < 0.001, P = 0.001, and P = 0.017, respectively). The BB+BT treatment achieved the best FCR, followed by the BB treatment, whereas the control and BT groups generally exhibited poorer FCR. The survival rate did not differ (P = 0.069) among treatments, while the EPEF was markedly improved (P < 0.001), with the BB+BT treatment producing the highest value in comparison with the individual probiotic treatments and the control.

Table 2.

Live body weight (LBW) and body weight gain (BWG) of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
LBW (g)
At hatch	42.32 ± 0.351	42.01 ± 0.276	41.76 ± 0.386	41.97 ± 0.240	0.673
Week 1	167.5 ± 1.678c	178.5 ± 1.637ab	174.1 ± 2.118bc	186.3 ± 3.094a	< 0.001
Week 2	452.3 ± 4.888	457.2 ± 8.497	453.0 ± 8.727	470.5 ± 6.820	0.292
Week 3	933.0 ± 12.90b	971.7 ± 19.03ab	932.4 ± 18.62b	1006 ± 22.48a	0.025
Week 4	1521 ± 17.48b	1571 ± 22.32ab	1554 ± 20.81b	1643 ± 23.76a	0.003
Week 5	2030 ± 28.85b	2158 ± 23.86ab	2049 ± 36.28b	2273 ± 40.98a	< 0.001
BWG (g/bird/day)
Weeks 0–1	17.88 ± 0.229c	19.50 ± 0.228ab	18.90 ± 0.306bc	20.62 ± 0.444a	< 0.001
Weeks 1–2	40.69 ± 0.588	39.81 ± 1.090	39.84 ± 1.220	40.60 ± 1.024	0.882
Weeks 2–3	68.68 ± 1.760	73.50 ± 2.616	68.49 ± 2.552	76.50 ± 2.950	0.087
Weeks 3–4	84.02 ± 0.983b	85.64 ± 1.651b	88.78 ± 1.032ab	91.01 ± 1.390a	0.003
Weeks 4–5	72.65 ± 1.710b	83.77 ± 1.567a	70.68 ± 2.795b	90.01 ± 2.599a	< 0.001
Weeks 0–5	56.78 ± 0.823c	60.44 ± 0.678ab	57.34 ± 1.035bc	63.75 ± 1.175a	< 0.001

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

Table 3.

Feed intake (FI), feed conversion ratio (FCR), survival rate, and European Production Efficiency Factor (EPEF) of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
FI (g/bird/day)
Weeks 0–1	22.63 ± 0.261	22.18 ± 0.329	21.82 ± 0.385	21.78 ± 0.235	0.205
Weeks 1–2	49.25 ± 0.588	50.32 ± 0.764	49.57 ± 0.676	50.68 ± 1.032	0.554
Weeks 2–3	93.11 ± 0.803	94.96 ± 1.407	92.63 ± 1.998	95.86 ± 1.602	0.403
Weeks 3–4	133.0 ± 4.033	135.2 ± 2.719	134.2 ± 1.998	136.7 ± 2.327	0.823
Weeks 4–5	191.5 ± 5.267	195.5 ± 4.914	194.8 ± 2.736	198.3 ± 3.648	0.726
Weeks 0–5	97.89 ± 1.717	99.64 ± 1.074	98.59 ± 0.947	100.7 ± 1.192	0.441
FCR (g/g)
Weeks 0–1	1.266 ± 0.018a	1.139 ± 0.027bc	1.157 ± 0.031b	1.059 ± 0.020c	< 0.001
Weeks 1–2	1.212 ± 0.022	1.270 ± 0.036	1.250 ± 0.033	1.256 ± 0.050	0.712
Weeks 2–3	1.363 ± 0.040	1.303 ± 0.046	1.367 ± 0.065	1.263 ± 0.042	0.395
Weeks 3–4	1.585 ± 0.054	1.584 ± 0.049	1.513 ± 0.033	1.502 ± 0.013	0.328
Weeks 4–5	2.641 ± 0.074ab	2.340 ± 0.076bc	2.791 ± 0.136a	2.216 ± 0.074c	0.001
Weeks 0–5	1.726 ± 0.034a	1.650 ± 0.029ab	1.725 ± 0.044a	1.582 ± 0.028b	0.017
Survival rate (%)
Week 5	96.25 ± 1.830	93.75 ± 1.830	96.25 ± 1.830	100.0 ± 0.000	0.069
EPEF
Week 5	324.6 ± 11.35b	351.3 ± 11.45b	328.7 ± 13.47b	412.1 ± 13.53a	< 0.001

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

Carcass traits

Table 4 presents the impact of different drinking water treatments on the carcass traits of 35-day-old broiler chicks. Overall, probiotic administration did not significantly affect relative weights of carcass (P = 0.941), dressing (P = 0.917), liver (P = 0.057), heart (P = 0.184), gizzard (P = 0.125), and total giblets (P = 0.505).

Table 4.

Carcass traits of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
Carcass (%)	74.41 ± 0.818	74.26 ± 0.486	74.88 ± 0.541	74.75 ± 1.162	0.941
Liver (%)	1.895 ± 0.043	1.927 ± 0.028	2.040 ± 0.042	1.970 ± 0.035	0.057
Heart (%)	0.430 ± 0.009	0.415 ± 0.008	0.407 ± 0.006	0.413 ± 0.005	0.184
Gizzard (%)	1.780 ± 0.043	1.671 ± 0.053	1.652 ± 0.039	1.760 ± 0.039	0.125
Giblets (%)	4.105 ± 0.070	4.013 ± 0.046	4.099 ± 0.068	4.143 ± 0.059	0.505
Dressing (%)	78.52 ± 0.813	78.27 ± 0.527	78.98 ± 0.583	78.89 ± 1.151	0.917

Data are presented as means ± SE.

Meat quality

According to the data presented in Table 5, probiotic supplementation via drinking water significantly affected several meat quality characteristics of broiler chickens. Moisture, lipid, crude protein, and ash contents were significantly influenced by the probiotic treatments (P < 0.001, P < 0.001, P = 0.005, and P < 0.001, respectively), with the combined probiotic treatment generally showing the most favorable meat composition. The BB+BT treatment resulted in the highest moisture and crude protein contents and the lowest lipid and ash contents, with significant differences from all other treatments, followed by the individual probiotic treatments. Lipid oxidation, as determined by TBA values, showed a significant reduction in birds receiving probiotics (P < 0.001), with the lowest value observed in the BB+BT group, with progressively higher values in the BB and BT groups and the highest value in the control group. In contrast, muscle pH measured 24 h postmortem and WHC were not significantly affected by the treatments (P = 0.141 and P = 0.070, respectively). Regarding meat color, significant effects were recorded in redness (a*; P < 0.001) and yellowness (b*; P = 0.001), whereas lightness (L*) remained unchanged (P = 0.633). The BB+BT treatment produced the lowest a* value and the highest b* value. Likewise, tenderness and juiciness scores were significantly improved in probiotic-supplemented groups (P = 0.001 and P = 0.002, respectively), with the BB+BT treatment recording the highest scores, followed by BB and BT treatments, whereas the lowest values were recorded in the control birds.

Table 5.

Meat quality of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
Chemical composition (%)
Moisture (%)	66.73 ± 0.207c	67.90 ± 0.222b	68.03 ± 0.220b	69.17 ± 0.269a	< 0.001
Lipids (%)	10.89 ± 0.106a	9.130 ± 0.119b	8.918 ± 0.089b	8.079 ± 0.127c	< 0.001
Protein (%)	20.43 ± 0.255b	21.72 ± 0.298a	21.07 ± 0.187ab	21.39 ± 0.184a	0.005
Ash (%)	1.130 ± 0.014a	0.908 ± 0.009b	0.890 ± 0.016b	0.730 ± 0.011c	< 0.001
Physicochemical traits
pH (24 h)	5.933 ± 0.145	6.221 ± 0.120	6.291 ± 0.065	6.150 ± 0.095	0.141
TBA (mg/kg)	0.410 ± 0.014a	0.300 ± 0.010b	0.280 ± 0.004b	0.230 ± 0.007c	< 0.001
WHC (%)	84.37 ± 1.344	89.11 ± 1.242	87.08 ± 0.909	87.75 ± 1.366	0.070
Color attributes (CIELAB)
Lightness (L*)	49.41 ± 0.781	48.73 ± 0.923	48.19 ± 0.685	47.85 ± 1.130	0.633
Redness (a*)	4.699 ± 0.114a	3.519 ± 0.074b	3.473 ± 0.068b	2.984 ± 0.101c	< 0.001
Yellowness (b*)	5.180 ± 0.085b	5.339 ± 0.107b	5.408 ± 0.126b	5.941 ± 0.152a	0.001
Sensory traits
Tenderness	8.511 ± 0.099b	8.961 ± 0.114a	8.694 ± 0.055ab	9.001 ± 0.059a	0.001
Juiciness	8.430 ± 0.087b	8.763 ± 0.076ab	8.811 ± 0.126a	8.960 ± 0.059a	0.002

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

TBA (thiobarbituric acid) and WHC (water-holding capacity).

Hematological parameters

The hematological parameters of broilers were significantly influenced by probiotic supplementation via drinking water (Table 6). The values of Hb, RBCs, WBCs, and PCV showed significant responses to drinking water treatments (P < 0.001, P < 0.001, P < 0.001, and P = 0.003, respectively). The BB+BT treatment generally produced the most favorable hematological profile, followed by the BB and BT groups, whereas the control group generally showed the least favorable values. In contrast, erythrocyte indices, including MCH, MCV, and MCHC, were not significantly affected by probiotic supplementation (P = 0.619, P = 0.317, and P = 0.815, respectively).

Table 6.

Hematological parameters of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
Hb (g/dL)	10.10 ± 0.227b	10.97 ± 0.235ab	11.19 ± 0.299a	11.74 ± 0.110a	< 0.001
RBCs (×106)	3.030 ± 0.080b	3.210 ± 0.114b	3.326 ± 0.076ab	3.639 ± 0.069a	< 0.001
WBCs (×103)	21.75 ± 0.647b	26.10 ± 0.718a	28.31 ± 1.012a	29.38 ± 1.054a	< 0.001
PCV (%)	37.16 ± 1.257b	41.20 ± 1.098a	42.20 ± 0.964a	42.49 ± 0.638a	0.003
MCH (pg)	33.46 ± 1.019	34.51 ± 1.506	33.71 ± 1.054	32.37 ± 0.858	0.619
MCV (FL)	123.5 ± 6.185	129.4 ± 5.553	127.3 ± 3.808	117.1 ± 3.239	0.317
MCHC (g/dL)	27.41 ± 1.153	26.74 ± 0.817	26.63 ± 1.023	27.68 ± 0.549	0.815

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

Hb (hemoglobin), RBCs (red blood cells), WBCs (white blood cells), PCV (packed cell volume), MCH (mean corpuscular hemoglobin), MCV (mean corpuscular volume), and MCHC (mean corpuscular hemoglobin concentration).

Liver and kidney function

Supplementing broiler drinking water with BB and BT significantly affected several serum biochemical, immunological, and antioxidant parameters (Table 7, Table 8, Table 9). Total protein concentration differed significantly (P = 0.003) among the drinking water treatments, with birds receiving the BB+BT treatment exhibiting the greatest value and the control group exhibiting the smallest value. The BB and BT treatments showed intermediate values and did not differ significantly from either group (Table 7). Likewise, significant reductions in serum ALT and AST activities were observed in all probiotic-treated groups (P < 0.001). However, serum albumin, globulin, albumin-to-globulin ratio, and creatinine were not significantly changed by drinking water treatments (P = 0.083, P = 0.431, P = 0.786, and P = 0.060, respectively).

Table 7.

Serum protein profile and liver and kidney function indices of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
Total protein (g/dL)	5.243 ± 0.061b	5.394 ± 0.077ab	5.468 ± 0.063ab	5.671 ± 0.092a	0.003
Albumin (g/dL)	3.271 ± 0.072	3.433 ± 0.031	3.368 ± 0.055	3.520 ± 0.093	0.083
Globulin (g/dL)	1.971 ± 0.077	1.961 ± 0.068	2.100 ± 0.081	2.151 ± 0.143	0.431
A/G ratio	1.684 ± 0.094	1.765 ± 0.062	1.625 ± 0.080	1.697 ± 0.134	0.786
Creatinine (mg/dL)	0.460 ± 0.006	0.470 ± 0.009	0.490 ± 0.013	0.500 ± 0.013	0.060
ALT (U/L)	12.93 ± 0.466a	11.17 ± 0.299b	10.98 ± 0.367b	10.31 ± 0.125b	< 0.001
AST (U/L)	100.3 ± 3.488a	83.94 ± 2.668b	84.99 ± 0.834b	88.08 ± 2.452b	< 0.001

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

A/G ratio (albumin-to-globulin ratio), ALT (alanine aminotransferase), and AST (aspartate aminotransferase).

Table 8.

Serum lipid profile (mg/dL) of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
TG (mg/dL)	115.0 ± 2.815	111.7 ± 3.429	109.5 ± 1.660	108.9 ± 4.251	0.523
TC (mg/dL)	131.6 ± 1.776a	118.9 ± 1.661b	122.3 ± 2.419b	117.3 ± 1.885b	< 0.001
HDL (mg/dL)	63.91 ± 1.270	58.83 ± 0.870	59.91 ± 1.973	61.18 ± 1.455	0.100
LDL (mg/dL)	44.64 ± 1.339a	37.69 ± 1.467b	40.49 ± 2.233ab	34.30 ± 1.480b	0.001
VLDL (mg/dL)	23.00 ± 0.563	22.35 ± 0.686	21.89 ± 0.332	21.78 ± 0.850	0.523

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

TG (triglycerides), TC (total cholesterol), HDL (high-density lipoprotein), LDL (low-density lipoprotein), and VLDL (very-low-density lipoprotein).

Table 9.

Immunological and antioxidant indices of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
Immunological parameters
IgA (mg/dL)	2.151 ± 0.046	2.275 ± 0.085	2.309 ± 0.034	2.319 ± 0.054	0.174
IgM (mg/dL)	2.449 ± 0.093b	2.898 ± 0.088a	2.850 ± 0.076a	3.101 ± 0.107a	< 0.001
IgG (mg/dL)	13.80 ± 0.208b	14.25 ± 0.235b	14.30 ± 0.347ab	15.25 ± 0.198a	0.003
Antioxidant parameters
SOD (U/mL)	148.0 ± 6.250b	160.0 ± 2.469ab	162.0 ± 7.013ab	175.0 ± 3.522a	0.010
MDA (nmol/mL)	5.866 ± 0.124a	5.466 ± 0.085b	5.418 ± 0.086b	5.235 ± 0.110b	0.001

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

IgA (immunoglobulin A), IgM (immunoglobulin M), IgG (immunoglobulin G), SOD (superoxide dismutase), and MDA (malondialdehyde).

Lipid profile

Supplementing broiler drinking water with BB, BT, or their combination significantly improved the blood serum lipid profile (Table 8). Total cholesterol and LDL cholesterol were reduced (P < 0.001 and P = 0.001, respectively) by probiotic supplements. The combined BB+BT treatment exhibited the most favorable values, followed by the individual probiotic treatments. In contrast, serum triglycerides, HDL, and VLDL were not changed (P = 0.523, 0.100, and 0.523, respectively) by probiotic treatments.

Immunity and antioxidant status

The broiler chicks’ immunity and antioxidant status were significantly enhanced by adding probiotics in drinking water (Table 9). Serum IgM concentrations were increased (P < 0.001) across all probiotic-treated groups relative to the control group. The BB+BT treatment produced the greatest serum value of IgG (P = 0.003), differing significantly from the control and BB groups, while the BT treatment showed intermediate values. However, IgA levels did not change significantly (P = 0.174) among experimental groups. Likewise, antioxidant status was significantly improved, characterized by increased SOD activity (P = 0.010) and reduced MDA concentration (P = 0.001). The BB+BT treatment exhibited the highest SOD activity, significantly exceeding the control treatment, whereas the BB and BT treatments showed intermediate values. On the other hand, all probiotic-treated groups had significantly lower MDA concentrations than the control group.

Economic efficiency

The economic evaluation demonstrated that probiotic supplementation significantly affected most economic parameters, except for feed cost per bird (P = 0.441)Table 10). As expected, probiotic cost differed significantly (P < 0.001) among the treatment groups, being highest in the BB+BT group, followed by the BB group and the BT group, while no probiotic cost was incurred in the control group. Consequently, total feed cost also differed significantly among treatments (P = 0.023), with the BB+BT group recording the highest value, significantly exceeding the control group, whereas the BB and BT groups showed intermediate values. Despite the higher supplementation cost, the BB+BT treatment achieved the greatest economic return, resulting in significantly higher total return, net return, EE, and REE than the control, BB, and BT groups (P < 0.001, P < 0.001, P = 0.013, and P = 0.013, respectively). In contrast, the BB and BT treatments failed to produce significant changes relative to the control group in total return, net return, EE, or REE.

Table 10.

Economic efficiency of broiler chickens as affected by drinking water supplementation with Bifidobacterium bifidum (BB), Bacillus toyonensis (BT), or their combination (BB+BT).

Items	Drinking water treatments				Sig.
	Control	BB (0.5 g/L)	BT (0.5 g/L)	BB+BT (1.0 g/L)
Feed cost($/bird)	1.610 ± 0.028	1.639 ± 0.018	1.622 ± 0.016	1.656 ± 0.020	0.441
Probiotic cost ($/bird)	0.000 ± 0.000d	0.027 ± 0.001b	0.020 ± 0.000c	0.051 ± 0.001a	< 0.001
Total feed cost ($/bird)	1.610 ± 0.028b	1.666 ± 0.018ab	1.642 ± 0.016ab	1.707 ± 0.021a	0.023
Total return ($/bird)	3.909 ± 0.105b	4.046 ± 0.092b	3.940 ± 0.082b	4.546 ± 0.082a	< 0.001
Net return($/bird)	2.299 ± 0.093b	2.379 ± 0.095b	2.298 ± 0.095b	2.839 ± 0.076a	< 0.001
EE (%)	142.9 ± 5.627b	143.0 ± 6.174b	140.4 ± 6.879b	166.4 ± 4.546a	0.013
REE (%)	100.0 ± 3.939b	100.1 ± 4.322b	98.25 ± 4.815b	116.5 ± 3.182a	0.013

Within a row, different superscript letters indicate significant differences (P ≤ 0.05; Tukey's HSD test).

Data are presented as means ± SE.

EE (economic efficiency) and REE (relative economic efficiency).

Discussion

The combined BB+BT treatment maximized (P < 0.05) broiler growth parameters (LBW, BWG, FCR, and EPEF) without affecting feed intake. Favorable blood biochemical profiles support these performance gains (Table 6, Table 7, Table 8, Table 9). Such responses indicate that the observed production benefits may be linked to underlying physiological and biochemical adaptations, as also reported by Salehi et al. (2025), who showed that nutritional strategies can modify physiological and quality-related parameters alongside production responses. Our findings are consistent with previous studies showing that multistrain probiotic preparations can be more effective than single-strain preparations in improving LBW, BWG, FCR, and production efficiency in broilers (Elleithy et al., 2023; Wang et al., 2025). Likewise, selected Bacillus-based probiotic combinations yielded improvements in growth performance better than individual probiotic preparations (Bromfield et al., 2024). Similar responses have also been reported with Bacillus subtilis supplementation, which enhanced LBW or BWG and improved FCR without significantly affecting FI (Cai et al., 2024; Šimunović et al., 2025). Previous studies using these probiotic strains also support our findings. In quail, supplementation with BB+BT improved final LBW and BWG, with the combined treatment proposed to exert additional benefits through complementary aerobic and anaerobic probiotic activities (Abou-Kassem et al., 2021). Similarly, BB and BT supplementation increased the activities of major digestive enzymes, specifically amylase and lipase, although improvements in FCR were not always observed, reflecting the strain-specific nature of probiotic responses (Nour et al., 2021).

The marked improvement in growth performance in the BB+BT treatment could be tentatively attributed to potential enhancements in intestinal efficiency and nutrient utilization. Previous investigations reported that Bacillus-containing probiotics stimulate digestive enzyme secretion, promote intestinal structural development by increasing villus morphometric parameters, and consequently improve the absorption of feed nutrients. (Cai et al., 2024; Chen et al., 2025a). Furthermore, previously reported effects of probiotics on the intestinal microbial ecosystem and enhanced SCFA generation may provide possible explanations for the observed improvements in productive performance. Numerous investigations stated that probiotic treatments enhance the gut microbial community by increasing the abundance of Bifidobacterium and Lactobacillus, accompanied by enhanced SCFA generation, and suppress pathogenic bacteria, thereby promoting intestinal development and nutrient utilization (Liao et al., 2020; Wang et al., 2025). The superior productivity of the BB+BT treatment may be related to complementary effects of the two probiotic strains. Bifidobacterium has been reported to contribute to carbohydrate fermentation, organic acid production, pathogen exclusion, and immune regulation (Nour et al., 2021; Wang et al., 2025), whereas Bacillus has been reported to stimulate enzyme activity, microbiota stability, and gut barrier integrity (Bromfield et al., 2024). Such complementary functions may contribute to improved nutrient utilization (Abdel-Raheem et al., 2024) and could partly explain the better feed utilization and overall productivity observed in the present study.

The nonsignificant changes in carcass yield, dressing percentage, and relative organ weights indicate that probiotic supplementation did not alter carcass partitioning in the present study. This finding agrees with previous reports, where probiotic treatments enhanced growth and nutrient utilization without substantially affecting carcass characteristics, suggesting that the improvement in overall growth was not accompanied by marked changes in the relative development of individual carcass components (Rehman et al., 2020). Similarly, supplementation with Bacillus coagulans, B. licheniformis, Lactobacillus plantarum, or Bacillus subtilis improved growth performance while producing little or no effect on carcass yield, dressing percentage, or the relative weights of the liver, heart, and gizzard (Elleithy et al., 2023; Gautam et al., 2024). Although some studies have reported improvements in eviscerated or breast yield following Bifidobacterium supplementation, carcass responses remain inconsistent and are likely influenced by probiotic strain, dosage, bird sex, and management conditions (Sjofjan et al., 2021). Furthermore, the unchanged relative weights of the liver, heart, and gizzard suggest that the probiotic treatments did not adversely affect visceral development, indicating no apparent adverse effects on the relative development of these visceral organs under the conditions of the present study (Salsabila et al., 2026).

The present findings demonstrate that probiotic supplementation improved meat quality by enhancing chemical composition, oxidative stability, color, and sensory characteristics, while muscle pH and WHC remained unchanged. These findings agree with previous reports indicating that probiotic supplementation more consistently improves lipid oxidation, meat color, and tenderness, whereas responses of postmortem pH and WHC are generally less consistent (Yibar and Uzabaci, 2024). These improvements are associated with changes in systemic biochemical and antioxidant markers observed in the present study (Tables 7, 8, and 9). While serum markers represent systemic physiological responses rather than localized muscle chemistry, these systemic responses may be associated with the improved oxidative status and meat quality observed in the present study. The higher moisture and crude protein contents, together with reduced lipid content, are consistent with a more favorable muscle chemical composition and may reflect differences in nutrient utilization. Similar improvements in muscle protein accretion and carcass leanness have been reported following supplementation with Bifidobacterium- and Bacillus-based probiotics, which have also been associated with reduced fat deposition in broilers (Ciurescu et al., 2020; Iakubchak et al., 2024). The decrease in ash may reflect changes in muscle chemical composition accompanying the higher moisture and protein fractions. However, this interpretation should be considered cautiously in the absence of direct mineral-status measurements, especially because Bacillus subtilis can improve tibial phosphorus deposition in broilers (Ciurescu et al., 2020).

The lower TBA values observed in probiotic-treated birds indicate improved oxidative stability, one of the most consistently reported benefits of probiotic supplementation. Although systemic blood profiles do not equate directly to postmortem muscle stability, a tentative relationship can be discussed with the findings of the present study (Table 9), in which the BB+BT treatment increased serum SOD activity while reducing serum MDA concentration, suggesting an improved systemic antioxidant status that may have contributed to lower oxidative stress before slaughter. Previous studies have shown that Bacillus subtilis and mixed probiotics reduce lipid peroxidation and enhance antioxidant enzyme activity, thereby improving meat oxidative stability (Bai et al., 2017; Abdel-Raheem et al., 2024). Previous studies have reported beneficial but variable effects of probiotics on meat color. Bacillus subtilis FMBJ altered the a* and b* color values, whereas compound probiotics increased breast meat redness, and high-dose B. subtilis improved meat color alongside changes in pH and WHC (Wang et al., 2024; Liu et al., 2026). In contrast, Bacillus-Bifidobacterium supplementation in quail reduced redness and TBA values, suggesting that color responses depend on probiotic strain, bird species, and experimental conditions (Abou-Kassem et al., 2021). The improvement in tenderness and juiciness despite unchanged pH and WHC suggests that factors beyond WHC may have contributed to the improved sensory characteristics. This interpretation is supported by previous studies demonstrating improved sensory quality and reduced shear force following Bacillus supplementation, with one study attributing enhanced tenderness to muscle fiber transformation mediated through the AMPK/SIRT1/PGC-1α signaling pathway (Bai et al., 2017; Mohammed et al., 2024).

The present findings demonstrate that probiotic supplementation improved the hematological status of broilers by increasing hemoglobin concentration, RBC count, WBC count, and PCV, while MCH, MCV, and MCHC remained unchanged. These results are consistent with previous studies showing that probiotics exert more pronounced effects on blood cell counts and hemoglobin than on erythrocyte indices (Deraz, 2018; Shah et al., 2021). The observed increases in hemoglobin, RBCs, WBCs, and PCV coincide with these treatments, which could tentatively reflect a positive shift within normal physiological ranges, though they do not conclusively demonstrate altered erythropoiesis or immune competence rather than changes in red blood cell morphology. A meta-analysis of 49 broiler studies similarly reported significant increases in RBC and WBC counts following probiotic supplementation, particularly at higher inclusion levels (Sjofjan et al., 2021). The hematological improvements may be associated with enhanced nutrient utilization and intestinal function. Probiotics have been reported to improve gut integrity, maintain microbial balance, and increase nutrient digestibility, which may contribute to the availability of nutrients involved in erythropoiesis (Jha et al., 2020; Sjofjan et al., 2021). In addition, the increased WBC count is associated with these probiotic groups, which might suggest a potential baseline modulation of the immune system, aligning with previous reports where probiotics were found to enhance immune cell populations, immunoglobulin production, cytokine responses, and overall hematobiochemical status in broilers (Shah et al., 2021; Younas et al., 2025). The absence of significant changes in MCH, MCV, and MCHC further suggests that probiotic supplementation increased the number of circulating erythrocytes and hematocrit without altering erythrocyte size or hemoglobin concentration per cell. Similar findings have been reported in recent broiler studies using multistrain probiotics, suggesting that the observed hematological changes were primarily related to erythrocyte number rather than marked changes in erythrocyte indices (Sarwar et al., 2019; Younas et al., 2025).

The serum biochemical profile indicates that probiotic supplementation altered liver- and kidney-associated biochemical indices. The increase in serum total protein together with the reductions in ALT and AST activities suggests potential shifts in hepatic metabolic parameters, whereas the unchanged creatinine concentration indicates stable baseline kidney-associated markers. Similar responses have been reported in broilers supplemented with probiotics, where serum total protein increased and liver enzyme activities decreased without significant changes in albumin, globulin, or creatinine (Biswas et al., 2019; Zhu et al., 2020). The higher serum total protein observed in the present study may be related to improved protein utilization and hepatic synthetic activity rather than dehydration, as albumin, globulin, and the A/G ratio remained unchanged. Previous studies have shown that probiotics enhance crude protein digestibility, intestinal absorptive efficiency, and amino acid utilization, thereby increasing circulating protein levels (He et al., 2019; Ogbuewu et al., 2022). The concurrent reductions in ALT and AST further indicate favorable changes in serum liver-associated enzyme activities, consistent with previous reports demonstrating that probiotic supplementation alleviates hepatocellular stress and modifies liver biochemical markers in broilers (Biswas et al., 2019; Zhu et al., 2020). These responses may be related to the previously reported effects of probiotics on gut health, intestinal barrier function, pathogen control, and antioxidant defense, although these mechanisms were not directly evaluated in the present study (Biswas et al., 2019). This interpretation is supported by the enhanced antioxidant status and immune response observed in the present study (Table 9).

The present results showed that probiotic supplementation reduced serum total cholesterol and LDL concentrations, whereas triglycerides, HDL, and VLDL remained unchanged. This pattern suggests that probiotics primarily influenced cholesterol metabolism rather than causing broad alterations in all circulating lipid fractions. Similar responses have been reported in broilers and other species, where probiotic supplementation consistently lowered total cholesterol and LDL, while changes in TG and HDL were less consistent (Yan et al., 2019; Zarezadeh et al., 2022). Several plausible mechanisms hypothesized in poultry literature may explain these effects (Mohamed et al., 2022; Susanti et al., 2023): First, probiotics with bile salt hydrolase activity may deconjugate bile salts, potentially increasing bile acid excretion and contributing to lower circulating cholesterol concentrations. Second, some probiotic strains may assimilate intestinal cholesterol or reduce its absorption, thereby potentially limiting its entry into the circulation. In addition, probiotic-induced modulation of the gut microbiota could presumably increase SCFA production, which is suggested to suppress hepatic lipogenesis (Wang et al., 2025). These hypocholesterolemic effects may reflect a functional action of probiotics rather than a direct nutritional contribution, consistent with the concept that bioactive feed supplements can induce physiological and metabolic effects beyond their basic nutritional contribution (Vakili et al., 2025). The present findings agree with previous broiler studies reporting lower serum total cholesterol and LDL following probiotic supplementation (Mohamed et al., 2022). Although some studies also observed reductions in TG and VLDL (Shamna et al., 2025), these responses are not consistently reported and appear to depend on probiotic strain, formulation, and experimental conditions.

In the present study, probiotic supplementation increased serum IgM, IgG, and SOD activity while reducing MDA concentration, whereas IgA remained unchanged, suggesting enhanced humoral immune response and antioxidant status. These responses were most pronounced in the BB+BT treatment, which may indicate a greater response to the combined probiotic treatment. Similar improvements in immune and antioxidant function have been widely reported in poultry following probiotic supplementation (Gavzy et al., 2023; Ashour et al., 2025). Bacillus probiotics have been reported to enhance immune responses through cytokine stimulation and immune modulation (Naeem and Bourassa, 2025), whereas Bifidobacterium promotes immune homeostasis by regulating immune cells and maintaining intestinal barrier integrity (Gavzy et al., 2023). In contrast, the unchanged IgA suggests that the effects were not uniform across all measured immunoglobulin classes, as mucosal IgA responses remain dependent on probiotic strain, dosage, and experimental conditions (Liu et al., 2025). The concurrent increase in SOD activity and reduction in MDA concentration further demonstrate enhanced antioxidant defense and reduced lipid peroxidation. Similar findings have been reported in broilers receiving probiotic supplementation (Liu et al., 2025). Bacillus-based interventions also reduced intestinal or tissue oxidative damage while improving barrier-associated and anti-inflammatory functions (Zou et al., 2022). Changes in gut microbiota have been proposed as one possible mechanism underlying the antioxidant effects of probiotics. Probiotics in poultry have been reported to increase beneficial bacteria, suppress coliforms and E. coli, and enhance SCFA-rich metabolic environments that may contribute to support barrier integrity, immune-metabolic balance, and oxidative stress status (Sultan et al., 2024; Oke et al., 2025). Recent reviews also emphasize that microbiota-derived metabolites regulate poultry immunometabolism, including oxidative stress resistance and immune cell activation (Oke et al., 2025).

The higher economic efficiency observed in the BB+BT group was mainly associated with its better growth performance. Although probiotic supplementation slightly increased production cost, it improved final LBW, overall BWG, FCR, and EPEF (Table 2, Table 3), indicating that the observed economic advantage was primarily associated with the improved weight gain and feed conversion efficiency. In addition, the improvements observed in hematological traits, liver function, lipid profile, immune response, and antioxidant status (Table 6, Table 7, Table 8, Table 9) may have supported better nutrient utilization and overall health, thereby enhancing production profitability. These results are consistent with previous studies showing that improvements in BWG and FCR are the main factors responsible for improving economic efficiency in broiler production (Zaghari et al., 2020; Bilal et al., 2026). Similar improvements in economic return following probiotic supplementation have also been reported by Mohammed et al. (2022), who attributed these responses to better growth performance with only small changes in production cost. The better economic performance of the BB+BT treatment may be explained by the combined action of these probiotics. Previous studies have shown that mixed probiotic products can improve BWG, FCR, gut health, and nutrient utilization more effectively than single strains (Abdel-Raheem et al., 2024). Since feed represents the largest proportion of broiler production costs, improving feed conversion can increase net return even when feed intake is unchanged (Bromfield et al., 2024).

Conclusion

The addition of probiotic preparation to broiler drinking water containing B. bifidum (0.5 g/L; 5 × 10⁸ cfu/L) and B. toyonensis (0.5 g/L; 5 × 10⁸ cfu/L) improved growth performance, feed conversion, hematological parameters, liver function, lipid profile, immune response, antioxidant status, and meat quality. Notably, the combined treatment (1.0 g/L; 5 × 10⁸ cfu/L of each probiotic) was more effective than using either probiotic alone. In addition, although probiotic supplementation slightly increased production cost, the BB+BT treatment resulted in the highest total return, net return, and economic efficiency. These results suggest that combining these probiotic strains represents a promising approach that may hold practical potential for optimizing broiler performance, health, meat quality, and economic return, although its applicability under large-scale commercial production conditions warrants further industry-scale validation. Further studies are also needed to clarify the potential interaction between B. bifidum and B. toyonensis and to evaluate the consistency of these responses under different management and environmental conditions.

Author contributions

A.I.I. conceived and designed the study, established the research objectives, interpreted the findings, and drafted and critically revised the manuscript; A.A.R. designed the experimental procedures, conducted the experimental work and laboratory analyses, evaluated the data, and critically revised the manuscript; A.A.M.G. planned the experimental workflow, performed the experimental procedures and laboratory analyses, analyzed the data, and critically revised the manuscript; D.E.A.-K. developed the experimental plan, conducted the laboratory investigations, interpreted the experimental results, and drafted and critically revised the manuscript; N.K.A. established the scientific framework, evaluated the biological significance of the findings, and critically revised the manuscript for important intellectual content; A.S.A. formulated the scientific rationale, integrated the major findings, and critically revised the manuscript for important intellectual content; N.A.-H. developed the conceptual framework, evaluated the consistency of the findings with the study objectives, and critically revised the manuscript; K.M.A. evaluated the experimental findings, addressed their physiological interpretation, and critically revised the manuscript for important intellectual content; S.C.D. assessed the scientific interpretation of the findings, examined the coherence of the results and conclusions, and critically revised the manuscript; F.A. reviewed the scientific content, refined the interpretation of the findings, and critically revised the manuscript for important intellectual content. All authors reviewed and explicitly approved the final version of the manuscript before submission and accept personal and public accountability for all aspects of the work, including its accuracy and integrity and the appropriate investigation and resolution of any questions concerning the work.

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.

Data availability

Data will be made available on request.