Abstract

Introduction:

Neonatal calves exhibit immature digestive and immune systems, rendering them susceptible to environmental stressors such as cold temperatures, which exacerbate gastrointestinal dysfunction and diarrhea incidence. Antibiotic use for mitigation poses risks, including microbiota disruption and resistance development, necessitating safe probiotic alternatives.

Methods:

This study evaluated the effects of Bacillus megaterium supplementation on growth performance, diarrhea occurrence, serum biochemical, immune, and antioxidant parameters, and rectal microbiota composition in neonatal calves under Xinjiang’s cold climate. Fifty crossbred calves were randomly assigned to five groups (n = 10): basal diet (Group I), basal plus 50 mg/day gentamicin (Group II), or basal plus 250, 500, or 1,000 mg/day B. megaterium (Groups III–V). Supplementation occurred via milk over 28 days, with assessments of growth performance, fecal scores, serum indices, and rectal microbiota.

Results and discussion:

The 500 mg/day B. megaterium treatment (Group IV) significantly increased average daily gain (ADG) and reduced feed-to-gain ratio (F/G) and diarrhea frequency compared to control (p < 0.05). Serum IgG increased, whereas pro-inflammatory cytokines (IL-1β, TNF-α, IFN-γ) decreased in the probiotic group compared with controls (p < 0.05). Antioxidant capacity improved significantly, with GSH-Px and CAT elevated and MDA reduced (p < 0.05). Rectal microbiota Shannon index was significantly higher in Group IV compared to the Group II (median: 2.7 vs. 3.8; p < 0.05). The relative abundance of Firmicutes increased, and beneficial genera (Lactobacillus, Faecalibacterium, Ruminococcaceae_UCG-014) were enriched, whereas Escherichia–Shigella decreased in Group IV (p < 0.05). Beneficial taxa were positively associated with immune and antioxidant markers and negatively associated with pro-inflammatory cytokines. Overall, these findings suggest that B. megaterium is a promising antibiotic alternative for promoting calf health, productivity, and beneficial gut microbiota under cold stress, with implications for more sustainable ruminant production systems.

Introduction

In modern cattle production systems, ensuring the health and efficient rearing of neonatal calves is a critical prerequisite for long-term herd productivity and profitability. Neonatal calves represent a critical developmental stage and are born with immature digestive and immune systems (Meale et al., 2017; Du et al., 2023). The structure and function of immune organs in neonatal calves differ significantly from those in adult cattle, characterized by fewer immune cells and reduced cellular functionality (Cangiano et al., 2024). Moreover, the integrity of the intestinal barrier is compromised due to loose tight junctions between intestinal epithelial cells, making calves highly susceptible to environmental stressors such as temperature fluctuations (Yu et al., 2024; Zhang L. et al., 2025; Zhang Z. et al., 2025). Xinjiang, China, provides extensive grasslands suitable for herbivore production. However, its harsh continental climate, characterized by long, cold winters, frequent strong winds, and pronounced diurnal and seasonal temperature fluctuations, imposes considerable stress on young animals (Guan et al., 2022). Beyond general stress, cold exposure can directly compromise the intestinal epithelial barrier by disrupting tight junctions and promoting mucosal inflammation, and it can also remodel gut microbial metabolism in ruminants (e.g., propionate and butyrate pathways), which may increase susceptibility to enteric dysfunction and diarrhea in neonatal calves (Guo et al., 2022; Cheng et al., 2025). These environmental constraints render neonatal calves vulnerable to gastrointestinal dysfunction, immunosuppression, and diarrhea (Chen et al., 2022; Yu et al., 2024). Neonatal diarrhea impairs growth performance by reducing average daily gain (ADG) and feed efficiency, increases veterinary and management costs, and disrupts intestinal development, with adverse consequences for long-term health and production efficiency (Chen et al., 2022; Du et al., 2023). Antibiotics are still widely used to control calf diarrhea, but their application is associated with disruption of the gut microbiota, impaired intestinal and immune development, the emergence of antimicrobial resistance and concerns about drug residues. Therefore, there is an urgent need to develop safe and effective alternatives to in-feed and therapeutic antibiotics in calf rearing (Du et al., 2023; Zhang L. et al., 2025).

Bacillus megaterium (B. megaterium) is a rod-shaped, gram-positive, strictly aerobic, and motile spore-forming bacterium (Etesami et al., 2023). It produces a wide range of extracellular enzymes that enhance nutrient digestion and absorption (Chen et al., 2018; Elisashvili et al., 2018). Certain strains produce bacteriocins and extracellular polysaccharides, and cell wall glycolipids and glycoproteins have been suggested to influence intestinal development (Chen et al., 2018). In addition, B. megaterium can activate immune cells, enhance host immune responses, reduce inflammation, and secrete antimicrobial substances that inhibit pathogens, thereby modulating gut microbiota composition (Avdiyuk and Varbanets, 2023). Evidence from young monogastric models provides supportive but indirect indications of these functions in vivo: dietary supplementation with B. megaterium has been reported to improve growth performance, optimize gut microbiota composition, and enhance intestinal barrier function in piglets (Bakun et al., 2021), and to be associated with improved immune status and a lower incidence of enteric disorders (including diarrhea) in broiler chickens (Luise et al., 2022). However, because gastrointestinal physiology and microbial ecology differ substantially between monogastrics and ruminants, the efficacy, optimal dosage, and underlying mechanisms of B. megaterium in neonatal calves remain largely unclear, particularly under environmental stress conditions.

Therefore, the present study aimed to evaluate the effects of different dietary doses of B. megaterium on growth performance, diarrhea incidence, serum biochemical, immune, and antioxidant indices, and rectal microbial community in neonatal calves raised under cold stress conditions in Xinjiang. By integrating growth, health, and microbiota, this research seeks to provide mechanistic insights into how B. megaterium modulates the gut–serum axis in neonatal calves and to generate scientific evidence and practical guidance for sustainable and efficient calf rearing in cold regions.

Materials and methodsMaterials preparation

B. megaterium (R&D center of COFCO Grain and Oil Industry Co., Ltd., Changji, China, ccj-bac-meg1801) was cultured in liquid enrichment medium at 37 °C for 48 h with agitation, then precipitated at 4 °C for 24 h. The precipitate was centrifuged at 4,000 rpm for 3 min, and the supernatant was discarded. The pellet was mixed with bacterial suspension, Tween-80, skimmed milk powder, and wheat bran (1:1:2:1, w/w) and lyophilized at −80 °C for 72 h to obtain freeze-dried powder. The freeze-dried powder was confirmed to be primarily in the spore form (>95% spores via microscopic count), and spore counts remained stable (±5%) when stored at 4 °C for 30 days. Prior to the trial, the viability of the powder after reconstitution in warm milk (42 °C) was verified, with a viability loss of <10% over a 30 min period. The final product had a survival rate of 88.47%, yielding a concentration of 2.6 × 1010 CFU/g. During the trial, the actual CFU delivered per calf was verified weekly by plating serial dilutions of the milk-probiotic mixture immediately after preparation. Gentamicin was obtained from Ruicheng Lvman Biopharmaceutical Co., Ltd.

The experiment was conducted from November to December 2024, when the average ambient temperature was −13.5 °C and wind speeds of beaufort scale 4–8 occurred 4–6 days per week. The trial was performed at a cooperative calf facility in Emin County, Tacheng, Xinjiang (46°65′N, 83°25′E). The region has a continental temperate climate with short hot summers, rapid cooling in autumn, and long cold dry winters. Average winter temperatures range from −6 °C to −7 °C, with extreme lows reaching −30 °C and heavy snowfall. In spring, the mean temperature is about 7 °C, with large diurnal variation and frequent strong winds.

Animals and diets

This study employed a single-factor randomized experimental design. Fifty healthy neonatal crossbred calves (Simmental × Xinjiang Brown), blocked by birth weight (40.8 ± 2.6 kg) and birth date, were randomly assigned using a number generator in Microsoft Excel to five groups (n = 10 per group; one calf per replicate). Housing, feeding, and immunization followed the cooperative’s standard protocols. Calves were individually housed in separate calf hutches (1.50 × 2.50 m; 3.75 m2) within a calf shed, each bedded with clean wheat straw. The temperature in the pen is 6 °C, with a humidity of 62%. Calves received 4 L of warm colostrum within 1 h after birth. From birth to 7 days, calves were fed 6 L/day of milk at 40 °C. From 8 to 28 days, calves received 8 L/day of milk divided into three meals (08:30, 14:30, and 20:30). B. megaterium powder was mixed into the milk immediately before feeding. Starter was offered from 7 days of age, and alfalfa was available ad libitum.

B. megaterium was provided as freeze-dried powder (2.6 × 1010 CFU/g) and mixed with milk warmed to 42 °C. Group I received only the basal diet; Group II received the basal diet plus 50 mg/day gentamicin per calf, a dose chosen based on long-term use at the cooperative. Groups III, IV, and V received 250, 500, and 1,000 mg/day of B. megaterium per calf, respectively. The experimental period was 28 days (from 1 to 28 days of age). The diet for neonatal calves consisted of milk, starter feed, and alfalfa. The composition of the starter feed is presented in Table 1. Nutritional levels of each diet are detailed in Table 2.

ItemsContent (%)Soybean meala25Expended soy13Whey powder5Corn25Expended corn17.9Wheat bran10CaHPO40.8NaCl0.5Limestone1.8Premixb1Total100

Composition of starter feed.

a

Soybean meal: 89.1% dry matter and 42.6% crude protein.

b

The premix provided the per kg of diet as follows: VA 15,000 IU, VD 5,000 IU, VE 50 mg, Fe 90 mg, Mn 60 mg, Cu 12.5 mg, Zn 100 mg, I 2.0 mg, Co 0.5 mg, Se 0.3 mg.

ItemsMilkStarterAlfalfaDry matter (%)12.6391.2794.63Crude protein (%)3.1918.0214.67Ether extract (%)3.924.191.53Crude ash (%)0.697.579.35Calcium (%)0.121.091.47Phosphorus (%)0.090.580.31Total energy (MJ/kg)2.7217.6517.98

Milk, starter feed and alfalfa nutrition.

The nutritional levels were measured.

Sampling

The amounts of feed offered and residual feed were recorded daily throughout the experimental period. Dry matter intake (DMI) was calculated for days 1–14, 15–28, and 1–28. Calves were weighed on days 1, 14, and 28 before the morning feeding. Average daily gain (ADG) was then calculated for days 1–14 days, 15–28 days, and the overall period (1–28 days). The feed-to-gain ratio (F/G) was calculated as DMI/ADG.

On 14 and 28 days, blood samples were collected from the jugular vein of neonatal calves using sterile vacuum tubes containing 0.1 mL of 0.04% EDTA anticoagulant before feeding. Samples were centrifuged at 3,000 r/min for 15 min at 4 °C. The serum was then separated and stored at −80 °C for subsequent analyses of physiological, biochemical, immune, and antioxidant indices.

One day before the end of the trial, fresh rectal fecal samples (~20 g) were collected using sterile long-arm gloves. Samples were immediately transferred into cryotubes with protective solution and snap-frozen in liquid nitrogen for microbial analysis.

Fecal scoring

As detailed in Table 3, a hierarchical scoring system was used to assess the presence and severity of diarrhea. Fecal consistency of the calves was evaluated twice daily (07:00 and 20:00 h) by two trained veterinarians following the method of Larson et al. (1977). Consistency was assessed using a 3-point scale, where 0 indicated firm but not hard and 3 indicated watery feces. A fecal consistency score ≥2 was classified as diarrhea (Renaud et al., 2020). Interobserver agreement among veterinarians was evaluated prior to the study using Fleiss’ kappa (0.81). Following the experiment, the average fecal score and diarrhea frequency were calculated using the following formulas:

ScoreTrait1Without fetid odor and hard firm feces2Without fetid odor but slightly soft feces3With a light fetid odor and soft, partially formed feces4With a distinct fetid odor and loose, semiliquid feces5With a pungent fetid odor and watery, mucous-like fecesBlood parameter analysis

Serum samples were thawed at room temperature and gently vortexed before analysis. Biochemical parameters were measured at the Clinical Chemistry Laboratory of the Third People’s Hospital of Xinjiang Uygur Autonomous Region (a tertiary-care facility) using an automated biochemical analyzer (Cobas 8000, Roche Diagnostics, Switzerland). The measured parameters included blood urea nitrogen (BUN), glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALT/AST ratio, total bilirubin (TBIL), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), globulin (GLOB), ALB/GLOB ratio, triglycerides (TG), total cholesterol (TC), and creatinine (CRE).

Serum IgG (Kit No. H106-1-1), IgA (Kit No. H108-1-1), IgM (Kit No. H109-1-1), IL-2 (Kit No. H003-1-1), and IFN-γ (Kit No. H025-1-1) and antioxidant indices [superoxide dismutase (SOD, Kit No. A001-3-2), glutathione peroxidase (GSH-Px, Kit No. A005-1-2), total antioxidant capacity (T-AOC, Kit No. A015-2-1), catalase (CAT, Kit No. A007-1-1), and malondialdehyde (MDA, Kit No. A003-1-2)] were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Microbiota diversity analysis

Based on growth, immune, antioxidant, and diarrhea data, 500 mg/day per calf was selected as the optimal B. megaterium dose for microbiota analysis. Thus, in this study, Groups I and II served as control groups, while Group IV (500 mg/day B. megaterium) served as the treatment group. This design enables mechanistic exploration of the optimal-dose phenotype but does not resolve dose–microbiota relationships across all probiotic levels. Rectal content samples were thawed, and DNA was extracted using a kit (Tiangen Biochemical Technology, Beijing, China) under aseptic conditions. DNA concentration was assessed via 0.8% agarose gel electrophoresis, and purity was determined using a multimode microplate reader (Infinite M200, Tecan, Switzerland). To ensure representativeness and avoid selection bias, six samples per group were selected for 16S rRNA gene amplicon sequencing (Beijing Novogene Bioinformatics Co., Ltd.) using a stratified random sampling method based on diarrhea incidence and initial body weight. Qualified DNA served as the template for PCR amplification of the bacterial 16S rRNA gene V3–V4 region, using the barcoded primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were purified and analyzed by 2% agarose gel electrophoresis in 1× TAE buffer. Target bands were excised, and PCR products were purified and recovered using the GeneJET PCR Purification Kit (Thermo Fisher Scientific). DNA libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Inc.), quantified with a Qubit fluorometer, and sequenced on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, United States).

Raw sequences were processed using a 97% similarity operational taxonomic unit (OTU) clustering pipeline. Briefly, primer sequences were trimmed, and paired-end reads were merged using USEARCH v7.0. Quality filtering was applied to remove reads with an expected error threshold >1.0. Chimeric sequences were identified and removed using the de novo mode of the UCHIME algorithm as implemented in USEARCH. High-quality, non-chimeric sequences were then clustered into OTUs at a 97% similarity threshold using the UPARSE-OTU algorithm within USEARCH. From each OTU, the most abundant sequence was selected as the representative sequence. Taxonomic assignment of OTU representative sequence was analyzed by RDP Classifier (version 2.2) against the Silva database (version 138.1) using a confidence threshold of 0.7.

Microbiota α-diversity indices (observed OTUs, Shannon, Simpson, and Chao1) were calculated using on rarefied OTU tables. β-diversity was assessed using unweighted UniFrac distances. The statistical significance of group differences in microbial community composition was tested using PERMANOVA with 999 permutations. Results were visualized via principal coordinate analysis (PCoA). Venn diagrams depicting OTU overlap among groups were generated using the Novogene online tool. Centered log-ratio (CLR) transformed genera abundances were correlated with serum indices via Spearman analysis; p-values were FDR-corrected (Benjamini–Hochberg, q < 0.05). The corrplot package visualized results in R (Wang et al., 2025).

Statistical analysis

Raw data were managed using Microsoft Excel. Statistical analyses were performed in IBM SPSS Statistics 31.0. Statistical differences among groups were determined by one-way ANOVA followed by Duncan’s multiple range test for post-hoc comparisons. This test was selected due to its high sensitivity in detecting potential differences across multiple groups, which aligns with the exploratory aim of this study to identify candidate microbial taxa associated with dietary intervention (Yang et al., 2024; Ma et al., 2025). Differences were considered significant at p < 0.05.

ResultsGrowth performance and diarrhea in neonatal calves

The effects of B. megaterium supplementation on growth performance are shown in Table 4. No significant difference in body weight (BW) was observed among the groups at the initial stage (1 day, p > 0.05). At day 28, BW in Groups II–V was higher than in Group I (p < 0.05). However, no significant difference was found between Groups III, IV, V, and Group II (p > 0.05). There were no significant differences in DMI between treatment groups at each period. Calves in Group II, IV, and V showed significantly higher ADG from 1 to 14 days compared to Group I (p = 0.029). From days 15 to 28, ADG was higher in Groups II–V than in Group I (p = 0.041). Over days 1–28, ADG was higher in the supplemented groups than in Group I (p = 0.038), with Group II showing the numerically highest value (Table 4). It is noteworthy that Group III, Group IV, and Group V showed no significant differences compared to Group II (p > 0.05). Consistent with these gains, F/G was greater in Group I than in the supplemented groups during days 1–14, 15–28, and 1–28 (p = 0.048, 0.041, and 0.037, respectively).

ItemsDaysGroup IGroup IIGroup IIIGroup IVGroup VSEMp-valueBW (kg)1 day38.3337.6738.2137.9538.096.180.88114 days41.38b42.96a42.05a43.02a42.86a6.100.47328 days47.80b50.21a49.32a50.14a49.87a2.360.038DMI (g/day)1–14 days830.62802.37807.12801.06799.3426.930.62415–28 days1098.721085.441090.531082.261089.6670.200.3111–28 days964.67947.51943.47941.66958.219.130.774ADG (g/day)1–14 days218.01b357.86a274.30ab362.33a341.17a92.540.02915–28 days458.36b517.79a519.08a508.41a502.92a81.080.0411–28 days338.19b417.86a400.76a428.37a421.17a29.270.038F/G1–14 days3.81a2.12b2.94ab2.21b2.34b0.190.04815–28 days2.40a2.10b2.10b2.19b2.16b0.280.0411–28 days2.85a2.12b2.35b2.20b2.280.200.037

Growth performance of neonatal calves supplemented with B. megaterium.

BW, body weight; ADG, average daily gain; DMI, dry matter intake; F/G, ratio of DMI to ADG. a,bDifferent lowercase letters indicate significant differences (p < 0.05). Group I: control group (basal diet); Group II: antibiotic group (basal diet + 50 mg/day per calf gentamicin); Group III: basal diet + 250 mg/day per calf B. megaterium; Group IV: basal diet + 500 mg/day per calf B. megaterium; Group V: basal diet + 1,000 mg/day per calf B. megaterium.

Dietary treatments significantly affected fecal score and diarrhea frequency (Table 5). Average fecal score varied among groups (p = 0.026). Calves in Group I had a higher fecal score than those in Groups II, III, IV, and V (p < 0.05). Diarrhea frequency also differed among treatments (p = 0.045). Groups II, IV and V had lower diarrhea frequency (9.97–12.38%) than Groups I and III (14.96–15.45%; p < 0.05).

ItemsGroup IGroup IIGroup IIIGroup IVGroup VSEMp-valueAverage fecal score2.50b1.63a2.09a1.69a1.72a0.2380.026Diarrhea frequency (%)15.45a9.97b14.96a10.86b12.38b2.8600.045

Effect of B. megaterium on diarrhea in calves.

a,bDifferent lowercase letters indicate significant differences (p < 0.05). Group I: control group (basal diet); Group II: antibiotic group (basal diet + 50 mg/day per calf gentamicin); Group III: basal diet + 250 mg/day per calf B. megaterium; Group IV: basal diet + 500 mg/day per calf B. megaterium; Group V: basal diet + 1,000 mg/day per calf B. megaterium.

Serum biochemical parameters

Serum biochemical parameters in calves supplemented with B. megaterium are presented in Table 6. Serum GLU concentrations differed significantly among groups (p = 0.029). Group II had the lowest GLU level (3.86 mmol/L), which was significantly lower than other groups (p < 0.05). ALP also varied significantly (p = 0.048), with higher values in Groups I and IV, compared to Groups II, III, and V. ALB concentrations differed significantly among the groups (p = 0.025). Group IV had the highest ALB level (35.74 g/L), which was higher than those in other groups, Group I had intermediate levels (33.20 g/L). TG levels showed significant differences (p = 0.033), with Group IV higher than Groups I, II, III, and V. No significant differences were observed in other parameters.

ItemsGroup IGroup IIGroup IIIGroup IVGroup VSEMp-valueBUN (mmol/L)5.236.844.103.554.520.910.624GLU (mmol/L)4.28b3.86c4.84b5.15a4.32b0.690.029ALT (U/L)28.4732.5629.2126.8327.249.210.081AST (U/L)85.40111.6235.2134.8534.209.940.066TBIL (μmol/L)5.847.246.546.265.9251.680.511ALP (U/L)199.77a199.38b188.55b202.32a192.12b55.910.048TP (g/L)63.5561.5460.6065.6761.5211.980.052ALB (g/L)31.54b31.20b30.46b35.74a33.20ab11.270.025GLOB (g/L)29.5430.2730.4229.7030.4560.760.056ALB/GLOB1.131.040.971.181.040.480.349TG (mmol/L)0.55b0.52b0.50b0.62a0.52b0.350.033TC (mmol/L)3.353.153.203.513.151.200.931CRE (μmol/L)62.7460.9059.2265.2461.7214.580.065

Effect of B. megaterium on serum biochemical parameters in calves.

a–cDifferent lowercase letters indicate significant differences (p < 0.05). Group I: control group (basal diet); Group II: antibiotic group (basal diet + 50 mg/day per calf gentamicin); Group III: basal diet + 250 mg/day per calf B. megaterium; Group IV: basal diet + 500 mg/day per calf B. megaterium; Group V: basal diet + 1,000 mg/day per calf B. megaterium.

Serum immune parameters

Serum immunoglobulin and cytokine concentrations in calves supplemented with B. megaterium are presented in Table 7. IgG concentrations differed significantly among the groups (p = 0.024). Group I had the lowest level, significantly lower than other groups (p < 0.05). IL-1β concentration was lowest in Group IV, significantly lower than in other groups (p = 0.038). IL-10 levels showed significant differences (p = 0.045), with the lowest concentrations in Group III, which were lower (p < 0.05) than those in Groups I, II, IV, and V. TNF-α concentrations differed among groups (p = 0.036), with Group IV exhibiting the lowest levels, lower than those in Groups I, II, III, and V (p < 0.05). Similarly, IFN-γ levels varied significantly (p = 0.023), with higher concentrations in Group I compared to other groups.

ItemsGroup IGroup IIGroup IIIGroup IVGroup VSEMp-valueIgG (g/L)6.54c8.20b