Effects of Bacillus subtilis C-3102 addition on nutrient digestibility, faecal characteristics, blood chemistry and faecal Lactobacilli spp., Enterococci spp., and Escherichia coli in healthy dogs

Abstract

The benefits of probiotics for animal health are widely recognised. In this study, we investigated the effects of dietary supplementation with Bacillus subtilis C-3102 on diet digestibility, faecal characteristics, intestinal shedding of microbiota, and hematological characteristics in healthy dogs. Adult Golden Retriever dogs (n = 18) were divided into three groups: the control group (no B. subtilis), the BS1 group (1 × 10⁹ CFU/g of B. subtilis), and the BS2 group (2 × 10⁹ CFU/g of B. subtilis). Dogs were fed for a 30-day administration period, and total faeces were collected in the last five days. On the first and last days of the study, blood samples were taken to determine hematological characteristics. In addition, the dry matter content, faecal consistency score, faecal output, pH, ammonia level, and quantity of short- and branched-chain fatty acids (SCFAs and BCFAs) in faecal samples were also evaluated. Results revealed that the dry matter, fibre, organic matter, and protein digestibility coefficients were higher in BS1 and BS2 (p < 0.05) compared to the control. Lower pH and higher acetate, propionate, and isobutyrate levels were detected in BS1 and BS2 (p < 0.05). Lower ammonia levels and firmer stools were observed in BS2 (p < 0.001). The numbers of faecal enterococci and lactobacilli were higher in BS1 and BS2 (p < 0.05). Furthermore, B. subtilis increased the concentrations of WBCs, RBCs, and granulocytes in BS1 and BS2 (p < 0.05). Data in the present study suggest that dietary supplementation with B. subtilis improved faecal quality, enhanced nutrient digestibility, and contributed to the dogs’ gut health by reducing gut ammonia, increasing SCFAs, and improving the number of Lactobacillus and Enterococci.

HIGHLIGHTS

• Probiotics are one of the most commonly used feed additives in dog food.

• Bacillus subtilis boosts the immunity and overall gastrointestinal health of companion animals.

• B. subtilis dietary supplementation improved dogs’ gut health, immunity, and digestibility of food.

• B. subtilis increased the concentration of WBCs, RBCs, and granulocytes.

• B. subtilis reduced gut ammonia and increased SCFAs and the number of lactobacilli and enterococci in faeces.

Keywords:

• Bacillus subtilis C-3102
• blood profile
• nutrient digestibility
• faecal characteristics
• faecal microbial count

Introduction

The contribution of functional additives in diets to dogs’ health has attracted attention recently (Rossi et al. 2020). The dogs’ normal intestinal microflora rapidly changes in response to gastrointestinal infections, oral administration of medicines (Zeng et al. 2017), and dietary changes (Pilla and Suchodolski 2021). Probiotics are one of these supplements that can improve the dogs’ health status and welfare. Probiotics are living microorganisms that promote intestinal health by maintaining, adjusting, or restoring the optimal balance of the gut microflora (Paap et al. 2016). In addition, probiotics can neutralise the harmful effects of pathogenic organisms by producing fatty acids through fermentation (Schauf et al. 2019). For this reason, diet supplementation with probiotics is a practical way to maintain a healthy gut by preventing or controlling pathogens in the gastrointestinal tract (GIT) and promoting the population of beneficial GIT microflora (Craig 2021).

Among the bacterial genera utilised as probiotics, the genus Bacillus stands out. Bacilli exhibit the advantage of sporulation (Bastos et al. 2020), which allows their stability during food preparation and storage. They can also withstand gastric acidic environments during digestion (Biourge et al. 1998; Coppola and Gil-Turnes 2004). The dried spores of bacilli can germinate in the intestine when reintroduced to proper nutrients and water, initiating proliferative cell development (Moir 2006). Previous studies have shown that dietary supplementation with Bacillus subtilis reduced gas formation (Paap et al. 2016) and ammonia concentration (Bastos et al. 2020) in dogs’ intestines. It was also reported to promote eubiosis and decrease the generation of inflammatory modulators in mice’s gut (Zhang et al. 2016).

Despite the known benefits of probiotics, data on their influence on dogs’ health is scarce. Some of the available studies claimed that B. subtilis dietary addition improved intestinal health by enhancing faecal characteristics and consistency and altering the dogs’ microbiota (Félix et al. 2010; Rychen et al. 2017; Bastos et al. 2020; de Lima et al. 2020). On the other hand, there is some controversy regarding the effect of the dietary addition of B. subtilis on nutrient digestibility. Some studies suggested that B. subtilis did not affect the nutrient digestibility of dogs’ diets (González-Ortiz et al. 2013; de Lima et al. 2020), while Schauf et al. (2019) reported improved fat digestibility in dogs offered a B. subtilis dietary supplement. Additionally, scientific studies on the effect of B. subtilis on dogs’ blood profiles are lacking.

Therefore, this study aimed to investigate the effects of dietary supplementation with B. subtilis C-3102 on the faecal characteristics, nutrient digestibility, blood parameters, including the levels of cholesterol and triglycerides, and the counts of white blood cells (WBCs), lymphocytes, monocytes, granulocytes, and the red blood cells (RBCs), and faecal microbiota, including Lactobacillus sp., enterococci, and Escherichia coli in healthy dogs.

Materials and methods

Bacterial strain, animals and experimental design

The B. subtilis C-3102 studied here was purchased from Nutrilab^® (Nutriflora-Canine, Konya, Turkey) as spray-dried endospores. The product contained 5.0 × 10⁸ CFU/g of B. subtilis. Eighteen spayed/neutered adult (nine male and nine female) Golden Retriever dogs (age: 5 –6 years, body weight: 22.5 ± 1.2 kg, body condition score: 5–6) were used. The dogs were divided into three groups with similar average body weights and genders (three males and three females). They were housed individually in kennels at the College of Veterinary Medicine Research and Application Farm, Selçuk University, and were served the same commercial dry food. The three animal groups were as follows: the control group (served only dry food), the BS1 group (provided with dry food supplemented with 1 × 10⁹ CFU/g of B. subtilis), and the BS2 group (received dry food with 2 × 10⁹ CFU/g of B. subtilis). The experimental diet lacked any other probiotics or prebiotics except its soluble fibre fraction. The probiotic powder (B. subtilis C-3102) was added daily to the weighed dry food and mixed well for each dog. According to the observation, no dog refused food, and no residue of probiotic powder was found in the bowls. The experiment lasted for 30 days. All dogs were evaluated clinically and physiologically, vaccinated, dewormed, and received no antibiotics before the study. The kennels had open (510 × 230 cm) and closed (190 × 190 cm) areas for dogs, including a bunk and free access to clean drinking water. The kennel’s climate temperature fluctuated from 16 to 28° C, with a 12-hour side-by-side light-dark cycle from 6 pm to 6 am. The provided diet was an extrusion dry food designed to match the energy and nutritional requirements of an adult dog, as suggested by the European Pet Food Industry Federation, FEDIAF (FEDIAF 2019). The total dietary fibre (TDF), insoluble dietary fibre (IDF), and soluble dietary fibre (SDF) levels, as well as other chemical compositions of the diet, are shown in Table 1. Food was offered once a day in a sufficient amount to meet the metabolisable energy (ME) requirements of the dogs, according to the following equation (NRC 2006): ME (MJ/day) = 95 × Body weight^0.75.

Table 1. Ingredient and chemical composition of dry extruded food, %.

Ingredient composition
Poultry meal	22
Barley	26
Maize	10
Maize gluten meal	10
Rice	26.6
Potassium chloride	0.5
Salt	0.1
Vitamin/mineral pre-mixture^a	0.4
Sunflower oil	2.5
Beef tallow	1.9
Determined Chemical Composition
Dry matter, %	91.5
Ash, %	5.28
Acid hydrolysed ether extract, %	9.21
Crude fibre, %	6.45
Crude protein, %	18.45
Metabolizable energy (ME), kcal/kg^b	3242.5
Total dietary fibre, %	12.54
Insoluble dietary fibre, %	9.28
Soluble dietary fibre, %	3.26
Calcium, %	1.3
Phosphorus, %	0.85
Sodium, %	0.35
Vit-A vitIU/kg	12 000
Vit-D IU/kg	1100
Vit-E mg/kg	60

^aVitamin-mineral premixture (mg/kg): biotin 250 mg, folate 2 mg, niacin 50 mg, pantothenic acid 20 mg, riboflavin 8 mg, thiamine 4 mg, Vit. B6 5 mg, Vit. B12 25 mg, Vit. A 3.6 mg, cholecalciferol 125 mg, Vit. E 50 mg, Mn proteinate 70 mg, Zn proteinate 100 mg, Fe proteinate 70 mg, Cu proteinate 14 mg, Iodine 1 mg, Co proteinate 350 mg, Se proteinate 140 mg, Mo proteinate 700 mg, Mg oxide 35.000 mg.

^bCalculated using the NRC (2006) equations.

Chemical composition of food and faecal samples

To determine the chemical composition of food samples, the dry matter (DM), ash, acid hydrolysed ether extraction (EE), crude fibre (CF), and crude proteins (CP) were analysed according to the methods of the Association of Official Agricultural Chemists, AOAC (2019). In addition, the chemical composition of the collected faecal samples was also analysed to determine nutrient digestibility. All analyses were carried out in duplicate. The ME level of the food was calculated using the following formula, according to the guidelines of the National Research Council, NRC (2006): $$\text{I}.\text{Gross}\text{energy}\left(\text{GE}\right):\text{GE}\left(\text{kcal}\right)=\left(\text{5}.\text{7}\times \text{CP }\%\right)+\left(\text{9}.\text{4}\times \text{EE}\%\right)+\left[\text{4}.\text{1}\times \left(\text{NFE}\%+\text{CF}\%\right)\right]$$ $$\text{Nitrogen}-\text{free}\text{extract},\text{NFE}\left(\%\right)=\text{DM}\%–\left(\text{EE}\%+\text{CP}\%+\text{ash}\%+\text{CF}\%\right)$$ $$\text{II}.\text{Energy digestibility}\left(\%\right)=91.2–\left(1.43\times \text{CF}\%\right)$$ $$\text{III}.\text{Digestible}\text{energy}:\text{kcal}\text{DE}=\left(\text{kcal}\text{GE}\times \text{energy}\text{digestibility}\right)/\text{1}00$$ $$\text{IV}.\text{Metabolizable}\text{energy}:\text{ME}\left(\text{kcal}\right)=\text{kcal}\text{DE}–\left(\text{1}.0\text{4}\times \text{CP}\%\right)$$

The total dietary fibre (TDF) and insoluble dietary fibre (IDF) contents of dog food were analysed using a Megazyme assay (cat. no. K-TDFR100A/K-TDFR-200A 04/17; Megazyme, Wicklow, Ireland). The soluble dietary fibre (SDF) content (as % DM) of the samples was determined by calculating the difference between the TDF and the IDF contents. These analyses were performed according to the procedure described by Kara (2021).

Determination of faecal consistency scores

Before collecting faeces in the last four days of the experiment, daily faecal consistency scoring was performed by three different authors (academic staff) of this study. The faecal consistency was scored according to a 1–5 grading system as follows: (1) diarrhoea-like and watery, (2) soft and slightly shaped, (3) soft and shaped, leaving marks on the floor, (4) well-formed, undispersed, (5) very well-formed and dry (Strickling et al. 2000).

Determination of nutrient digestibility of dogs’ diet

The nutrient digestibility of the dry food with or without the B. subtilis supplementation was determined using the total collection method (AOAC 2019). The experiment was conducted for 30 days, and all freshly excreted faeces were collected daily for the last five days. The average of the total faecal output collected daily during the collection phase was used to determine the daily faecal output (g as-is/d). After weighing the faeces, they were kept in a freezer (-18 °C). Subsequently, the faeces of each dog were thawed at the laboratory temperature (23–25 °C) and thoroughly mixed. Afterward, the faecal samples were dried in an oven (VWRVenti-line, USA) at 55 °C for 48 h. Dry faeces were ground using a laboratory mill (Retsch SM100, Germany) and passed through a 1-mm sieve. The apparent total tract digestibility (ATTD) of nutrients was calculated using the following formula based on the results of food and faeces: $$\text{ATTD}\%=\left[\frac{\left(\text{g of nutrient intake}–\text{g of nutrient excretion}\right)}{\text{g of nutrient intake}}\right]\times 100$$

Determination of short- and branched-chain fatty acids, ammonia and pH of faeces

On days 28, 29, and 30 of the experiment, faecal samples (three samples for each dog) were collected within 15 min after excretion to determine the short-chain fatty acids (SCFAs), branched-chain fatty acids (BSFAs), ammonia, and pH. Samples were analysed in duplicate separately after collection. The fresh faecal samples were mixed 1:5 with distilled water, shaken and incubated for 1 min at laboratory ambient tempreture (24–25 °C. The faecal pH was measured with a digital pH metre (Chuppava et al. 2023). The fatty acid profiles of fresh faeces collected during the last three days of the experiment (Abd El-Wahab et al. 2022) were determined by gas chromatography (Agilent 6890 N, USA). The SCFA and BCFA levels were determined in fresh faeces obtained up to 15 min after defaecation. Ten grams of faeces were weighed in a correctly labelled plastic jar with a cover and mixed with 30 mL of 16% formic acid. This suspension was blended and stored at 4 °C for three to five days. Before the examination, the mixtures were centrifuged for 15 min at 5000 rpm x g (2 L21 centrifuge, Sigma, Osterodeam Hans, Germany). For faecal ammonia, 2 g of fresh faeces were diluted 1:5 with distilled water, and 1 mL of samples were mixed with 20 µL of sulphuric acid. The ammonia levels were then determined by a spectrometric method (Weatherburn 1967).

Blood sampling

On the experiment’s first day (day zero) and last day (day 30), blood samples were collected from each dog to study hematological changes. The counts of monocytes, granulocytes, white blood cells (WBCs), lymphocytes, and red blood cells (RBCs) were determined in whole blood samples using an automated analyser (ADVIA 120, USA). Serum was separated from blood cells by centrifugation at 3000 RPM for 15 min at 4 °C and kept at −18 °C until use. Subsequently, the cholesterol and triglyceride levels were determined using a biochemistry analyser (Hitachi 747, Japan) at the Faculty of Veterinary Medicine laboratory, Selçuk University.

Counting of faecal bacteria

Fresh faecal samples (within 15 min of excretion) were collected in 2-mL sterile propylene cryotubes (Biosigma S.r.l., Cona, Italy), placed on an ice cube containing a foam box, transported immediately, and kept at −80 °C until plating. The collected samples (1 g each) were diluted with 9 mL of 1% peptone broth (Becton, Dickinson & Co., Franklin Lakes, NJ) and homogenised. The samples were plated on de Man-Rogosa-Sharpe (MRS) agar medium (no. 288130; Difco) with vancomycin and bromocresol green, which supports the growth of lactobacilli. The Petri dishes were then incubated at 37 °C for 48 h (Fu et al. 2006). The violet-red bile agar was employed to enumerate enterococci using the double pour method at 37 °C for 48 h. The tryptone bile x-glucuronide medium, TBX (Oxoid, CM945, England), was used for Escherichia coli enumeration at 37 °C for 24 h. Suspected colonies were confirmed with the Kovacs reagent. The colonies of E. coli and lactobacilli were counted immediately after incubation. Bacterial counts were expressed as log₁₀ CFU/g of faeces.

Statistical analysis

The microbiological parameters and faecal score data were compared using the Kruskal-Wallis test concerning the group and time of data collection. If the results of the Kruskal-Wallis tests were significant, a multiple comparison analysis based on a pairwise two-sample Wilcoxon test was performed. The differences between the bacterial counts were assessed by the Wilcoxon signed-rank test (nonparametric). For the blood profile data, the normal distribution and homogeneity of variances were checked with the Shapiro–Wilk test and evaluated with the Bonferroni test. The bacterial count data were also analysed using a complete randomised design with dose, time, and dose × time interactions. The digestibility and faecal characteristics data were analysed by Tukey’s HSD test in ANOVA. Data with a p-value of < 0.05 were considered statistically significant.

Results

Nutrient digestibility

As shown in Table 2, the coefficients of total tract apparent digestibility (CTTAD) for dry matter (DM), crude fibre, organic matter, and crude proteins were higher in the BS1 and BS2 groups, which received the B. subtilis C-3102-supplemented diet, compared to those of the control group, CON (p < 0.05). However, the dietary supplementation with B. subtilis C-3102 did not affect the CTTAD of fat from extruded dog food (p > 0.05).

Table 2. Apparent nutrient digestibility coefficients of the groups, % (Mean  ±  SEM).

	CON	BS1	BS2	p Value
DMD	71.52 ± 1.19^b	75.52 ± 1.44^a	75.06 ± 0.57^a	0.031
OMD	78.11 ± 0.92^b	83.33 ± 1.15^a	83.57 ± 0.65^a	0.012
EED	90.78 ± 0.55	91.28 ± 1.22	92.29 ± 0.92	0.589
CFD	61.60 ± 1.02^b	64.94 ± 0.88^a	65.92 ± 0.99^a	0.001
CPD	81.84 ± 0.73^b	85.78 ± 0.97^a	85.30 ± 0.58^a	0.001

DMD: dry matter digestibility; OMD: organic matter digestibility; EED: fat digestibility; CFD: crude fibre digestibility; CPD: crude protein digestibility; CON: control; BS1: B. subtilis supplementation group 1 receiving 1 × 10⁹ CFU/g of B. subtilis; BS2: B. subtilis supplementation group 2 receiving 2 × 10⁹ CFU/g of B. subtilis; SEM: standard error of the mean;^abcvalues in the same row that do not share the same superscripted letter differ significantly (p < 0.05).

Faecal parameters

The faecal consistency scores were higher in the B. subtilis supplementation groups, BS1 and BS2, than in the control group (p = 0.04), consistent with the higher faecal DM contents observed in these groups. In parallel with the increased digestibility level, faecal pH was lower in the BS1 and BS2 groups (p = 0.001) than in the control group. In addition, more elevated faecal concentrations of acetate, propionate, and isobutyrate (p = 0.001) were observed in the faeces of dogs fed with B. subtilis C-3102-supplemented diets. Total SCFAs of BS1 and BS2 were higher (p = 0.001) The BS2 group showed lower concentrations of faecal ammonia (p = 0.01) than the BS1 and control groups (Table 3).

Table 3. Faecal parameters of the control and B. subtilis groups of dogs (Mean  ±  SEM).

	CON	BS1	BS2	p Value
Faecal consistency score	3.78 ± 0.13^b	4.15 ± 0.14^a	4.23 ± 0.12^a	0.041
Faecal pH	7.11 ± 0.15^a	6.81 ± 0.08^b	6.78 ± 0.11^b	0.001
Faecal dry matter	45.59 ± 0.57^b	50.86 ± 0.69^a	51.37 ± 0.43^a	0.001
Faecal output (g as-is/day)	101.97 ± 5.72	96.86 ± 7.39	95.97 ± 6.66	0.372
Faecal NH3 (µmol/g DM)	0.71 ± 0.35^a	0.64 ± 0.50^a	0.49 ± 0.15^b	0.011
Acetate (µmol/g DM)	153.15 ± 24.13^b	204.45 ± 25.88^a	195.37 ± 32.01^a	0.001
Propionate (µmol/g DM)	92.93 ± 23.24^b	118.11 ± 26.74^a	104.92 ± 30.87^a	0.001
Butyrate (µmol/g DM)	18.01 ± 2.09	21.31 ± 4.53	20.49 ± 1.99	0.068
Isobutyrate (µmol/g DM)	2.26 ± 0.19^b	3.61 ± 0.72^a	1.91 ± 0.21^b	0.001
Isovalerate (µmol/g DM)	2.42 ± 0.15	3.02 ± 0.20	1.87 ± 0.06	0.074
Valerate (µmol/g DM)	0.76 ± 0.11	0.52 ± 0.04	0.65 ± 0.1	0.186
Total SCFAs (µmol/g DM)	254.11 ± 11.61^b	343.83 ± 13.41^a	320.78 ± 16.31^a	0.001
Total BCFAs (µmol/g DM)	6.37 ± 0.39^ab	7.79 ± 0.76^a	5.26 ± 0.42^b	0.012

CON: basal diet; BS1: CON + 1 × 10⁹ CFU/g of B. subtilis; BS2: CON + 2 × 10⁹ CFU/g of B. subtilis; SCFAs: short-chain fatty acids; BCFAs: branched-chain fatty acids;^a,b,cvalues in the same row that do not share the same superscripted letter differ significantly (p < 0.05).

Faecal bacterial counts

Bacterial numbers of the first (day 0) and day 30 which did not show normal distribution were evaluated with the Wilcoxon test. The bacterial counts of Lactobacillus spp. and Enterococcus spp. increased in the faeces of dogs in the BS1 and BS2 groups on the 30^th day compared with the control (Table 4). An increased count of E. coli was found in the BS1 group after 30 days (p = 0.03). The highest numbers of Lactobacillus spp. (3.14 ± 0.56 CFU/g, p = 0.01) and of Enterococcus spp. (3.58 ± 0.32 CFU/g, p = 0.001) were observed in the BS2 group on day 30 of the experiment (Table 4). Notably, the dosage and time combination significantly affected the counts of Lactobacillus spp. and Enterococcus spp. (Table 4).

Table 4. Effects of dietary supplementation with Bacillus subtilis on faecal microbiota in dogs, log₁₀ CFU/g.

Group	Dose (gram)	Time (day)	n	Lactobacillus spp.	E. coli	Enterecoccus spp.
CON	0	zero	6	1.40±0.2	1.28±0.17	2.04±0.1
		30	6	1.93±0.15	1.50±0.12	2.27±0.21
P				0.068	0.181	0.109
BS1	2	zero	6	1.33±0.07	1.27±0.16	2.19±0.22
		30	6	2.36±0.13	1.86±0.1	3.46±0.04
P				0.024	0.042	0.031
BS2	4	zero	6	1.54±0.54	1.31±0.05	2.75±0.05
		30	6	3.14±0.56	1.51±0.15	3.58±0.32
P				0.028	0.061	0.021
CON (day 30)			6	1.93^b	1.50	2.27^b
BS1(day 30)			6	2.36^b	1.86	3.46^a
BS2 (day 30)			6	3.14^a	1.51	3.58^a
P				0.012	0.112	<0.001
Tukey HSD
SEM				0.19	0.07	0.11
Dose				<0.001	0.342	<0.001
Time				<0.001	0.245	<0.001
dose*time				<0.001	0.064	<0.001

CON: basal diet; BS1: CON + 1 × 10⁹ CFU/g of B. subtilis; BS2: CON + 2 × 10⁹ CFU/g of B. subtilis; SEM: standard error of the mean; ^a,bvalues with different superscripted letters are significantly different (p < 0.05).

Blood profile

The blood profile results in Table 5 indicate that the WBCs, RBCs, and granulocyte concentrations were affected by dietary supplementation with B. subtilis. The lymphocyte and RBCs counts were increased in the BS2 group at day 30 (p < 0.05) compared to the control, whereas the WBCs and RBCs concentrations were higher in both the BS1 and BS2 groups (p < 0.05). Moreover, the granulocyte count in the BS1 group at day 30 was higher than that of the control and BS2 groups (p = 0.043). On the contrary, the B. subtilis supplementation did not affect the levels of blood cholesterol and triglycerides (Table 5).

Table 5. Effects of dietary supplementation with Bacillus subtilis on blood profiles of dogs.

Group	Time (day)	N	Cholesterol, mg/dL	Triglycerides, mg/dL	WBCs, 10^3/µL	Lymphocytes^c, %	Monocytes^c, %	Granulocytes^c, %	RBCs, 10^3/µL
CON	zero	6	223.16±32.1	84.33±3.8	8.37±0.61	25.87±1.20	3.81±0.15	59.34±1.4	7.57±1.67
	30	6	219.5±16.09	87.83±4.85	8.66±0.54	26.16±1.15	3.51±0.11	58.98±1.05	8.01±1.56
		p-value	0.865	0.114	0.563	0.665	0.255	0.842	0.291
BS1	zero	6	228.83±14.1	93.16±2.91	7.8±0.52	27.05±0.1	3.66±0.1	60.65±2.40	8.08±0.78
	30	6	203.84±15.5	88.1±3.1	7.97±0.65	27.1±0.1	3.76±0.12	62.98±1.93	7.66±0.58
		p-value	0.264	0.17	0.856	0.971	0.152	0.401	0.678
BS2	zero	6	232.33±12.8	87.1±4.01	8.81±0.66	21.62±0.7	2.98±0.1	60.51±1.27	6.7±0.4
	30	6	204.1±10.5	82.1±4.23	10.65±0.53	24.23±6	3.33±0.2	59.8±1.02	9.72±0.3
		p-value	0.119	0.269	0.039	0.027	0.67	0.945	0.001
CON	30	6	219.5±16.09	87.83±4.82	8.66±0.54^b	26.16±1.15	3.51±0.1	58.98±1.05^b	8.01±0.63^b
BS1	30	6	203.84±15.5	81.1±7.11	7.97±0.65^b	27.1±0.51	3.76±0.12	62.98±1.13^a	7.66±0.58^b
BS2	30	6	204.1±10.5	80.1±10.5	10.65±0.55^a	24.23±6.01	3.33±0.2	59.8±1.02^ab	9.72±0.3^a
		p-value	0.684	0.178	0.017	0.081	0.095	0.043	0.048
		Reference range	136–392	23–102	6.0–17.0	10.0–30.0	2.0–10.0	50.0–80.0	5.5–8.5

^a,bValues with different superscripted letters are significantly different (p < 0.05); RBCs, red blood cells; WBCs: white blood cells; ^cvalues are presented as a percentage of the total WBCs count; CON, basal diet; BS1, CON + 1 × 10⁹ CFU/g of B. subtilis; BS2, CON + 2 × 10⁹ CFU/g of B. subtilis.

Discussion

Probiotics are one of the most commonly used feed additives in dog food. In this study, we investigated the effect of dietary supplementation with B. subtilis C-3102 on dogs’ health and welfare. All dogs did not refuse to eat the food offered with or without the B. subtilis addition. Data suggests that the inclusion of B. subtilis improved the nutrient digestibility of dry food. According to several studies, there is no difference in the nutritional digestibility of diets enriched with B. subtilis, B. licheniformis, or B. amyliquefaciens in dogs (Félix et al. 2010; González-Ortiz et al. 2013; de Lima et al. 2020). However, evidence suggests that dietary supplementation with Lactobacillus acidophilus fructooligosaccharides promoted higher DM digestibility in dogs (Swanson et al. 2002). In a recent study, dogs offered a diet containing B. subtilis C-3102 showed a higher apparent digestibility of fat and nitrogen-free extract (Schauf et al. 2019). In addition, the effect of B. subtilis as a single probiotic on nutrient digestibility was reported in dogs (Félix et al. 2010; Schauf et al. 2019). Likewise, the study of Giang and colleagues revealed enhanced apparent total tract digestibility of protein, fibre, and organic matter due to B. subtilis in pigs (Giang et al. 2011). B. subtilis also improved the apparent ileal digestibility of protein in broilers (Boroojeni et al. 2018). The increased digestibility could be due to the difference in the B. subtilis dosages and supplementation method used in this study. For instance, in a study by de Lima et al. (2020), B. subtilis was added at a cell density of 1 × 10⁶ CFU/g in poultry fat by spraying post-extrusion. Félix et al. (2010) added 1 × 10¹⁰ CFU/g of B. subtilis in 300 mL of soybean oil at 0.01%. Biourge et al. (1998) included B. subtilis CIP 5832 in an extruder with other ingredients and found no effect on the digestibility of dog food. In the present investigation, we studied B. subtilis dosages of 1 × 10⁹ and 2 × 10⁹ CFU/g by directly supplementing the dry food.

The digestibility level is always a crucial criterion for assessing the quality of dogs’ food (Deschamps et al. 2022). The extrusion process in making kibble food is a good way of enhancing digestibility (Montegiove et al. 2022). However, the quality of the extrusion-formed dry food used in the present study was not premium, as its digestibility coefficients were not satisfactory. We assume that the reason for undesirable digestibility coefficients was its high contents of TDF and IDF (Table 1). Therefore, this study could provide evidence for the enhancing effect of B. subtilis on the apparent digestibility of OM, DM, CP, and CF in the high IDF-contained diet. Microbial enzyme decomposition can solubilise certain fibre constituents (de-Oliveira et al. 2012). Bacillus can produce enzymes, such as α-amylase, cellulase, dextranase, alkaline protease, and β-glucanase (Hentges 1992). Soluble fibre is a dietary component required for the proliferation of lactobacilli and is therefore associated with the thriving of beneficial microflora (Guan et al. 2021). In this study, the level of soluble fibre in the diet could support the flourishing of the gut microflora. This is another piece of evidence that B. subtilis increased the diet’s digestibility by secreting enzymes that decomposed dietary fibre and other nutrients. Also, ingested lactobacilli could provide enzymes such as sucrase, lactase and tripeptidase, contributing to improved digestibility (Giang et al. 2011).

In addition to nutrient digestibility, faecal characteristics should be considered when assessing the quality of dogs’ food. Faecal characteristics reflect intestinal functionality, and dog owners typically favour foods that improve consistency. In this study, B. subtilis supplementation ensured drier and well-shaped dog stools compared to the control diet although faecal scores were in the ideal range (3–4) in all groups, the BS1 and BS2 groups showed improved faecal consistency scores. These results are consistent with other studies using the same probiotic (Félix et al. 2010; Schauf et al. 2019). Moreover, faecal DM was higher in the BS1 and BS2 groups. Higher faecal consistency scores coincide with higher faecal DM content (Zentek et al. 2004). In other studies in dogs with diarrhoea, B. subtilis improved faecal consistency and DM content (Herstad et al. 2010; Paap et al. 2016). B. subtilis is believed to enhance faecal scores and DM content in dogs with gastrointestinal diseases.

The data in this study also suggested that the levels of faecal SCFAs (acetate and propionate) increased in dogs fed the B. subtilis diet compared to the control group. The total SCFAs can regulate sodium and fluid absorption in the colon (Scheppach 1994). Therefore, the higher acetate and propionate levels in the faeces of dogs in the BS1 and BS2 groups are consistent with their firmer faecal consistency scores. An earlier study revealed that B. subtilis did not change the level of total SCFAs but increased propionate in dog faeces (de Lima et al. 2020). In the present study, acetate, propionate, and the total SCFAs significantly increased in the BS1 and BS2 groups. In agreement with our results, Schauf et al. (2019) reported higher SCFAs in dogs’ faeces fed the same probiotic. It has been suggested that low levels of SCFAs are produced in the large intestine from digestible foods because of low amounts of undigested nutrients reaching there (Chen et al. 2006). The fact that the diet used in this study was of low digestibility and therefore was fermented by B. subtilis in the large intestine could explain the high levels of SCFAs and BCFAs in the BS1 and BS2 groups. Additionally, B. subtilis used in this study could effectively reach the colon due to its ability to form spores and its resistance to gastric pH levels (Chen et al. 2006).

The results also indicated a decline in faecal pH in the BS1 and BS2 groups. This decrease in fresh stool pH could be explained by the increased levels of SCFAs in these treatment groups. Although not tested in this study, lactic acid production could also explain the decrease in faecal pH (Swanson et al. 2002). Lactic acid is a major end product of lactobacilli, whose number increased in the BS1 and BS2 groups due to B. subtilis. Decreased luminal pH could be harmful to several pathogenic species (Swanson et al. 2002). Feliciano et al. (2009) observed a decline in the faecal pH of dogs fed a diet containing Lactobacillus spp. On the other hand, no faecal pH changes were reported in other studies (Feliciano et al. 2009; Bastos et al. 2020). The decreased faeces pH is consistent with the higher protein digestibility coefficients in the BS1 and BS2 groups. Probiotics, such as B. subtilis C-3102 used here, can modify protein digestibility by decomposing undigested protein in the intestine and regulating the microflora involved in proteolysis (Wang and Ji 2019).

In this investigation, lower ammonia levels were detected in dogs’ faeces in the BS2 group. Similarly, previous studies reported a reduction in ammonia (Wang and Ji 2019), biogenic amine concentrations (Bastos et al. 2020), and odour (de Lima et al. 2020) in the faeces of dogs fed a diet supplemented with B. subtilis or B. licheniformis. In this study, the alteration in faecal microbiota caused by B. subtilis C-3102 might occur in such a way that hindered bacteria that produce phenols, biogenic amines, and ammonia (Bastos et al. 2020). When in excess, these end-products could contribute to intestinal inflammation (dos Santos Felssner et al. 2016). The enhanced faecal lactobacilli observed here could explain the decreased faecal ammonia. Additionally, the improved nutrient digestibility might also result in a decline in substrate availability for microbiota in the large intestine, which consequently lowers ammonia production by microbial fermentation.

We used plating methods to enumerate faecal bacteria instead of a molecular DNA technique. This could be the reason for the low number of bacteria we evaluated. Even though some studies that used plating methods have provided evidence that the administration of probiotics has the ability to manipulate the gastrointestinal microbiota of dogs (Weese and Arroyo 2003; Strompfová et al. 2012), these traditional plating techniques quantify a limited number of bacteria. Herewith, those techniques do not eliminate concerns about bias caused by inefficient recovery. Recently, high-throughput DNA sequencing techniques are implemented to determine the microbiomes of canines (Sivamaruthi et al. 2021). Therefore, DNA techniques will be used in future microbiota studies. B. subtilis increased faecal lactobacilli counts in the BS1 and BS2 groups. Contrary to our findings, de Lima et al. (2020) reported lower lactobacilli in dogs fed B. subtilis. Yet, they did not report any deleterious effects on dogs’ health. In agreement with our results, Gagné et al. (2013) found a significant increase in Lactobacillus spp. and Bifidobacterium spp. In addition, Biagi et al. (2007) reported that Lactobacillus influenced the metabolism and composition of the intestinal microbiota of dogs.

The faecal abundance of Lactobacillus spp. and Enterococcus spp. in this study could be attributed to the probiotic doses used. B. subtilis spore concentrations of 1 × 10⁹ CFU/g and 2 × 10⁹ CFU/g effectively increased these bacteria in the dogs’ faeces. The effect of probiotics cannot always be explained by different strains having diverse functions and survivability along the intestines (Ahasan et al. 2015). A combination of dosage and time was a significant factor that affected the numbers of lactobacilli and enterococci. In a previous study, adding 5 × 10⁹ CFU/g of Lactobacillus spp. to a prebiotic preparation significantly increased the count of Enterococcus spp. in faeces (Garcia-Mazcorro et al. 2011). A similar finding was reported by Sauter et al. (2006), who fed the dogs with 1 × 10⁶ CFU/g of L. acidophilus for 12 weeks and found an increased number of Lactobacillus spp. in faeces. Dissimilarly, the addition of 1.5 × 10⁷, 1.5 × 10⁸, and 1.5 × 10⁹ CFU/g of Bifidobacterium animalis did not increase the number of Lactobacillus spp. in faeces of healthy dogs (Kelley et al. 2012). This emphasises that the duration of treatment and dosage of the probiotic are significant factors affecting the faecal microbial population.

Notably, we observed similar E. coli counts in all test groups. The previous studies that tested probiotics and prebiotics simultaneously as symbiotics reported an increased number of enterococci but lower numbers of E. coli in the faeces of dogs (Swanson et al. 2002; Garcia-Mazcorro et al. 2011; Gagné et al. 2013). This might be due to the beneficial impact of B. subtilis even without supplementation with prebiotics. Using Weissella cibaria JW15 as a probiotic in beagle dogs, Sun et al. (2019) revealed improved faecal lactic acid bacteria with increasing doses of W. cibaria without adding a prebiotic to the food. There is still much to learn about the antagonistic effect of probiotics on pathogens, but it is understood that B. subtilis could inhibit the growth of pathogenic bacteria such as E. coli. The selection of suitable probiotic strains is essential to supporting animals’ well-being. Therefore, comprehensive studies are needed to determine the distinct advantages of B. subtilis on total microbiota composition.

The probiotic recovery analysis was not performed here, but it is believed that B. subtilis C-3102 could survive passage through the intestinal tract of dogs, leading to microflora enrichment and local and systemic effects. Increased numbers of lactobacilli and Enterococci spp., were seen as local effects and systemic changes were observed in several haematologic parameters. Blood lymphocytes and WBCs reflect the physiological and immunologic status of dogs, and increased concentrations of granulocytes, WBCs, and RBCs were observed in the present study. It was reported that dietary probiotics may have interactive actions with the immune system (Collado et al. 2009; Sun et al. 2019). Increased concentrations of granulocytes, WBCs, and RBCs in the BS1 and BS2 groups proved the stimulant effect of B. subtilis. The changes were small, which was consistent with the health status of the dogs. Therefore, beneficial effects may occur only in dogs with gastrointestinal pathogens (Baillon et al. 2004). The dogs used in this study were healthy and did not have any intestinal disorders. This could be the reason that most of the blood parameter changes were not significant.

Conclusion

We examined the effects of dietary supplementation with B. subtilis C-3102 on dogs’ health. This is the first study conducted on Golden Retriever dogs. Compared to the control, animals receiving 1 × 10⁹ or 2 × 10⁹ CFU/g of B. subtilis showed improved nutrient digestibility, improved faecal consistency scores, higher short-chain fatty acid (acetate and propionate) levels, lower faecal pH, and increased abundance of lactobacilli and enterococci. The application of B. subtilis in dog diets could be functional without an adverse effect on the health status of adult dogs. Taken together, the findings of this study suggest that B. subtilis can be used regularly in healthy dogs as a supportive supplement to the gastrointestinal system. However, more studies on dogs of different ages and breeds and fed different types of foods and in various health and physiological conditions are needed to define the effectiveness of probiotics on animal health and the quality of gut microbiota.

Ethical approval

This work was approved by the Ethics Committee of the College of Veterinary Medicine Experimental Animals Production and Research Centre,the Selçuk University, protocol number 2021/01.

Author contributions

All authors contributed equally to the conception and writing of the manuscript. All authors critically revised the manuscript and approved the final version.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data presented in this study are available from the corresponding author upon reasonable request.