Effects of Bacillus Subtilis C-3102 addition to dry dog food on nutrient digestibility faecal characteristics, faecal microbiota and blood chemistry in healthy dogs

doi:10.21203/rs.3.rs-2450446/v1

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Effects of Bacillus Subtilis C-3102 addition to dry dog food on nutrient digestibility faecal characteristics, faecal microbiota and blood chemistry in healthy dogs

https://doi.org/10.21203/rs.3.rs-2450446/v1

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This study evaluated the effects of Bacillus subtilis C-3102 supplementation on digestibility, faecal characteristics, some blood chemistry parameters and microbiota of dogs. Eighteen adult Golden retriever dogs were assigned to three treatments: control (no B. subtilis), 2g B. subtilis (5 × 108 CFU/g, BS1) and 4g B. subtilis (5 × 108 CFU/g, BS2) added groups. Dogs were fed 25 days of adaptation period followed by 5 days of faecal collection. Blood samples were taken from all dogs on the first and last day of the study. Fecal characteristics were evaluated on the last 3 days of the study by dry matter content, fecal score, fecal output, pH, ammonia, short (SCFA) and branched chain fatty acids. Improved dry matter, fibre, organic matter and protein digestibility coefficents were determined in BS1 and BS2 (P < 0.05). Lower pH, higher dry matter, acetate, propionate and isobutyrate levels in feaces were determined in BS1 and BS2 (P < 0.05). Lower ammonia and firmer stools were observed in BS2 (P < 0.001). Counts of CFU of faecal Enterococci, Lactobacilli were improved BS1 and BS2 (P < 0.05). Serum cholesterol and triglyceride levels were not affected (P > 0.05). Higher values of WBCs, RBCs and granulocyte were recorded in B.subtilis groups (P < 0.05). In conclusion, supplementation of B. subtilis in dog food improved faecal quality, enhanced digestibility of diet and contributed to the health by supporting immune functions of dogs, reducing gut ammonia, increasing SCFA and number of Lactobacillus and Enterecocci.

Bacillus subtilis

blood profiles

nutrient digestibility

faecal characteristics

gut health

A nutritiously balanced and diets are essential for the well-being of dogs. Various type of commercial diet, foods, treats and additives are produced for the healthy nutrition of dogs that used to be fed only with table scraps. The interest of usage of functional additives have raised in recent years in dog diets to contribute to the welfare of dogs (Rossi et al. 2020). Because of the developed strong bonds between human and dog, people want to keep their dogs healthy as well as themselves. Probiotics are among the additives, which may contribute to health status and welfare of dogs. Optimal composition of dog’s normal intestinal microflora can be rapidly altered by gastrointestinal infections, oral administration of medications and especially dietary changes. Probiotics are organisms such as bacteria that can be used a supplements to the food. They are living microorganisms that support intestinal health by maintaining, regulating or restoring the ideal balance of the gut flora (Paap et al. 2016). In addition, probiotics have been shown to have an suprasive effect on harmful and pathogenic organisms by producing fatty acids through fermentation (Schauf et al. 2019). For this reason, feeding of probiotics to dogs may be a practical way to support health and reduce the risks of diseases (Biagi et al. 2007). Probiotics provide benefits by promoting an action that maintains health or by preventing negative effects (Craig 2021). Besides these useful benefits, there are just a few studies on the effects of B. subtilis supplementation in dogs. Some of the available studies claim that B. subtilis supplementation improve intestinal health by improving faecal characteristics and alters the microbioata of dogs (Félix et al. 2010; Bastos et al. 2020; de Lima et al. 2020). On the other hand, effects of addition of B.subtilis on nutrient digestibility of diet are contradictive (González-Ortiz et al. 2013; Schauf et al. 2019; de Lima et al. 2020). Also, there are not any scientific data on the effects of addition of B.Subtilis on blood parameters in dogs. Thus, the objective of this comprehensive study was to evaluate the effects of B.subtilis addition on faecal characteristics, nutrient digestibility, faecal microbiota and some blood parameters of dogs.

The B. subtilis C-3102 product was obtained from a commercial company and is composed of spray-dried spore-forming endospores. The B. subtilis product contain 5.0 × 10⁸ cfu/g bacteria.

Animals and diet

Eighteen adult (age = 5–6 years, body weight = 22.5 ± 1.2kg) Golden retriever dogs (9 male, 9 female) were used for the presented study. Dogs were divided into 3 groups having similar body weight average and same number of gender. They were housed individually in the kennels at Selçuk University, Faculty of Veterinary Medicine Research and Application Farm. The kennels had open (510 × 230 cm) and closed (190 × 190 cm) parts. All dogs were submitted to physical examination and de-wormed before the trial. One chicken and rice containing commercial dry dog food was tested and it’s determined chemical composition was given in Table 1.

Table 1

Determined Chemical Composition of Commercial Dry Food Fed to Dogs (%DM)
Dry matter, %	91.5
Ash, %	5.28
Acid hydrolyzed ether extract ,%	9.21
Crude fibre, %	6.45
Crude protein, %	18.45
ME, kcal/kg*	3242.5
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
*Calculated using NRC equations.

Chemical analysis

Dry matter (DM), ash, acid hydrolyzed ether extraction (EE), crude fibre (CF) and crude protein (CP) analyzes were performed according to AOAC (2019)methods. In order to determine digestibility, chemical analysis were also performed on the collected faeces. The metabolizable energy (ME) level of the food was calculated using the following formula according to NRC (2006):

I. Gross energy (GE): GE (kcal) = (5.7 × CP%)+(9.4×EE%) + [4.1× (NFE%+ CF%)]

Nitrogen-free extract, NFE (%) = DM% – (EE% + CP% + ash% + CF%)

II. Energy digestibility (%) = 91.2 – (1.43 × CF%)

III. Digestible energy: kcal DE = (kcal GE × energy digestibility)/100

IV. Metabolizable energy: ME (kcal) = kcal DE – (1.04 × CP%)

Diet was offered once a day in sufficient amount to meet the ME requirements of the dogs, according to the following equation (FEDIAF 2019): ME (MJ/day) = 95 × Body weight^0.75. Drinking water was available ad libitum.

Determination of Faecal Consistency Score

Faecal consistency scoring was performed by three different researchers before collection in the last foura days of the trial. Faecal consistencies were scored according to 1–5 system. 1. diarrhea-like 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

The digestibility of the nutrients of dry food with and without supplemented B. Subtilis was determined using the total collection method (AAFCO 2004). Control group (n = 6) was given only dry food. BS1 group was given dry food + 2g B.subtilis and BS2 group received dry food + 4gr B.subtilis. Probiotic was poured on the dry food and mixed. Trial lasted for 30 days, the all freshly excreted faeces were collected daily on last 5 days. After weighing the feaces, they were kept in a freezer (-18°C). The faeces of each dog were thawed at laboratory temperature (23–25°C) and mixed homogeneously. Samples were dried in oven set at 55°C for 48 h (VWRVenti-line, USA). Dry faeces were ground via a laboratory mill (Retsch SM100, Germany) passed through a 1 mm sieve. The apparent total tract digestibility (ATTD) of nutrients were calculated based on the analysis results of food and faeces.

ATTD% = [(g of nutrient intake – g of nutrient excretion)/g of nutrient intake] × 100

Determination of short and branched chained fatty acids ammonia and pH of faeces

The fresh feces samples were mixed 1:5 with distilled water, shaken and waited for 1 min. pH measured with a digital pH meter. Spectrometric method was used to determine fecal ammonia levels (Weatherburn 1967). For ammonia analysis, 2gr fresh faeces sample diluted 1:5 with distilled water and 1 ml of sample was taken and mixed with 20 µl of sulfuric acid. Fecal fatty acid profiles were determined by gas chromatography (Agilent 6890N, USA) in fresh faeces collected last three days of the study according to procedure of El Wahab et al (2022).

Blood sampling

At the beginning (day 0) and end of the experiment (day 30), blood samples were taken from each dog to obtain serum and whole blood. Monocyte, granulacyte, white blood cells (WBC), lymphocyte and red blood cells (RBC) counts of samples were determined using an automatic analyzer (ADVIA 120, USA). Serum was separated by centrifugation at 3000 RPM for 15 min at 4 \(^\circ\)C and kept at -18\(^\circ\)C until analysis. Cholesterol and triglyceride levels were determined by using a biochemistry analyzer (Hitachi 747, Japan) at Selçuk University, Faculty of Veterinary Medicine laboratory.

Fecal Bacterial Counts

Collected Faecal samples were placed on ice and transported to a -80°C freezer immediately. Samples were plated on Sharpe, deMan, Rogosa agar (MRS; Difco), as well as Rogosa SL agar plates. In order to evaluate the anaerobic bacteria counts, the fecal samples were also plated on blood base plate (CDC-BAP) agar. After 48 to 72 h of anaerobic incubation at 37 C°, bacteria colonies confirmed (Lai et al 2019). Membrane filtration method was used for determination of Enterococci and Escherichia coli (E. coli) (Lee et al. 1994). Lactobacillus, Escherichia coli and Enterococci counts were expressed as log₁₀ CFU/g faeces.

Statistical Analysis

Microbiological parameters and faecal score were compared using Kruskal-Wallis test in relation to the group and time of data collection. If the results of Kruskal-Wallis tests were significant, a multiple comparison analysis based on pairwise two-sample Wilcoxon comparisons was performed. The differences between the bacterial counts were assessed by the Wilcoxon signed-rank test (nonparametric). In the blood results, the normal distribution and homogeneity of variances were checked with the Shapiro-Wilk test and evaluated with the Bonferroni test. Bacterial count data were also analysed using a randomised complete design with dose, time and dose × time interactions. Digestibility and faecal characteristics were evaluated by Tukey HSD test in ANOVA. Values of P < 0.05 were considered significant.

None of the dogs showed any side effects during the trial and remained healthy. CTTAD of dry matter(DM), crude fibre, organic matter and crude protein were improved in the BS1 and BS2 groups than the control group (P < 0.05). The inclusion of B. subtilis C-3102 had no effect on CTTAD of fat of extruded dog food (P > 0.05) (Table 2).

Table 2

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	0.03
OMD	78.11 ± 0.92^b	83.33 ± 1.15^a	83.57 ± 0.65^a	0.01
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 digestibiliy, CFD: Crude fibre digestibility, CPD: Crude protein digestibility; CON: control; BS1: 2g B.subtilis suplemented group; BS2: 4g B.subtilis suplemented group; SEM: standart error of mean; a,b,c; Values in the same row that are not sharing a common superscript differ significantly (P < 0.05).

Faecal consistency scores were improved by B.subtilis addition in BS1 and BS2 groups (P = 0.04) which are consistent with higher faecal DM content determined in these groups. In parallel with the increased digestibility level, faecal pH was lower in BS1 and BS2 groups (P = 0.001). A greater fecal concentration of acetate, propionate and isobutyrate (P = 0.001) was observed for dogs fed B. subtilis C-3102. BS2 group had lower concentrations of fecal ammonia (P = 0.01) than BS1 and control groups (Table 3).

Table 3

Faecal parameters of 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	0.04
Fecal pH	7.11 ± 0.15^a	6.81 ± 0.08^b	6.78 ± 0.11^b	0.001
Fecal DM	45.59 ± 0.57^b	50.86 ± 0.69^a	51.37 ± 0.43^a	0.001
Fecal output(g/day)	101.97 ± 5.72	96.86 ± 7.39	95.97 ± 6.66	0.37
Faecal NH₃ (µmol/g DM)	0.71 ± 0.35^a	0.64 ± 0.50^a	0.49 ± 0.15^b	0.01
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 SCFA (µmol/g DM)	254.11 ± 11.61^b	343.83 ± 13.41^a	320.78 ± 16.31^a	0.001
Total BCFA (µmol/g DM)	6.37 ± 0.39^ab	7.79 ± 0.76^a	5.26 ± 0.42^b	0.01
CON: Basal Diet; BS1: CON + 2 g of B.subtilis with 5.0×10⁸ cfu/g; BS2: CON + 4 g of B.subtilis with 5.0×10⁸ cfu/g; SCFA: short chained fatty acids; BCFA: Branched chained fatty acids; ^a,b,c; Values in the same row that are not sharing a common superscript differ significantly (P < 0.05).

Lactobacillus and Enterecocci bacterial counts increased at the 30th day feaces’ in BS1 and BS2 groups (Table 4). The increased count of E.coli was detected in BS1 after 30 days (P = 0.03). The highest number of Lactobacillus was determined in BS2 (P = 0.01) and Enterecoccus in BS1 and BS2 groups ( P < 0.001) (Table 5).

Table 4

Wilcoxon test results of within-group bacterial counts (log10 CFU/g) on the first (day 0) and day 30 (Mean ± SD)
		Lactobacillus	E.coli	Enterococci
CON	0 day	1.45 ± 0.15	1.28 ± 0.17	2.04 ± 0.1
CON	30 day	1.83 ± 0.15	1.5 ± 0.12	2.27 ± 0.21
	P	0.108	0.18	0.109
BS1	0 day	1.33 ± 0.07	1.22 ± 0.14	2.12 ± 0.15
BS1	30 day	2.36 ± 0.17	1.80 ± 0.1	3.35 ± 0.16
	P	0.028	0.03	0.027
BS2	0 day	1.51 ± 0.33	1.4 ± 0.14	2.75 ± 0.11
BS2	30 day	3.05 ± 0.42	1.57 ± 0.17	4.31 ± 0.37
	P	0.017	0.06	0.02
SD: Standart deviation; CON: Basal Diet; BS1: CON + 2 g of B.subtilis with 5.0×10⁸ cfu/g; BS2: CON + 4 g of B.subtilis with 5.0×10⁸ cfu/g

Table 5

Effects of dietary supplementation of Bacillus subtilis probiotic on faecal microbiata in dogs, log10 CFU/g
Groups	dose(gram)	time (day)	n	Lactobacillus	E.coli	Enterecocci
CON	0	0	6	1.40 ± 0.2	1.28 ± 0.17	2.04 ± 0.1
CON	0	30	6	1.93 ± 0.15	1.50 ± 0.12	2.27 ± 0.21
P				0.068	0.18	0.109
BS1	2	0	6	1.33 ± 0.07	1.27 ± 0,16	2.19 ± 0.22
BS1	2	30	6	2.36 ± 0.13	1.86 ± 0.1	3.46 ± 0.04
P				0.024	0.042	0.03
BS2	4	0	6	1.54 ± 0.54	1.31 ± 0.05	2.75 ± 0.05
BS2	4	30	6	3.14 ± 0.56	1.51 ± 0.15	3.58 ± 0.32
P				0.028	0.06	0.02
CON(day 30)			6	1.93^b	1.5	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				0.01	0.112	< 0.001
Tukey HSD
SEM				0.19	0.07	0.11
dose				< 0.001	< 0.001	< 0.001
time				< 0.001	< 0.001	< 0.001
*dosetime**				< 0.001	< 0.001	< 0.001
CON: Basal Diet; CON: Basal Diet; BS1: CON + 2 g of B.subtilis with 5.0×10⁸ cfu/g; BS2: CON + 4 g of B.subtilis with 5.0×10⁸ cfu/g; SEM: standart error of mean; ^a,b: values with different superscript letters are significantly different (P < 0.05).

Results of blood characteristics showed that WBC, RBC and granulocyte concentrations were affected by supplementation of B.subtilis (Table 6). Lymphocyte and RBC’s counts were increased in BS2 group at day 30 (P < 0.05). In day 30 results comparison of all groups WBC and RBC concentrations were higher in BS1 and BS2 (P < 0.05). In blood serum, the concentration of cholesterol and triglyceride were not affected during the experiment. Granulocyte level of BS1 at day 30 was higher than control and BS2 (P = 0.043) (Table 6).

Table 6

Results of evaluated blood profiles of dogs
Groups	time(day)	n	Cholesterol,mg/dl	Triglyceride,mg/dl	WBC, 10³/µL	Lymphocyte¹,%	Monocyte¹,%	Granulocyte¹,%	RBC, 10³/µL
CON	0	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
CON	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	0	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
BS1	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	0	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
BS2	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 ± 4.8	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 ± 7.1	7.97 ± 0.65^b	27.1 ± 0.5	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	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
^a,b: values with different superscript letters are significantly different (P < 0.05). RBC: red blood cell; WBC: white blood cell. ¹ Values are presented as a percentage of the total WBC count. CON: Basal Diet; CON: Basal Diet; BS1: CON + 2 g of B.subtilis with 5.0×10⁸ cfu/g; BS2: CON + 4 g of B.subtilis with 5.0×10⁸ cfu/g

In the experiment all dogs ate all the food presented with and without B. Subtilis without refusal. The inclusion of 2 gr and 4 gr of B. subtilis (5 × 10⁸ CFU/g C-3102) altered nutrient digestibility of the dry food. In some studies, it has been reported that there is no change in nutrient digestibility of diets supplemented with B. subtilis, B. licheniformis and B. amyliquefaciens in dogs (Félix et al. 2010; González-Ortiz et al. 2013; de Lima et al. 2020). However, other studies demonstrated that the dietary supplementation of Lactobacillus acidophilus with fructooligosaccharides promoted higher DM digestibility in dogs (Swanson et al. 2002). In another study, dogs fed the B. subtilis (C-3102) included diet showed a higher apparent digestibility of fat and nitrogen free extract (Schauf et al. 2019). Also, Giang et al. (2011) determined improved apparent total tract digestibility of protein, fibre and organic matter with inclusion of B.subtilis probiotic in pigs. B.subtilis inclusion also improved ileal apparent digestibility of protein in broilers (Boroojeni et al. 2018). The increased digestibility may be due to high dosages and different addition methods used in this study. For instance; de Lima et al. (2020) added B. Subtilis (1 x 10⁶ CFU/g) in poultry fat by spraying post-extrusion. Felix et al (2010), added 1x10¹⁰ CFU/g B. Subtilis in 300 ml of soybean oil at a level of 0.01%. On the other hand, Biorgue et al (1998) added B. subtilis CIP 5832 in extruder with the other ingredients and found no effect on digestibility. In this study, B. Subtilis supplementation at the levels of 0.4%(2g) and 0.8%(4g) was added directly to the dry food. Although Gonzalez-Ortiz et al. (2013) suggested that improved digestibility rates in dog food are not so necessary considering the high rates of obesity prevalence in dogs. However, digestibility level is always the first criteria of assesment of quality of a dog diet and it is a indicator of optimal gastrointestinal health of a dog (Deschamps et al. 2022). Extrusion process to make kibble foods is a good way to enhance digestibility (Montegiove et al. 2022). But, the extruded formed dry food used in this study was not a premium quality one as it’s digestibility coefficients were not desirable (Table 2). Therefore, the enhancer effect of Bacillus subtilis on the apparent digestibility of OM, DM, CP and CF seems to be evidenced by this study when the diet is in poor quality or economical class. Probiotics may be suggested to be used to optimise the digestive tract of the dogs fed poor quality dry foods. Bacillus can produce some useful enzymes like α-amylase, cellulase, dextranase, alkaline protease and β-glucanase (Hentges 1992).

Besides digestibility of nutrient contents, faecal characteristics also need to be taken into consideration in assessment of quality of dog food. Faecal characteristics reflect intestinal functionality and dog owners prefer the foods that improve consistency. When compared to control diet, B subtilis supplementation ensured drier and well-shaped stools of the dogs in this study. Although faecal scores were considered in ideal range (3–4) in all groups, the improved faecal consistency score found in BS1 and BS2 group. This result was expected and in accordance with the other studies used the same probiotic (Félix et al. 2010; Schauf et al. 2019). Faecal dry matter was higher in BS1 and BS2 groups. Higher fecal consistency scores coincided with higher fecal DM content (Zentek et al. 2004). In other studies, B. subtilis was tested in dogs with diarrhea and observed positive effects on faecal consistency and DM content (Herstad et al. 2010; Paap et al. 2016). It seems B.subtilis positively affects faecal scores and DM content in dogs that do not show the signs of gastrointestinal diseases.

The faecal SCFA levels showed an increase of acetate and propionate in dogs fed B. subtilis diet compared to the control group. Total SCFA are known to stimulate and regulate sodium and fluid absorption in colon (Scheppach 1994). Therefore, the higher acetate and propionate content in faeces determined in BS1 and BS2 groups were consistent with the firmer faecal consistency score observed in the entire study. In earlier studies B. Subtilis did not change the level of total SCFA in dog feaces (de Lima et al. 2020). Absorbsion of SCFA could be the reason. Because the SCFA, produced from intestinal fermentation of carbohydrates, tend to be absorbed into the lumen, and before reaching the distal colon, most of SCFA are absorbed. But in this study, acetate, propionate and total SCFA were determined significantly higher in BS1 and BS2 groups. Also, increased faecal propionate in dogs was also observed by de Lima et al (2020). Schauf et al (2019) found higher SCFA in dogs of probiotic group similar to this study. It has been previously stated that low level of SCFA production can be expected in the large intestine for highly digestible diets because of undigested nutrient as a substrate reaching the large intestine for fermentation (Chen et al. 2006). The fact that the dog food used in this study did not have high digestibility and therefore undergoes higher fermentation with B. subtilis in the large intestine may explain the high SCFA and BCFA values in BS1 and BS2 groups. Also, B.subtilis used in this study may be effectively reached the colon, considering it’s forming spore ability and resist to gastric pH levels (De et al. 2004).

The decrease in fresh stool pH can be explained by the increased SCFA in BS1 and BS2 groups. Although not determined in this study, the decrease in pH may be due to lactic acid production (Swanson et al. 2002). Because lactate is a major end product of lactobacillus which determined increased in this study. This result was a positive effect of B. subtilis. Because, decreased luminal pH is a potential antimicrobial substance to several pathogenic species (Swanson et al. 2002). Feliciano et al. (2009) observed a reduction in faecal pH of dogs fed a diet containing Lactobacillus spp. On the other hand, no faecal pH changes were reported in dogs in other studies (Félix et al. 2010; Bastos et al. 2020). Decreased pH in faeces was expected because protein digestibility coefficients were higher in BS1 and BS2 groups. Low protein digestibility causes high levels of ammonia production in hindgut especially when there is not available fermentable carbohyrate reached in large intestine. Also, probiotics can modify protein digestibility and the breakdown of the undigested protein in the intestine and regulate the microflora related to proteolysis (Wang and Ji 2018). In present study, reduced pH might have been observed due to B. subtilis regulated the dogs intestinal flora via affecting protein breakdown.

Reduces ammonia level was determined in faeces of dogs in BS2 group. Other studies also reported a reduction in ammonia (Félix et al. 2010), biogenic amine concentrations (Bastos et al. 2020) and odor (de Lima et al. 2020) in faeces of dogs fed a diet with B. subtilis or B. licheniformis. Microbiota have been altered by the inclusion of B. subtilis C-3102 in such a way that hindered bacteria that produce compounds like phenols, biogenic amines and ammonia (Bastos et al. 2020). When these end-products of protein fermentation in excess, they contribute to the development of inflammatory intestinal problems (dos Santos Felssner et al. 2016). The possible explanation for decreased faecal ammonia may be due to the improved lactobacillus bacteria count in dog feces. Also improved digestibility of nutrients might result in less substrate for microbiota in large intestine which consequently decreases ammonia production by microbial fermentation.

The improved faecal lactobacillus counts were detected in BS1 and BS2 groups, an indication of increased number of lactobacillus in the gut. Ingested lactobacillus can increase useful enzymes such as sucrase, lactase and tripeptidase along the intestine which could have contributed to the improved digestibility (Giang et al. 2011). In contrast to this study, de Lima et al (2020) found lower lactobacillus in B.subtilis supplemented dogs. But, they did not report any negative effects on dogs’health. Gagné et al (2013) found significant rises Lactobacillus and Bifidobacterium spp, which is in agreement with this study. Biagi et al. (2007) reported that Lactobacillus probiotic influenced the metabolism and composition of the intestinal microbiota of dogs. Rises in Lactobacillus and enterecoccus spp. in this study could be related to doses used. Amounts of 2 gr and 4 gr of B.subtilis were effective to rise these bacteria in faeces of dogs. Also effects of probiotics can not always be explained due to different strains having various functions and survivability along the intestines (Asml et al. 2015). In another study use of Lactobacillus spp. with prebiotics showed significant rise of Enterecoccus counts like in this study (Garcia-Mazcorro et al. 2011). Werner et al (2021) isolated Enterococci spp. and Escherichia coli (E.coli) bacteria in healthy dogs faeces. E.coli counts were similar in all groups of this study. Some studies that used probiotic and prebiotic at the same time as symbiotics showed Enterococci counts increased and E.coli decreased significantly in the feces of dogs (Swanson et al. 2002; Garcia-Mazcorro et al. 2011; Gagné et al. 2013). This might be the results of power of the B.subtilis without using prebiotics. Sun et al (2019) used Weissella Cibaria JW15 as probiotics in Beagle dogs and found improved faecal lactic acid bacteria count with increasing levels of Weissella Cibaria without prebiotic addition to the diet. According to faecal characteristics results, B.subtilis supplementation helps to maintain the balance of microbiota in healthy dogs’ intestines that do not suffer gastrointestinal diseases.

Another effect of probiotics is the stimulation of the immune system (Baillon et al. 2004). B.subtilis probiotic used in this study was able to survive passage through intestinal tract of dog, leading to enrichment of the microflora, local and systemic effects. Local effects included increased numbers of lactobacilli and Enterococci spp whereas systemic changes were observed in several hematologic parameters. Blood lymphocytes and WBCs reflect the physiological and immunologic status of dogs and increased concentration of granulocyte, WBCs, RBCs in present study was observed. It was reported that dietary probiotics may have interactive actions with immune system (Collado et al. 2009). Increased concentrations of granulocyte, WBC and RBC in BS1 and BS2 groups proved the stimulant effect of B.subtilis. Changes were small which were consistent with the healthy status of the dogs. Therefore, beneficial effects may occur only in dogs with gastrointestinal pathogens (Baillon et al. 2004). Dogs used in this study were healthy and did not have any intestinal disorders. This could be the reason of most of the blood parameter changes were not significant.

The concentration of serum triglyceride and cholesterol level were not affected by B.subtilis at the end of treatment despite an increase of acetate concentrations in feaces, the primary substrate for cholesterol synthesis (Pereira and Gibson 2002). Some SCFA decrease lipid concentrations by reducing acetate utilization and cholesterol synthesis (Strompfová et al. 2014). But most of SCFA produced in colon are absorbed into the blood and the faecal concentrations of SCFA could be misleading. In another study, Sun et al (2019) used Weissella Cibaria WJW15 as probiotics in dogs and found lower levels of trygliceride. This result was probably about dosage. Because Sun et al (2019) used 50g of WJW15 with 3 ×10⁸ cfu/g but 2g(BS1) and 4g(BS2) 5×10⁸ cfu/g of B. subtilis were supplemented in this study. However, Zhang et al (Zhang et al. 2012) reported that blood triglyceride were not affected in laying hens by lactobacillus probiotics which is consistent with the result of our experiment. Another reason of blood lipid results of this study may be alteration of the fermentation of indigestible carbohydrates by B.subtilis which allowed redistribution of cholesterol.

The effects of probiotics on dogs are still unclear. Different strains of bacteria have varying effects on dogs of different breeds. Supplementation of dry dog food with 5 × 10⁸ CFU of B. subtilis C-3102/g altered digestibility, increased fecal SCFA concentrations, improved fecal consistency and reduced fecal ammonia. It also altered the gut microbiota of Golden retriever dogs by increasing beneficial bacterial counts. The increase in WBCs, RBCs and granulocyte counts indicates that B.subtilis also has positive effects on the general health status and well-being of dogs. This study is the first study conducted with Golden retriever dogs. More studies are needed to reveal the probiotics that affect both diet digestibility and health status in dogs. In order to determine effective probiotics, studies should conducted with dogs of different ages, breeds, fed different types of diets and in various health and physiological conditions.

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

CONFLICT OF INTEREST

The authors declared that there is no conflict of interest. Authors are responsible for all content and writing of this paper.

CONSENT FOR PUBLICATION

All authors give consent for publication.

FUNDING

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

AUTHORS’ CONTRIBUTIONS

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

DATA AVAILABILITY

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Effects of Bacillus Subtilis C-3102 addition to dry dog food on nutrient digestibility faecal characteristics, faecal microbiota and blood chemistry in healthy dogs

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Abstract

Introduction

Materials And Methods

Animals and diet

Chemical analysis

Determination of Faecal Consistency Score

Determination of nutrient digestibility

Determination of short and branched chained fatty acids ammonia and pH of faeces

Blood sampling

Fecal Bacterial Counts

Statistical Analysis

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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