Research Article

Effect of Probiotic Bacillus coagulans on Performance and Blood Metabolites of Dairy Cows with Subclinical Mastitis

Sudeb Saha1,2*, Jay Prakash Ray3, Apurbo Kumar Mondal4, Md. Irtija Ahsan5, Marina Debnath6, Dilruba Afrin7, Syed Sayeem Uddin Ahmed5, Jayanta Datta Gupta3, Nivedita Datta8, Hugo O. Toledo-Alvarado9, Haruki Kitazawa1

1Food and Feed Immunology Group, Laboratory of Animal Food Function, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8576, Japan; 2Department of Dairy Science, Faculty of Veterinary, Animal and Biomedical Sciences, Sylhet Agricultural University, Sylhet-3100, Bangladesh; 3Square Pharmaceuticals PLC., Dhaka-1212, Bangladesh; 4Department of of Physiology and Pharmacology, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh; 5Department of Epidemiology and Public Health, Faculty of Veterinary, Animal and Biomedical Sciences, Sylhet Agricultural University, Sylhet-3100, Bangladesh; 6Ministry of Public Administration, Government of the People’s Republic of Bangladesh, Dhaka-1000, Bangladesh; 7Department of Animal and Fish Biotechnology, Faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University, Sylhet-3100, Bangladesh; 8Associate Editor, International Journal of Dairy Technology, Wiley-Blackwell, UK; 9Department of Genetics and Biostatistics, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico, Mexico City, Mexico.

Received | September 11, 2023; Accepted | December 30, 2023; Published | January 26, 2024

*Correspondence | Sudeb Saha, Food and Feed Immunology Group, Laboratory of Animal Animal Food Function, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8576, Japan; Email: [email protected]

Citation | Saha S, Ray JP, Mondal AK, Ahsan MI, Debnath M, Afrin D, Ahmed SSU, Gupta JD, Datta N, Toledo-Alvarado HO, Kitazawa H (2024). Effect of probiotic Bacillus coagulans on performance and blood metabolites of dairy cows with subclinical mastitis.

Adv. Anim. Vet. Sci., 12(2):318-326.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.2.318.326

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Bovine mastitis is a widespread and critical problem in the dairy industry, negatively impacting the economic profitability and the well-being of dairy cattle (Cheng and Han, 2020; Kober et al., 2022). Mastitis is generally indicated as an inflammation of the mammary gland in either clinical or subclinical form. A devastating feature of mastitis is its recurrent nature (Jamali et al., 2018). The recurrent rate of mastitis is approximately 50%, causing poor milk quality, reducing milk yields, increasing the culling rate, and shrinking the animal’s longevity in the farm (Wente et al., 2020). Mastitis is caused by various microorganisms such as bacteria, virus, protozoa, and fungi (Dalanezi et al., 2020). These pathogens most frequently affect the mammary gland of animals during the dry off and transition period and in the immunosuppression conditions of animals, resulting in progression of inflammation during early lactation phase (Cobirka et al., 2000). This infection starts in the subclinical form and can progress into clinical mastitis, which continues to the early stage of lactation (Bradley and Green, 2000). A study by Rajala-Schultz et al. (2011) revealed that the subclinical mastitis of animals in the dry period were progressed in to clinical mastitis in the early lactation in 50% cases.

The conventional treatment for controlling mastitis is based on antibiotic therapy (Cheng et al., 2008). However, suceess of the mastitis treatment relies on the pathogen specific therapy, and any irrational use of antibiotics can establish the development of antimicrobial resistance, resulting in a major threat to the well-being of cattle and public health (Dalton, 2006; Kober et al., 2022). In recent years, some countries have been using the vaccine for the control of bovine mastitis. However, the efficacy of vaccination is a questionable as well because multietiological microorganisms are involved to cause mastitis (Tashakkori et al., 2020; Urakawa et al., 2022). However, the low frequency of cure rate and high probability of antimicrobial resistance, and composition dependent of the target of vaccine application resulted in for exploration of an innovative and sustainable approach for treating mastitis in animals.

Recent studies suggested employment of probiotics seems to be a good choice for the treatment/control of mastitis (Shkromada et al., 2022; Spaniol et al., 2015). Bacillus coagulans, a gram-positive, spore-forming, lactic acid-producing bacteria, has been proved as a probiotic (Özüsağlam, 2010). After the administration, of the Bacillus coagulans, it can survive in gastrointestinal conditions and exert health benefits on the host. As a result, Bacillus coagulans have been widely used as a probiotic feed in animal husbandry worldwide (Zhou et al., 2020). Literature showed that supplementation of Bacillus coagulans can improve growth performance and reduce diarrhea in piglets (Zhang et al., 2018). Additionally, Bacillus coagulans exhibits a growth enhancing effect in broiler and in aquatics. e.g. shrimp (Zhou et al., 2020). However, so far, very limited numer of researches have been conducted to evaluate the effect of Bacillus coagulans on dairy farming. Thus, the present study was undertaken to evaluate the effects of Bacillus coagulans MTCC 25250 on the body reserves, blood metabolites, yield, and milk composition of cows with subclinical mastitis.

Materials and Methods

The care and treatment of the experimental animals were performed by the guidelines and regulations of Faculty of Veterinary, Animal and Biomedical Sciences, Sylhet Agricultural University, Bangladesh. The animal experiment protocol was approved by the ethics committee, Sylhet Agricultural University, Bangladesh (SAU/Ethical committee/AUP/22/19).

Farms and animals

A positive test using the California Mastitis Test (CMT; DeLaval, Tumba, Sweden) to detect subclinical mastitis was used as the criteria to select cows for their the mastitis history during the previous lactation period. A total of 73 milking cows were present in a dairy farm located in the Sylhet region of Bangladesh. Machine milking was performed twice a day (08:00 h and 17:00 h). All milking cows were tested for California Mastitis test. Twenty four cows were showed positive results in California Mastitis test. We considered twenty (20) multiparous (2-4 lactations) crossbred cows (Local × Holstein) for this study, and the rest four cows were excluded due to different crossbred cows. The cows in this experiment had an average parity of 2.9 and days in milk (DIM) were 135 days, respectively. All animals were randomly allocated to four (4) treatment groups, and 5 cows were included in each treatment. Four dietary treatments were used in this trial and the description of the diet is shown as:

1. Control: Animals were fed a total mixed ration without probiotic supplementation;
2. T1: Animals were fed a total mixed ration with 15 g/d/cow probiotic supplementation;
3. T2: Animals were fed a total mixed ration with 30 g/d/cow probiotic supplementation;
4. T3: Animals were fed a total mixed ration with 45 g/d/cow probiotic supplementation. Probiotics (Bacilllus coagulans MTCC 25250 6.0 × 109 cfu/g) was supplied by the Square Pharmaceuticals, PLC., Bangladesh. The cows were fed adlibitum access to feed and water at 7.30 and 16.00 hours daily. Total mixed rations were prepared by mixing of concentrate mixture (38%), green grass (20.5%), and rice straw (41.5%). Diets were prepared according to the NRC recommendations (NRC, 2001). The cows were housed in a tie-stall barn and remained in an identical manner through out the entire experimental period.

Sample collection and laboratory analysis

The total study period was 67 days with 7 days of the adjustment period. The following data were recorded, and sample collection was performed three times (start, 0 day; mid, 30 day; and end, 60 day) during the entire experiment period.

• Individual daily milk yields (MY, kg/d).
• Milk samples (100 ml per cow) were collected from individual cows at the evening milking and immediately transferred to the laboratory of Dairy Science, Sylhet Agricultural University, Bangladesh, and refrigerated at 4o C without preservative.
• Live body weight (BW), were measured using digital cattle weighing scale (Model: NVK-SCS-A12, NVK Electric Weighing Scale, China).
• Body Condition score (BCS), were measured by an experienced operator on the same day of milk sample collection based on the method described by Edmonson et al. (1989), using a 1 to 5 scale with 0.25 increments.

Detection of subclinical mastitis using California mastitis test

Milk samples were tested with the California Mastitis Test for showing the presence of subclinical mastitis, using the techniques described by Dingwell et al. (2003). This test was performed twice, at the start (0 d) and end (60 d) points.

Milk quality parameters

Milk samples were analysed to investigate the composition (fat, protein, lactose, and total solids) using an ultrasonic milk analyzer (MT-25, Wincom company Ltd, China). The somatic cell count (SCC) was determined using Eko-milk Scan (Somatic Cell Analyzer, Bulgaria) and the data were transformed into the logarithmic somatic cell score (SCS), according to Ali and Shook (1980) method.

Microbial analysis of milk samples

The standard plate count (SPC) and Coliform counts were determined in the collected milk samples, on the same test day of milk quality traits using the method of American Public Health Association (Saha and Ara, 2012), The plate count agar for SPC and EMB agar for the Coliform count were used and the methods described by Saha et al. (2022) were utilized for this microbial study.

Blood collection and analysis

Blood samples (50 ml) were collected at the start (0 days) and end (60 days) date from the jugular vein of cows into tubes containing sodium heparin. To determine blood parameters, blood samples were quickly shifted to the laboratory of Physiology, Sylhet Agricultural University, Bangladesh. Red blood cells (RBC) and White blood cells (WBC) were counted using Neubauer haemocytometer method. The Microhaematocrit technique were used to determine the packed cell volume according to Islam et al. (2014). Differential leukocyte test was performed using thin blood films stained with Giemsa stain by counting 100 white cells from each slide, and the relative abundance in percent of each white cell type was calculated.

Statistical analysis

The somatic cell score was calculated as SCS= (log)2 (SCC⁄100000) + 3. The statistical analyses were conducted using R software (R Core Team, 2016). Data were analyzed using a linear mixed model using the following equation:

Where yijkl is the response trait (BW, BCS, MY, Fat, Protein, Lactose, Total solids, Fat: Protein, SCC, SCS, SPC, Coliform, WBC, RBC, Hb, PCV, Neutrophil, Eosinophil, Lympocyte, Monocyte); μ is the general mean; β1i is the partial regression coefficient on DIMi; Parityi is the effect of parity number j(j= 2,3,4); (Period)k× (Group)l is the effect of the interaction between the Period k(k=start, middle, end) and the Group j(j=C, T1, T2, T3); Animalm is the random effect of the animal; and εijklm~NID(0,σe2) is the residual. An analysis of variance of the linear mixed model was performed, and then multiple comparisons of means were made using a Tukey test. In addition, the estimated marginal means of the periods and groups were calculated, and their 95% confidence intervals were obtained for the data analysis.

Results

Table 1 present the descriptive statistics for body weight (BW), body condition score (BCS), blood parameters, yields, traits, and microbial parameters of milk produced from treatment group cows during the experimental period. On average, the BW and BCS of treatment group animals were 420 kg and 3.17, respectively. The average milk yields was 9.6 kg/d, containing 4.18% fat, 3.57% protein, 4.97% lactose, and 13.0% total solids. The mean value of SCS was 5.06. The average amount of standard plate count (SPC) and coliform counts were 94.03 × 103 CFU/ml and 30.17 CFU/ml, respectively. Results from blood samples indicated that the mean value of White blood cell (WBC), Red blood cell (RBC), and hemoglobin (Hb) were 161.8 × 109/l, 6.26× 1012/l, and 11.26 g/100 ml, respectively. The observed packed cell volume (PCV), neutrophil, eosinophil, lymphocyte, and monocyte were 23%, 35.98%, 0%, 33.39%, and 4.29%, respectively.

 

Table 1: Descriptive statistics for body weight (BW), body condition score (BCS), blood parameters, yield, traits, and microbial parameters of milk.

Traits1	n	Mean	SD	Median	Minimum	Maximum
BW, Kg	60	420	71.75	438	260	521
BCS	60	3.17	0.21	3.25	2.75	3.75
MY, Kg	60	9.57	2.11	9.45	5.6	15.5
Fat, %	60	4.18	0.47	4.12	3.47	5.51
Protein, %	60	3.57	0.22	3.58	3.03	4.08
Lactose, %	60	4.97	0.22	4.96	4.51	5.59
Total solids, %	60	13	0.37	13	12.23	14.21
Fat:Protein	60	1.17	0.14	1.19	0.91	1.46
SCC thous./ml	60	442.4	137.1	473	78	661
SCS	60	5.05	0.58	5.24	2.64	5.72
SPC, CFU/ml × 103	60	94.03	165.6	93.4	66.7	123.1
Coliform, CFU/ml	60	30.17	10.7	28.5	12	69
WBC, 109/L	60	161.76	28.28	156.7	121.6	231.9
RBC, 1012/L	60	6.26	0.73	6.18	5.11	7.69
Hb, g/100ML	60	11.26	1.67	11.75	6.75	13.4
PCV, %	60	23	4.0	23	17	40
Neutrophil, %	60	35.98	11.19	36.17	18.75	57.31
Eosonophil, %	60	0	0	0	0	0
Lympocyte, %	60	33.39	8.15	33.33	17.67	47.29
Monocyte, %	60	4.29	0.82	4	3.23	6.21

 

1BW, Body weight; BCS, Body condition score; MY, Milk yield; WBC, White blood cell; RBC, Red blood cell; Hb, Hemoglobin; PCV, Packed cell volume; SPC, Standard plate count; SCC, Somatic cell count; SCS, Somatic cell score [SCS = 3 + log2 (SCC/100,000)].

 

The sources of variation in the statistical model are presented in Table 2. Days in milk (DIM) and parity exerted a smaller effect than the tested period, group, and period × group. DIM affected the milk-related traits (fat, fat: protein, SCS, and SPC), while no effects were found on blood parameters except RBC. Similarly, parity effect was significant for milk-related traits (fat, fat: protein, SCS, Total solids, and SPC), whereas a little effect observed on blood parameters (RBC, WBC, and hemoglobin). The variation included period, group, and period × group affected most of the studied traits are displayed in Table 2.

Effect of probiotic feeding on body and milk traits

The addition of Bacillus coagulans showed no effect on BW (Figure 1). On the other hand, the groups had received probiotic showed apparent beneficial effect on BCS and it is more pronounced for T2 and T3 groups. There was a trend towards increasing for the T2 group (p < 0.05) after 30 days of supplement and was better prominent after 60 days. The T3 group had little effect on BCS but did not significant different from the control and other treatment groups.

 

Table 2: Results from ANOVA (F-value and significance) for body weight (BW), body condition score (BCS), blood parameters, yield, traits, and microbial parameters of milk.

Traits1	DIM2	Parity	Period	Group	Period × Group
BW, Kg	0.20	4.04 *	0.00	0.04	0.02
BCS	1.36	2.50	0.46	3.98 *	4.57 **
MY, Kg	9.48 **	5.44 **	25.83***	2.78	0.95
Fat, %	5.25 *	12.66***	3.94 *	1.21	1.72
Protein, %	0.00	2.09	2.27	12.04***	2.71 *
Lactose, %	006	0.50	4.66 *	1.28	2.48 *
Total solids, %	1.23	2.75 †	1.46	1.81	1.76
Fat:Protein	3.06 †	7.78 **	5.57 **	1.84	0.61
SCC	3.01 †	0.89	41.21***	20.89***	6.22 ***
SCS	5.11†	1.72	29.9***	12.77***	6.48***
SPC, CFU/ml × 103	15.07***	14.99***	95.34***	40.39***	18.57***
Coliform, CFU/ml	0.16	1.36	14.67***	1.16	2.44 *
WBC, 109/L	0.4	5.83 **	2.58 †	18.61***	6.18 ***
RBC, 1012/L	8.65 **	10.89***	22.20***	19.51***	6.04 ***
Hb, g/100ML	0.35	4.88 *	4.29 *	11.79***	3.51 **
PCV, %	1.07	1.45	0.15	2.26 †	0.78
Neutrophil, %	0.09	0.83	13.63***	12.81***	2.67 *
Eosonophil, %	1.60	1.93	2.56 †	2.30 †	2.56 *
Lympocyte, %	0.27	2.88 †	2.02	28.16***	8.18 ***
Monocyte, %	2.82	1.02	21.93***	8.65 ***	3.78 **

 

1BW, Body weight; BCS, Body condition score; MY, Milk yield; WBC, White blood cell; RBC, Red blood cell; Hb, Hemoglobin; PCV, Packed cell volume; SPC, Standard plate count. 2DIM: Days in milk. Significant codes: †= 0.05; *= 0.01; **= 0.001; ***= 0; SCC, Somatic cell count; SCS, Somatic cell score [SCS = 3 + log2 (SCC/100,000)].

 

As expected, milk yield was decreased over the experimental period due to the advancement of lactation, whereas the decreasing rate was lower for the T3 group than other groups and indicated a significant difference (p < 0.05) after 60 days of supplementation (Figure 1). Fat percentage in milk did not differ among treatment groups, but protein percentage in milk was higher for T2 and T3 groups at the end of 60 days of supplementation. The supplementation of probiotic did not influence the other milk components (lactose, total solids, and fat: protein). Somatic cells are considered one of the major defense components of mammary gland found in milk. Our findings showed that supplementation of probiotic reduced the SCC on day 30 of this study (p<0.05) and showed more pronounced reduction on day 60 (Figure 1). Regarding microbiological characteristics, probiotic supplementations decreased the total bacterial count in milk compared to the control (Figure 2). There was a decrease of bacterial count observed after 30 days of usage and more noticeable after 60 days of probitic usage.

 

Effect of probiotic feeding on blood metbolites

The results of blood metabolites are presented in Figure 3. Our study reveals a significant improvement of RBC and Hb in blood by adding probiotics in the diet. There was a tendency to increase RBC and Hb after 30 days of spplementation, as wll as the increasing trend was observed after 60 days of the study. However, the probiotic suplement groups showed decrease in WBC counts after 60 days of the trial. In the context of differential WBC count parameters (neutrophil, lymphocyte, and monocyte), a significant decrease in neutrophil count was observed after 60 days of supplementation for all probiotic-treated groups, whereas lymphocyte and monocyte were increased significantly in probiotic treated groups, which highlights that probiotic supplement improves the immune system. Thus, probiotic supplement in the subclinical mastitis of cow diet can be considered as a critical component for mitigating the mastitis.

 

 

Discussion

Dairy cattle health, milk yields, and quality are the significant factors for sustainable and profitable dairy farming. During the last two decades, several studies have focused on improving the cattle health, milk quality, and disease control by adding probiotics to the dairy ration (Lambo et al., 2021). However, studies related with the efficacy of probiotic against subclinical mastitis in cow are scarce. In the present study showed probiotic supplementation particularly use of Bacillus coagulans had a positive effect on the body condition score and similar to our study Fredebeul-Krein (2022) found that supplementation of some Bacillus spp. can improve the body condition, and assist in reducing the clinical mastitis in dairy cows. In addition, some researchers have observed improved body condition of dairy cows that have received Bacillus spp. as a single or multispecies probiotic (Choonkham et al., 2021; Merati and Tawhidi, 2022). Dietary probiotic supplement may help improve animal health status by improving nutrient digestion and absorption through the gastrointestinal tract (Mahesh et al., 2021).

The probiotic supplement has notable effects on daily milk yields and composition (Merati and Tawhidi, 2022). In the current study, with the advancement of lactation, the reduction of daily milk yields was as expected, but reduced the decreased rate for T3 probiotic supplement group as compared to the control. Souza et al. (2017) corroborated a positive response to milk yields treated with Bacillus subtilis spores. In order to illucidate this beneficial effect of probiotics on milk production, Spaniol et al. (2015) reported that probiotics may influence milk yields by improving the digestion, preventing ruminal acidosis, boosting the immune system and reducing the somatic cell count. Therefore, the supplementation with probiotics similarly influencing the protein content of milk from T2 and T3 group cows after 60 days of the study. On the other hand, the probiotic supplementation did not influence the fat and lactose percentage in milk. Like our study, Souza et al. (2017) reported that B. subtilis spores supplementation affected the protein metabolism in the rumen resulted an increased protein content in milk, whereas fat and lactose contents were not altered. Several literatures suggest that ruminal ammonia accumulation increased by the supplementation of B. subtilis in the diet which may contribute to the increase of protein content in milk (Qiao et al., 2010; Peng et al., 2012).

Generally, milk SCC (SCS) was increased in subclinical mastitis cows. This higher SCC not only detrimental to udder but also produces severe affects in the quality of raw milk which are prepared with the high SCC raw milk by enhanced protease and lipase enzyme concentrations, which are highly heat resistant and had a detrimental effect in the processing of milk and other milk products (Barbano et al., 2006). Milk SCC (SCS) was decreased in the current study by probiotic supplementation which indicating a beneficial effect. Sun et al. (2013) reported that supplementation of Bacillus subtili in natto reduced SCC in milk, but a crystal clear mechanism remained unclear, and their observation was supported by our findings.

The present study showed that the feeding of probiotic decreased the total bacterial count in milk, but coliform counts were comparable. There is no sufficient evidence in the literature on Bacillus spp. supplementation lowering the bacterial counts in milk in in subclinical studies. In the current investigation, a possible explanation of reduction numer of bacterial count in milk withthe probiotic suplement could be include the inhibitory effects of probiotics on the growth of pathogenic bacteria. The probiotics have exerted the inhibitory effects by the production of acetic, lactic and other acids, bacteriocin, nisin and other antimicrobial compounds for destroying pathogenic and spoilage bacteria present in raw milk.

In general, the health status of the animals could be reflected by blood parameters. The supplementation of probiotic may have the capacity to act on the blood parameters of animal (Choonkham et al., 2021). In this study, probiotic supplementation showed the increament of Hb concentration and RBC count, similar results were obtained by Kabir et al. (2022) and Ghazanfar et al. (2015) in fattening cattle and dairy heifers fed probiotic-supplemented diet, respectively. The probable reasons of elevated Hb concentration and RBC count in blood is that supplementation of probiotics can increase the absorption of iron salt and vitamins B from the small intestine which exerted a positive effect in the blood cells formation process (Ghazanfar et al., 2015). On the otherhand, control group recorded a higher leukocyte (WBC) count compared to probiotic supplement groups, however the presence of normal range of WBC in the blood reflecting absence of negative effects of immunity by WBC which also ensuring providing the regular immunity by WBC. Sarvesha et al. (2017) reported that circulating leukocytes content were increased in the blood and its value reached above the reference value in cows with clinical and subclinical mastitis. In this investigation, a decreased trend for the neutrophils counts was observed for probiotic supplementation groups. Neutrophils are the first cells to act as a defense against any inflammation in the body, and the high neutrophil count is an indication of inflammation. A decrease in blood neutrophils in probiotic supplemented group may be indicated by the diminished mastitis inflammation in the animals which enhancing the beneficiary list. Additionally, a probiotic supplementation causes an increased in the lymphocyte and monocyte counts in blood, which might indicate gut microbiota stimulating intestinal immune system responses. Our findings corroborated with Mousa et al. (2019) results, who reported that supplement of Bacillus spp. to lamb significantly improved leukocytes and monocytes counts in the blood. Elevated lymphocyte and monocyte counts can play an essential role by improving body immune system with eventual destroying invading disease-producing agents.

Conclusions

In conclusion, our study reveals that addition of a probiotic (Bacillus coagulans MTCC 25250) in the diet of dairy cows had positive effects to mitigate subclinical mastitis in cows, influencing body condition score, daily milk yields, protein percentage and decrease the SCC and bacterial count in milk by the adding of 30 and 45 g of probiotic per day per cow. Moreover, blood parameters such as RBC, Hb, lymphocyte, and monocyte contents were improved in cows fed probiotics causing changes in local and systemic immunity for alleviating subclinical mastitis. The present study demonestrated that the probiotic (Bacillus coagulans MTCC 25250) supplementation to the diet can be a new alternative prophylaxis for control of subclinical mastitis in cow. Further studies with larger sample size and an establisment of mechanistic approach of probiotics will be important aspects in order to control subclinical mastitis precisely.

Acknowledgements

We are grateful to the Square Pharmaceuticals PLC., Bangladesh for providing the bacterial strain Bacillus coagulans MTCC 25250 (Trade Name: MastiAid Powder). Also, the authors are thankful to Hlamrasong Marma, Md. Nazmul Islam Bappy, Md. Mashfiq Hossain and Md. Khademul Islam for their technical assistance, data, and sample collection.

A special thanks to Md. Fokruddin Raji (farm owner) for his cooperation and support.

Novelty Statement

This is a novel study for evaluating the dietary supplement of probiotic Bacillus coagulans MTCC 25250 against subclinical mastitis in dairy cows. The research findings highlighted that application of Bacillus coagulans MTCC 25250 strain can be a new alternative to mitigate subclinical mastitis in dairy cows.

Author’s Contribution

Conceptualization: SS, JPR and HK. Methodology: SS, AKM, MIA. Software: SS, MD, DA. Validation: SS, JPR, JDG, HK. Formal analysis: HOTA, SS, JPR, SSUA. Investigation: JDG, HK. Resources: HK, SS. Data curation; SS, AKM. Writing original draft preparation: SS. Writing review and editing: SS, JPR, MIA, MD, SSUA, HK. Visualization: MD, AKM, DA. Supervision: HK, JDG, SS. Project administration: SS, HK. Funding acquisition: HK, JPR, SS. Scientific corrections and editing for English: ND. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the research grant (VetMed/003/2205) from the Square Pharmaceuticals PLC., Bangladesh to Sudeb Saha. This research was also supported by the JSPS Research Fellow (22F22080) to Haruki Kitazawa.

Data availability

Data may be shared upon reasonable request to the corresponding author.

Conflicts of interest

The authors have declared no conflict of interest.

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