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Effects of Bacillus Subtilis on Growth Performance, Metabolic Profile, and Health Status in Dairy Calves at the First 90 Days of Age

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12 August 2024

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14 August 2024

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Abstract
: This study focused on assessing whether the inclusion of probiotics (B. subtilis) as feed additives during the preweaning stage can enhance the body weight and metabolic condition of neonatal calves. A total of 50 Holstein calves, all born on the same farm, were randomly divided into two homogeneous treatment groups after birth. The probiotic was administered 24 hours post-birth, signifying the start of the experimental period. The Control Group (CG), consisting of 25 calves, was fed with a milk replacer, while the Treatment Group (TG), also with 25 calves, received milk replacer supplemented with 7.5 mL/calf/day of B. subtilis probiotic, in accordance with the manufacturer's guidelines. It took one month to collect the animals. Body weight was measured at birth for all animals. A local veterinarian, working on the farm, conducted daily health checks of the calves, recording health parameters and any antibiotic treatments. Blood samples were collected from each calf at birth and again after 30 days by puncturing the jugular vein, using 10 mL evacuated serum tubes before morning feeding. Significant differences in body weight were observed at 30 days of age (p < 0.05). The CG's average body weight (BW) was 52.71 kg (±10.68), compared to 58.49 kg (±14.21) in the TG, a difference of 5.78 kg or an increase of 9.88%. Additionally, the TG exhibited significantly lower aspartate transaminase (AST) activity at 30 days of age (p < 0.001), with an average of 51.02 IU/L (±5.16) compared to 72.00 IU/L (±12.68) in the CG, a 41.12% decrease. The TG also had a significantly higher average phosphorus (P) concentration (p < 0.001) of 3.27 mmol/L (±0.22), a 9.36% increase over the CG's 2.99 mmol/L (±0.27). A significant increase in gamma-glutamyl transferase activity was observed in the TG compared to the CG (p < 0.001), with a 64.48% (or 26.29 IU/L) increase. The TG's average GGT activity was 40.64 IU/L (±22.43), while in the CG it was 14.35 IU/L (±3.45). Additionally, there was a significant 17.7% decrease (or 10.02 g/L) in total protein (TP) concentration in the TG compared to the CG, with the TG averaging 46.32 g/L (±17.43) and the CG 56.34 g/L (±9.81). Overall, dairy calves who were given conventional milk replacer along with a daily dose of 7.5 mL of B. subtilis probiotic experienced enhanced growth performance and a more favorable metabolic profile. Their body weight exhibited a 9.88% growth, but the activity of AST decreased by 41.12%, suggesting a decreased likelihood of liver damage. Additionally, their protein digestibility was 17.7% greater compared to the control group. In addition, B. subtilis had a positive impact on the overall concentration of phosphorus.
Keywords: 
Subject: Biology and Life Sciences  -   Animal Science, Veterinary Science and Zoology
Simple Summary. This study evaluated the effects of adding the probiotic Bacillus (B.) subtilis to the feed of preweaning neonatal calves. Fifty Holstein calves from the same farm were split into two groups: a Control Group (CG) fed with milk replacer and a Treatment Group (TG) receiving milk replacer plus 7.5 mL/calf/day of B. subtilis. The study began 24 hours after birth, with calves' health monitored daily. Significant growth differences emerged by 30 days. The TG showed a 9.88% higher body weight and a 41.12% reduction in aspartate transaminase activity, indicating lower liver damage risk. Phosphorus concentration was 9.36% higher in the TG, while gamma-glutamyl transferase activity was 64.48% higher, suggesting enhanced protein metabolism. Total protein concentration decreased by 17.7% in the TG, indicative of better protein utilization. The inclusion of B. subtilis in the calves' diet led to improved growth and a better metabolic profile, suggesting its potential as a beneficial feed additive in dairy farming.

1. Introduction

Probiotics and prebiotics can enhance gut immunity, generate local antimicrobials, reduce pathogen levels, and inhabit the gastrointestinal tract to avoid colonisation. Probiotics are provided to newborn ruminants to avoid diarrhoea by controlling the balance of microorganisms in the intestines. Many germs, primarily lactic acid bacteria such as Lactobacillus spp. and Bifidobacterium spp., are recognised as probiotics, along with a smaller number of non-lactic acid bacteria including Bacillus (B.) licheniformis and B. subtilis [1]. Proper feeding management is crucial in preventing viral illnesses and promoting healthy growth in calves due to their immature immune system [2]. For probiotics to be successfully implemented in calf production, it is critical to identify probiotic bacteria that function as feed additives and to elucidate the mechanisms by which these probiotics influence the intestinal microbiota and immunity of the host to enhance health and performance [3]. Probiotics have been shown to have positive effects on health and the economy. Specifically, giving Lactobacillus (L.) animalis LA51 and Propionibacterium (P.) freudenreichii PF24 to calves before, during, and after a Clostridium (C.) perfringens challenge significantly reduced the frequency and severity of diarrhoea, improved their overall condition and appearance, and increased survival rates compared to controls [4].The importance of probiotics stems from the significant role a host’s microflora plays in determining susceptibility to diseases, influencing the health of the intestines either positively or negatively [5]. The small intestine’s normal microflora includes species such as Escherichia coli, Lactobacillus spp., and Streptococcus spp. [6]. Certain bacteria, like Lactobacillus spp. and Bifidobacterium spp., are particularly beneficial when present in the small intestine, making them probiotics of special interest [7]. Previous studies have revealed that probiotics demonstrate varying effectiveness against Clostridia infections [8]. It is reported that lactic acid bacteria (LAB) populations in the intestines can be sustained or even increased through the feeding of probiotics and synbiotics [9]. Specifically, Bifidobacterium spp., Lactobacillus spp., and B. subtilis are effective in maintaining beneficial bacterial populations within the intestine [10]. B. subtilis is utilized in the feed industry [11] due to its ability to foster favorable changes in intestinal microflora [12], aid in recovery from diarrhoea [13], and enhance average daily growth and feed efficiency [14]. B. subtilis is acknowledged as a potent probiotic in the livestock sector. Research by Jeong and Kim [15] highlighted its effectiveness for broilers, including reducing pathogens and boosting beneficial gut bacteria, alongside improving daily growth and feed efficiency. Kritas et al. [16] demonstrated its positive impact on swine, notably enhancing reproductive performance in sows, which includes improved body condition during pregnancy, minimized weight loss during lactation, and increased piglet birth weight. In the case of dairy cows, the use of B. subtilis has been linked to increased milk production, as noted by Souza et al. [17]. While the exact mechanism of its beneficial effects is not fully understood, it is theorized that B. subtilis may enhance the anaerobic conditions in the gastrointestinal tract by rapidly consuming oxygen during germination, as suggested by Hoa et al. [10]. Feeding B. subtilis to calves may lead to enhanced feed efficiency post-weaning through the development of beneficial bacteria in the rumen. More extensive research with a larger sample size is required to confirm this potential benefit [2]. Bacillus subtilis modifies the rumen microbiome, enhances digestion during weaning, and reduces the intensity of diarrhoea [18]. Bacillus species, unique in their ability to form spores that resist harsh environments and attract scientific interest, have been proposed for oral administration to improve feed efficiency, immune response, and growth performance [1]. Although probiotics derived from Bacillus spp. have been used for more than seven decades, scientific attention has increased dramatically in recent decades [19]. Administering colostrum and milk replacer containing a combination of probiotics and phytobiotics to dairy calves during the preweaning period improved their health, intake, growth, and metabolic status, in contrast to separate additions of phytobiotics (with rosmarinic acid) and multi-strain Lactobacillus probiotics, which showed no significant impact on feed intake, growth, and physiological indices, suggesting a synergistic effect of the combined treatment that requires further investigation in larger field trials [20]. The blend of probiotics and phytobiotics enhanced the metabolic health of dairy calves [20]. An in vitro study indicated the potential of Bacillus subtilis, likely through its production of catalase and subtilisin, as suggested by Hosoi et al. [21]. According to Wang et al. [22] future research is needed to confirm the effects of live Bacillus additives in vivo, to assess additional live Bacillus additives for enhancing rumen fermentation, and to elucidate the mechanisms by which Bacillus additives affect ruminants. The impact of feeding B. subtilis needs to be assessed to more clearly demonstrate the effects of these probiotics [1].
By examining the impact of probiotics on the diversity of intestinal microbiota, we can gain a deeper understanding of the correlation that exists between probiotics, the structure of microbial communities, and overall health [23]. Bacillus species have exhibited considerable probiotic potential because of the resistance of their spores to various environmental conditions, including heat, pH and pressure. These attributes ensure the preservation of viability throughout gastrointestinal transit and stability when it comes to feed pelleting, storage, and handling [24]. Bacillus subtilis is a thermophilic spore-producing Gram-positive, motile bacterium with rod-shaped morphology [25]. B. subtilis is a probiotic strain commonly utilized as a feed supplement for ruminant animals. It can enhance ruminal fermentation and generate a diverse array of enzymes that facilitate intestinal absorption and digestion when administered to cows. In addition to facilitating the digestion of ratio proteins and returning urea to the rumen, Bacillus subtilis stimulates protein synthesis, facilitates the absorption of amino acids and peptides generated during decomposition, and augments the protein count that reaches the intestine [26]. In addition, B. subtilis is capable of synthesising polypeptides that exhibit an antagonistic nature towards intestinal microorganisms, thereby significantly enhancing the digestibility of animal feed. That aerobic bacterium maintains the ecological equilibrium of the intestines by consuming oxygen and contributing to an anaerobic environment. This, in turn, promotes the reproduction of dominant bacteria in the intestines.
The present study focused on assessing whether the inclusion of probiotics (Bacillus subtilis) as feed additives during the first 90 days of age, can improve growth performance, metabolic profile, and health status of dairy calves.

2. Materials and Methods

2.1. Calves Management and Feeding

The research was executed from October 2023 to July 2024 at a dairy farm situated in the western region of Lithuania. The experimental protocols were executed in adherence to the legal requirements of Lithuania. The study received approval under the number PK012858. After birth, total 50 (25 males and 25 females). Holstein calves were randomly allocated into two homogeneous treatment groups. All calves came from the same farm. Upon birth, the calves were promptly isolated from their mothers and placed in individual calf hutches in a naturally ventilated barn. It took one month to collect the animals. Every day, the pens were cleaned, and dung was taken away, so they always looked clean and dry. A stomach tube was used to administer 4 L of colostrum to calves within the first hour of life and weighed within 2 h after birth. Culpable colostrum from the mother (≥ 23 BRIX) was administered to the calves; if the colostrum failed to meet the quality standards, frozen colostrum was used. Only calves in good health before the study were included in the research. The trial began after birth with the administration of probiotics and lasted until the calf reached 30 days old. Calves were fed individually two times a day. Calves received 3.5 liters of milk replacer at each feeding. Replacement milk (Vilomilk-50, Mørke, Denmark) was supplied at 6 A.M. and 6 P.M. The milk substitute was dissolved in hot water (50–60 °C) and administered once the temperature reached around 40 °C. In a commercial milk replacer (Vilomilk-50, Mørke, Denmark) containing vegetable fat (18. 0%), hydrolyzed wheat protein concentrate (7.5 %), dextrose (2 %), vitamins, and trace elements (2.5 %), 150 g of milk replacer are combined with one liter of water to make milk replacer and blended prior to each feeding in accordance with the guidelines provided by the manufacturer. The composition of the milk replacer is presented in Table 1. Throughout the investigation, the experimental feed additive (probiotic) 7.5 mL/calf/day was mixed into the milk substitute just prior to administration. Each calf ingested an equal daily portion of milk replacer. Calves had free access to water. From day 5 to day 30 of the trial, the calves were provided with unlimited access to pelleted starter feed (Table 2).
The weaning procedure was conducted gradually over a span of four days, commencing at 56 days and concluding at 60 days of age.

2.2. Creation of Groups

The animals were administered the probiotic twenty-four hours after birth, marking the beginning of the experiment period. The calves in the control group (CG) were fed with a milk replacer (n = 25) (13 females and 12 males) and treated group (TG), (n = 25) (13 females and 12 males) was fed with milk replacer with 7.5 mL/calf/day of B. subtilis probiotic (complied with the manufacturer’s guidelines). Following delivery, the calves were assigned to either of the two groups in a random manner. Calves in the TG were given probiotics until they were 30 days old, before being weaned. The probiotics were introduced into the morning milk feeding pail.

2.3. Measurements

Animals were measured for their weight upon birth, 15, 30, 60 and 90 days old using a scale (AGRETO Animal Scale, AGRETO Electronics GmbH, Raabs, Austria). The health status of the calves was checked daily by a local veterinarian working on the farm, health parameters and treatment events (with antibiotics for calves exhibiting clinical symptoms related to bacterial respiratory infections or diarrhoea) were recorded. Calves were monitored daily to assess their health condition. Calves experiencing diarrhoea were administered electrolyte solution, and in cases where it was deemed required, antibiotic treatment was administered. Health and nutritional characteristics, including fecal consistency, and body temperature, were assessed. These parameters were analysed based on the research methodology of Salah et al. [27]. We utilised fecal score as a criterion for diarrhoea, following the method outlined by Magalhães et al. [28]. In short, fecal consistency was rated as follows: 1 for hard, 2 for soft or moderate, 3 for runny or mild diarrhoea, and 4 for watery and profuse diarrhoea. Criteria for respiratory disorders were established in accordance with the Wisconsin Health Scoring Grid, as delineated by Vandermeulen et al. [29].
A body temperature over 39.5 °C was classified as a fever. Recorded were gastrointestinal symptoms such as loose stool and respiratory symptoms such as coughing and nasal discharge.
Blood samples were obtained from each calf at birth, 30, 60 and 90 days, through puncturing the jugular vein. 10 mL evacuated serum tubes were used to collect blood prior to morning feeding. Blood samples were taken from the jugular vein using an evacuated tube without any anticoagulant (BD Vacutainer®, Eysin, Switzerland). The blood samples were transported at a temperature of 4 °C to the Laboratory of Clinical Tests at the Large Animal Clinic of the Veterinary Academy of the Lithuanian University of Health Sciences within one hour of being collected for subsequent examination. Following this, in the laboratory, the blood samples underwent centrifugation for a duration of 10 to 15 minutes at a speed of 1500 x g. After feeding of colostrum a portable refractometer (RHC200, Yhequipment, Shenzhen, China) was used to measure the IgG level in the sample serum and look for passive transfer of immunity. This was done by putting 1-2 drops of the serum fraction onto a refractometer, following the method reported by Renaud et al. [30]. The activities of gamma-glutamyl transferase (GGT) and aspartate transaminase (AST), the quantities of iron (Fe), calcium (Ca), magnesium (Mg), phosphorus (P), albumin (ALB) and total protein (TP) were determined in blood serum using a Hitachi 705 analyzer (Hitachi, Tokyo, Japan) and DiaSys reagents (Diagnostic Systems GmbH, Berlin, Germany).

2.4. Statistical Analysis

The statistical analysis for this study was carried out using IBM SPSS Statistics for Windows, Version 25.0, developed by IBM Corp. in 2017 and based in Armonk, New York, USA. The Shapiro-Wilk test was utilized to ensure the data’s normal distribution. The mean values of the variables were analyzed using Student’s t-test. The Pearson’s correlation was calculated to define the statistical relationships between the evaluated traits. A repeated-measures analysis of variance (ANOVA) test was performed to compare the mean values across the investigated variables. The results were expressed as the mean and standard error of the mean (M S.E.M.), with a set significance level of 0.05 (p < 0.05) for determining probability.

3. Results

3.1. Impact of Bacillus subtilis on Growth in Dairy Calves at the First 90 Days of Age

Significant differences in body weight were observed at 30, 60, and 90 days of age (p < 0.05). At 30 days, the control group (CG) averaged 52.71 kg (±10.68), while the test group (TG) averaged 54.38 kg (±12.34), a difference of 1.67 kg, representing a 3.07% increase. At 60 days, the CG averaged 76.34 kg (±2.05) compared to 79.21 kg (±1.86) in the TG, a difference of 2.89 kg, or a 3.64% increase. At 90 days, the CG averaged 109.67 kg (±1.67), whereas the TG averaged 112.87 kg (±1.95), a difference of 3.2 kg, representing a 2.8% increase (Table 3).

3.2. Impact of Bacillus subtilis on the Fecal Score of Dairy Calves during the First 90 Days of Age

During the current study, we did not find any statistically significant differences in fecal scores between the investigated groups. The largest differences were observed at 15 days of age, but they were not significant (Table 4).

3.3. Impact of Bacillus subtilis on the Respiratory and Digestive Disorders of Dairy Calves during the First 90 Days of Age

According to our results, we found a higher incidence of digestive diseases in the CG group at 15 days of age and an increase in respiratory disorders at 30 days of age (Table 5).

3.4. Impact of Bacillus subtilis on Blood Biochemic Parameters of Dairy Calves during the First 90 Days of Age

AST activity. During this study, significantly lower AST activity was observed in the TG in calves at 30 days of age (p < 0.001). During this period, the average AST activity in the CG was 72.00 IU/L (±12.68), while in the TG it was 51.02 IU/L (±5.16). The difference in AST activity between the groups was 20.98 IU/L, representing a 41.12% decrease (Table 6).
GGT activity. In this study, we found a statistically significant (p < 0.001) increase in gamma-glutamyl transferase activity in the TG compared to the CG in 30 days of age. The GGT activity was 64.48% (or 26.29 IU/L) higher in the TG. The average GGT activity in the TG was 40.64 IU/L (±22.43), while in the CG it was 14.35 IU/L (±3.45) (Table 7).
Phosphorus. We detected that the average phosphorus concentration in the TG at 30 days of age was significantly (p < 0.001) higher by 0.28 mmol/L, corresponding to a 9.36% increase compared to the CG. In the TG, the concentration was 3.27 mmol/L (±0.22), while in the CG it was 2.99 mmol/L (±0.27) (Table 8).
Total protein. We observed a significant decrease of 17.7% (or 10.02 g/L) in TP concentration in the TG compared to the CG at 30 days of age. The average TP concentration in the TG was 46.32 g/L (±17.43), whereas in the CG it was 56.34 g/L (±9.81). Additionally, we found a significantly lower TP concentration in the TG group compared to the CG group, with a reduction of 30% at 60 days of age (Table 9).

4. Discussion

4.1. The Influence of Bacillus subtilis on Growth in Dairy Calves during the Initial 90 Days of Life

Statistically significant disparities in body weight were noted at 30, 60, and 90 days of age (p < 0.05). According to our results, we found a higher body weight in the TG group compared to the CG group, with differences of 3.07% at 30 days of age, 3.64% at 60 days of age, and 2.8% at 90 days of age.
Calf growth performance is a crucial metric for assessing the efficacy of feed additives utilized by producers. By functioning as feed additives, probiotics have the capacity to modulate intestinal flora, augment gastrointestinal digestion and absorption, subsequently enhance animal growth performance, and feed conversion rate [31]. Probiotics have the potential to promote intestinal flora balance in animals by stimulating the production of biologically active substances, increasing the abundance of beneficial bacteria, and decreasing the abundance of pathogenic bacteria [32]. Probiotics primarily work by restoring the balance of gut flora and outcompeting harmful bacteria for resources and space, therefore hindering their growth and colonization. Moreover, probiotics create antimicrobial compounds like organic acids and bacteriocins, regulate the immune system by promoting the production of cytokines and other immune agents, enhance the intestinal epithelial barrier, assist in digestion by producing enzymes that help break down complex molecules, and produce short-chain fatty acids associated with enhanced digestibility and nutrient uptake [33]. Probiotics, widely used as feed additives, play a significant role in forming protective microflora in the gastrointestinal tract of calves. Since the ruminal microorganisms in calves include various bacteria, the hypothesis is that compound probiotics could be beneficial. Therefore, a commercial compound probiotics product was used in this study to assess its impact on Holstein calves [26]. Bacillus spp. cultures are utilized in feed additives for enhancing feed digestibility and preventing diseases in industrial cattle husbandry. This practice aims to reduce antibiotic usage, which significantly contributes to animal preservation and productivity. The inclusion of yeasts in feed promotes the growth of anaerobic bacteria, as the respiratory activity of yeasts shields these bacteria in the rumen from damage caused by oxygen [34].
The findings of this study, along with insights from previous research [35,36,37,38,39], indicate that the management and health status of calves play a role in the effectiveness of probiotics on their growth performance. Sun et al. [40] found that the oral administration of B. subtilis to calves led to enhanced feed efficiency and an increase in their average daily weight gain. Administering B. subtilis natto directly to calves in the preweaning phase improved their growth by enhancing average daily gain and feed efficiency. Additionally, it led to earlier weaning of the calves, all without any negative effects [40]. According to the study of Wu et al., the treated groups had a higher total feed intake compared to the control group, likely due to the enhanced bioavailability of multispecies probiotics that produce organic acids, metabolites, enzymes, and essential nutrients during animal metabolism [41]. In addition, probiotics are thought to indirectly promote the developmental process and fermentation of the reticulorumen. This might lead to higher intake of feed, which may change the microbial population in the rumen [20]. Research on chickens has demonstrated that Bacillus subtilis can suppress the growth of pathogenic microorganisms and enhance digestive enzyme activity, leading to a reduction in ammonia production. These effects, in turn, have been shown to improve the growth performance of poultry [40].
Noori et al. [42] discovered that probiotics can enhance the withers height in Holstein calves. This improvement might be attributed to probiotics, which potentially increases the bioavailability of essential minerals like calcium, phosphorus, and magnesium, thereby contributing to the growth of body size. Bacillus species are thought to mitigate pathogen colonization by activating essential survival pathways and boosting the immune response in epithelial cells, as noted by Williams [43] and Piewngam et al. [44]. However, the impact of probiotics on the growth and health of calves remains uncertain and varies significantly. This variability is often due to differences in the species of probiotics used, the number of viable probiotic bacteria, and the methods of administration, as discussed by Diao et al. [45]. Our study focuses only on body weight data, but it might be improved by including data analysis on feed intake, rumen fermentation, or bacterial community for a more comprehensive analysis. We recommend include these data in future studies.

4.2. The Influence of Bacillus subtilis on the Fecal Score of Dairy Calves in the Initial 90 Days of Life

In the present study, no statistically significant disparities in fecal scores were seen across the groups under investigation. The most notable disparities were noted at 15 days of age, although they were not statistically significant. The fecal score system is a frequently employed method for predicting the occurrence of diarrhea in young animals [30]. According to Cho and Yoon [46], diarrhea in preweaning dairy calves is linked to illness, difficulty absorbing nutrients, and reduced productivity in the future. The current investigation revealed that calves experienced several ailments such as diarrhea, fever, pneumonia, and cough. Diarrhea is closely associated with inflammation of the gastrointestinal system and has a significant impact on the growth performance of calves [47]. The main problem during the first three weeks of birth is acute diarrhea, which is then replaced by respiratory disease about four weeks of age [48]. A study conducted by Medrano-Galarza et al. [49] found that the prevalence of diarrhea in calves varied between 16% and 27% depending on the season of birth. The average prevalence throughout all seasons was 23%. Furthermore, the prevalence of diarrhea inside a herd was greater than within a pen, with average rates of 23.75% and 17.75% respectively. The current study found that the incidence of diarrhea in calves was 12.54%, which was comparatively lower than previous studies. The health of calves was enhanced by a diet that included compound probiotics, as seen by a reduction in fecal score and the need for medical treatments. Therefore, it is advisable to administer a substantial amount of compound probiotics to enhance the growth and well-being of newly born Holstein calves [26].

4.3. The Influence of Bacillus subtilis on Blood Biochemical Parameters of Dairy Calves during the Initial 90 Days of Life

4.3.1. The Influence of Bacillus subtilis on AST Activity of Dairy Calves during the Initial 90 Days of Life

According to our results, the disparity in AST (aspartate aminotransferase) activity across the groups was 20.98 IU/L, indicating a reduction of 41.12% in the TG group. Blood AST activities are a crucial metric for assessing liver function, with heightened AST activity signaling liver impairment [50]. AST, an enzyme prevalent in various tissues and organs and particularly active in the liver [51], shows elevated activity in the serum as an indicator of liver or skeletal muscle injury [52]. The levels of hepatic AST rise during pathological events, leading to the release of these enzymes into the bloodstream [53]. Typically, in animals, the generation and neutralization of free radicals maintain a dynamic equilibrium under normal physiological circumstances, but an overabundance of free radicals can result in oxidative stress [22,54]. According to Wu et al. [41], results with Sprague-Dawley rats involved oral administration of probiotic Bacillus SC06, which decreased activities of alanine transaminase (ALT), AST, alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) and suppressed mitochondrial dysfunction. Studies with lambs and sheep demonstrated reduced activities of AST in the group that was fed Bacillus subtilis [55]. In another study by Choonkham et al. [56], it was discovered that cows given additional B. subtilis had reduced levels of creatinine and albumin and showed a tendency towards decreased AST and β-hydroxybutyrate concentrations. Additionally, these results clarify the processes through which probiotics mitigate oxidative stress and offer a potentially effective approach to averting liver diseases through the modulation of intestinal microbiota [41].

4.3.2. The Influence of Bacillus subtilis on GGT Activity of Dairy Calves during the Initial 90 Days of Life

Our study revealed a significant (p < 0.001) rise in GGT activity in the TG group compared to the CG group at 30 days of age. The GGT activity in the TG was 64.48% greater compared to the reference value of 26.29 IU/L. Mert et al. [57] found that serum GGT activity can be a reliable indicator for assessing passive transfer status in newborn calves. While total protein and urea levels on their own might not provide complete information, they could be useful when evaluated in conjunction with GGT activity. GGT is known to be produced by the cells lining the secretory ducts of the mammary glands. This has led to the understanding that GGT, much like immunoglobulins, is absorbed from the intestines. Previous studies have highlighted that the level of GGT activity in colostrum is significantly higher, about 400-800 times, than in the serum of adult animals. It has been reported that in calves that consume colostrum, the serum GGT activity is observed to be 60-160 times higher than in adult animals [57].

4.3.3. The Influence of Bacillus subtilis on Phosphorus Concentration of Dairy Calves during the Initial 90 Days of Life

Our analysis revealed that the mean phosphorus level in the TG group at 30 days old was significantly (p < 0.001) elevated by 9.36% rise compared to the CG group. Zhang et al. [1] also found a greater phosphorus concentration in calves fed with Lactobacillus plantarum and B. subtilis compared to the control group. Administering probiotics during the weaning stage may aid in the development of the ruminal bacterial community, as suggested by Krehbiel et al. [58]. This could potentially enhance the utilization of nutrients [59]. Hosoi et al. [21] observed that in vitro supplementation with B. subtilis could enhance the quantity and activity of lactic acid bacteria. However, due to the complex nature of the bacterial community in the rumen, influenced by diet, environmental factors, and the physiological and health status of ruminants, further investigation into rumen fermentation and bacterial community changes is needed. Additionally, assessing the effect of B. subtilis alone is crucial for a clearer understanding of the associative impacts of these probiotics [59].

4.3.4. The Influence of Bacillus subtilis on Total Protein Concentration of Dairy Calves during the Initial 90 Days of Life

At 30 days of age, we saw a substantial reduction of 17.7% in TP content in the TG when compared to the CG. In addition, we observed a significantly reduced concentration of TP in the TG group compared to the CG group, with a decrease of 30% at 60 days of age. The most probiotics in the milk replacer bypassed the rumen due to the closure of the oesophageal groove, affecting intestinal bacteria. Fuller [60] noted that probiotics are particularly effective in the presence of a microbial population, especially under stress. [59]. Administering probiotics during the weaning stage may assist in the growth of the bacterial community within the rumen, according to Krehbiel et al. [58]. This could potentially lead to better nutrient utilization [59].
Further research, sample collection, and experimentation could be implemented to broaden the scope of this study. It might be beneficial to evaluate blood urea nitrogen (BUN), since it is the primary product of protein metabolism. Higher protein intake results in increased protein breakdown into amino acids, which are then converted into urea and discharged into the bloodstream [61]. Weaned calves supplemented with Lactobacillus probiotics showed significantly elevated BUN levels compared to the control group on days 7 and 14, suggesting increased amino acid metabolism and delayed nitrogen excretion [62]. Meanwhile, the relationship between urea nitrogen levels in newborn calf serum and protein intake fluctuates. The fluctuation in blood urea nitrogen levels in calves is influenced by the interplay between their energy levels and protein consumption. Calves may deaminate amino acids to produce carbon skeletons for gluconeogenesis [63]. Exploring the use of plasma urea levels to evaluate protein utilisation in future studies, particularly in calves given probiotics, might be advantageous.
Studying has some limitations. Our study has the relatively small sample size, which might limit the generalizability of the findings. Additionally, the study primarily focuses on short-term outcomes, leaving the long-term effects of Bacillus subtilis supplementation on health and development of calves largely unexplored.

5. Conclusions

The findings indicate that dairy calves who were given conventional milk replacer along with a daily dose of 7.5 mL of B. subtilis probiotic experienced enhanced growth performance and a more favorable metabolic profile over the initial 90 days of their lives. More precisely, their body weight grew by 3.07%, and they experienced a decreased risk of liver injury, as seen by a 41.12% drop in AST activity. In addition, the total protein was 17.7% lower when compared to the control group (CG). Additionally, we observed that B. subtilis had a beneficial impact on the overall concentration of phosphorus.
According to the study, producers should include Bacillus subtilis in the meals of dairy calves, since it boosts body weight and enhances metabolic profiles. It is advisable to regularly evaluate health markers, such as body weight and nutrient digestibility. Additionally, monthly blood sampling for AST activity and total protein should be conducted to assess the efficiency of this supplementation.
Future study should prioritize increasing the sample size to ensure more reliable and conclusive findings, as well as investigating the enduring impacts of Bacillus subtilis supplementation on dairy calves. Studies should also take into account various environmental contexts in order to evaluate the adaptability and efficacy of the probiotic. Furthermore, by comparing Bacillus subtilis with other probiotics and completing a comprehensive nutritional study that includes examining nutrient absorption, changes in rumen fermentation, and bacterial population, we may gain a more profound understanding of the most effective ways for improving calf health and growth.

Author Contributions

R.A.: supervision of the whole study, writing—review and editing and software; K.D., J.K, S.A., A.G. and E.A.: data collection and investigation; W.B.: review and editing.

Funding

Ministry of Agriculture of the Republic of Lithuania. Project number MTE-23-10.

Institutional Review Board Statement

This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee (the study approval number is PK016965. 2017.06.06).

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Table 1. Analytical constituents and quantity of the milk replacer.
Table 1. Analytical constituents and quantity of the milk replacer.
Analytical composition
Crude protein 22.0 %
Crude fat 18.0 %
Fiber 0.0 %
Ash 6.5 %
Lysine 17.5 g/kg
Methionine 5.0 g/kg
Calcium 7.5 g/kg
Sodium 4.4 g/kg
Phosphorus 6.3 g/kg
Vegetable fat 18.0 %
Hydrolysed wheat protein concentrate 7.5 %
Dextrose 2 %
Vitamins and trace elements 2.5 %
Table 2. Composition of calf starter ingredients.
Table 2. Composition of calf starter ingredients.
Nutrient Dry Matter
DM (%) 88.82
CP (%) 21.10
SP (%CP) 27.83
NH3 (%) 0.00
ADIP (%) 0.75
NDIP (%) 1.79
ADF (% 10.89
aNDFom (%) 18.28
pef (%NDF) 37.81
Lignin (%) 1.93
NFC (%) 47.14
Sugar (%) 6.96
Starch (%) 23.20
Sol. Fiber (%) 6.97
EE (%) 4.04
Ash (%) 9.45
Ca (%) 1.01
P (%) 0.66
Mg (%) 0.38
K (%) 1.14
S (%) 0.26
Na (%) 0.50
Cl (%) 0.24
Fe (ppm) 132.59
Zn (ppm) 76.39
Cu (ppm) 44.90
Mn (ppm) 40.88
Se (ppm) 1.90
Co (ppm) 1.90
I (ppm) 3.65
Vit-A (IU/k) 31.63
Vit-D (IU/k) 13.53
Vit-E (IU/kg) 288.87
DM–dry matter; CP–crude protein; SP–soluble protein; NH3–ammonium; ADIP-acid detergent insoluble protein; NDIP-neutral detergent insoluble protein; ADF-acid detergent fiber; aNDFom–ammonia neutral detergent fiber on an organic matter basis; pef (%NDF)–physically effective fiber as a percentage of neutral detergent fiber; NFC–non-fiber carbohydrates; Sol. Fiber–soluble fiber; EE- ether extract; Ca–calcium; P-phosphorus; Mg-magnesium; K-potassium; S-sulfur; Na-natrium; Cl-chloride; Fe–iron; Zn–zinc; Cu–copper; Mn-manganese; Se–selenium; Co–cobalt; I–iodine; Vit-A–vitamin A; Vit-D–vitamin D; Vit-E–vitamin E; %-percentage; ppm–parts per million; IU/kg–international units per kilogram. .
Table 3. Body weight of the investigated groups throughout the study.
Table 3. Body weight of the investigated groups throughout the study.
Group Group N Mean Std. Error 95% Confidence Interval for Mean p
0 d. Minimum Maximum
CG 25 34.54 2.05 30.32 38.75 0.737
TG 25 35.48 1.86 31.63 39.33
Total 50 34.98 1.38 32.2 37.76
15 d. CG 25 44.21 2.05 39.99 48.42 0.966
TG 25 44.35 2.68 38.84 49.86
Total 50 44.28 1.67 40.92 47.63
30 d.
 CG 25 52.71 2.05 48.5 56.92 0.048
TG 25 54.38 2.687 52.98 64.01
Total 50 53.54 1.72 52.15 59.05
60 d.
 CG 25 76.34 2.05 65.67 85.09 0.045
TG 25 79.21 1.86 73.45 87.06
Total 50 77.77 1.955 69.56 86.07
90 d.
 CG 25 109.67 1.67 98.43 110.32 0.032
TG 25 112.87 1.95 101.65 123.34
Total 50 111.27 1.81 100.04 116.83
0 d.–body weight at birth; 15 d.–body weight at 15 days of age; 30 d.–body weight at 30 days of age; 60 d.–body weight at 60 days of age; 90 d.–body weight at 90 days of age. CG-control group; TG–test group.
Table 4. Fecal score of the investigated groups throughout the study.
Table 4. Fecal score of the investigated groups throughout the study.
Group N Mean Std. Error 95% Confidence Interval for Mean p
0 d. Minimum Maximum
CG 25 1.67 0.043 1 3 0.87
TG 25 1.56 0.042 1 3
Total 50 1.615 0.0425 1 3
15 d. CG 25 2.76 0.034 2 4 0.09
TG 25 2.25 0.095 1 4
Total 50 2.505 0.0645 1.5 4
30 d. CG 25 1.54 0.042 1 3 0.16
TG 25 1.67 0.049 1 4
Total 50 1.605 0.0455 1 3.5
60 d. CG 25 1.43 0.067 1 3 0.65
TG 25 1.54 0.043 1 3
Total 50 1.485 0.055 1 3
90 d. CG 25 1.54 0.076 1 2 0.56
TG 25 1.16 0.056 1 2
Total 50 1.35 0.066 1 2
0 d.–fecal score at birth; 15 d.– fecal score at 15 days of age; 30 d.– fecal score at 30 days of age; 60 d.– fecal score at 60 days of age; 90 d.– fecal score at 90 days of age. CG–control group; TG–test group.
Table 5. Respiratory and digestive disorders of the investigated groups throughout the study.
Table 5. Respiratory and digestive disorders of the investigated groups throughout the study.
Group Group N Number of respiratory disorders Number of digestive disorders
0 d. CG 25 0 2
TG 25 0 2
15 d. CG 25 0 11
TG 25 0 3
30 d. CG 25 5 2
TG 25 1 3
60 d. CG 25 3 1
TG 25 0 1
90 d. CG 25 0 1
TG 25 0 1
Table 6. AST activity of the investigated groups throughout the study.
Table 6. AST activity of the investigated groups throughout the study.
AST (U/L) N Mean Std. Error 95% Confidence Interval for Mean p
Minimum Maximum
0 d. CG 25 71.14 6.79 57.2 85.09 0.35
TG 25 54.89 2.99 48.73 61.05
Total 50 63.16 3.88 55.37 70.95
30 d. CG 25 72 2.53 66.76 77.24 <0.001
TG 25 62.02 0.97 49.01 53.02
Total 50 60.92 1.94 57.02 64.81
60 d. CG 25 83.78 2.53 66.76 77.24 0.543
TG 25 79.45 0.97 49.01 53.02
Total 50 81.615 1.75 57.885 65.13
90 d. CG 25 66.64 1.53 62.45 76.44 0.67
TG 25 65.67 1.32 50.2 75.76
Total 50 66.155 1.425 56.325 76.1
0 d.–AST activity at birth; 30 d.– AST activity at 30 days of age; 60 d.– AST activity at 60 days of age; 90 d.– AST activity at 90 days of age. CG–control group; TG–test group.
Table 7. GGT activity of the investigated groups throughout the study.
Table 7. GGT activity of the investigated groups throughout the study.
GGT (U/L) N Mean Std. Error 95% Confidence Interval for Mean p
Minimum Maximum
0 d. CG 25 349.11 81.26 182.07 516.16 0.168
TG 25 496.69 66.64 359.43 633.96
Total 50 421.51 53.24 314.68 528.34
30 d. CG 25 14.35 0.72 12.85 15.84 <0.001
TG 25 40.64 4.23 31.95 49.34
Total 50 28.78 2.97 22.81 34.76
60 d. CG 25 32.54 0.8 25.45 43.93 0.154
TG 25 37.45 1.55 29.34 47.54
Total 50 29.995 1.175 27.395 45.735
90 d. CG 25 25.54 0.8 25.45 29.45 0.143
TG 25 28.65 1.55 23.32 34.45
Total 50 27.095 1.175 24.385 31.95
0 d.–GGT activity at birth; 30 d.– GGT activity at 30 days of age; 60 d.– GGT activity at 60 days of age; 90 d.– GGT activity at 90 days of age. CG – control group; TG – test group.
Table 8. Phosphorus concentration of the investigated groups throughout the study.
Table 8. Phosphorus concentration of the investigated groups throughout the study.
P (mmol/L) N Mean Std. Error 95% Confidence Interval for Mean p
Minimum Maximum
0 d. CG 25 2.44 0.06 2.31 2.57 0.745
TG 25 2.91 0.1 2.71 3.12
Total 50 2.67 0.06 2.54 2.81
30 d. CG 25 2.99 0.05 2.88 3.1 <0.001
TG 25 3.27 0.04 3.18 3.36
Total 50 3.14 0.03 3.06 3.22
60 d. CG 25 3.22 0.08 2.77 3.12 0.54
TG 25 3.11 0.03 2.88 3.11
Total 50 3.165 0.055 2.825 3.115
90 d. CG 25 3.32 0.04 2.43 3.34 0.45
TG 25 3.22 0.06 2.97 3.45
Total 50 3.27 0.05 2.7 3.395
0 d.–phosphorus concentration at birth; 30 d.– phosphorus concentration at 30 days of age; 60 d.–phosphorus concentration at 60 days of age; 90 d.–phosphorus concentration at 90 days of age. CG–control group; TG–test group.
Table 9. Total protein concentration of the investigated groups throughout the study.
Table 9. Total protein concentration of the investigated groups throughout the study.
TP (g/L) N Mean Std. Error 95% Confidence Interval for Mean p
Minimum Maximum
0 d. CG 25 47.56 2.01 42.22 59.45 0.56
TG 25 49.33 2.29 36.32 51.33
Total 50 48.445 2.15 39.27 55.39
30 d. CG 25 56.34 1.96 52.29 60.39 0.014
TG 25 46.32 3.29 39.56 53.08
Total 50 51.05 2.07 46.89 55.21
60 d. CG 25 58.12 1.55 55.43 63.55 0.045
TG 25 48.32 2.29 44.56 61.22
Total 50 53.22 1.92 49.995 62.385
90 d. CG 25 60.22 3.55 45.44 65.43 0.055
TG 25 55.54 3.29 47.67 66.43
Total 50 57.88 3.42 46.555 65.93
0 d.– total protein concentration at birth; 30 d.– total protein concentration at 30 days of age; 60 d.–total protein concentration at 60 days of age; 90 d.–total protein concentration at 90 days of age. CG–control group; TG–test group.
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