Use of bean husk as an easily digestible fiber source for activating the fibrolytic rumen bacterium Fibrobacter succinogenes and rice straw digestion

A series of in sacco and in vitro studies were carried out to evaluate bean husks for activation of fibrolytic rumen bacteria and rice straw digestion. First, lablab bean husk, chickpea husk and rice straw were suspended in the rumen of sheep to analyze the bacterial consortium developed on each fiber source. Known members of fiber‐associating bacteria were found on both lablab bean husk and rice straw, but some of these bacteria were lacking on chickpea husk. Second, a pure culture study was carried out using six strains of Fibrobacter succinogenes. Both husks stimulated the growth of all tested strains, including a strain that did not grow on rice straw. The strain OS128 that showed the highest growth on rice straw displayed even higher growth on lablab bean husk without a time lag. Finally, two‐step incubations were carried out to determine whether prior incubation of rumen fluid with husks stimulates subsequent rice straw digestion. Higher digestibility of rice straw was recorded in the second‐round incubation following the first incubation with bean husks. These results suggest that the tested bean husks improve the digestion of rice straw by activating fibrolytic F. succinogenes and other associated bacteria.     

Characteristics of various fibrolytic isozyme activities in the rumen microbial communities of Japanese Black and Holstein Friesian cattle under different conditions

Rumen microorganisms produce various fibrolytic enzymes and degrade lignocellulosic materials into nutrient sources for ruminants; therefore, the characterization of fibrolytic enzymes contributing to the polysaccharide degradation in the rumen microbiota is important for efficient animal production. This study characterized the fibrolytic isozyme activities of a rumen microbiota from four groups of housed cattle (1, breeding Japanese Black; 2, feedlot Japanese Black; 3, lactating Holstein Friesian; 4, dry Holstein Friesian). Rumen fluids in all cattle groups showed similar concentrations of total volatile fatty acids and reducing sugars, whereas acetic acid contents and pH were different among them. Predominant genera were commonly detected in all cattle, although the bacterial compositions were different among cattle groups. Zymograms of whole proteins in rumen fluids showed endoglucanase activities at 55 and 57 kDa and xylanase activity at 44 kDa in all cattle. Meanwhile, several fibrolytic isozyme activities differed among cattle groups and individuals. Treponema, Succinivibrio, Anaeroplasma, Succiniclasticum, Ruminococcus, and Butyrivibrio showed positive correlations with fibrolytic isozyme activities. Further, endoglucanase activity at 68 kDa was positively correlated with pH. This study suggests the characteristics of fibrolytic isozyme activities and their correlations with the rumen microbiota.     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

Bean husks as a supplemental fiber for ruminants: Potential use for activation of fibrolytic rumen bacteria to improve main forage digestion

This study evaluated the suitability of easily digested fiber sources as a supplemental fiber to improve overall fiber digestion in ruminants. First, the degradation of five fibrous feedstuffs and the stimulatory effects on rumen bacteria were examined in situ. Chickpea and lablab bean husks were selected for their potential use due to their large degradable fraction (> 94%), which had a stimulatory effect on fibrolytic rumen bacteria such as Fibrobacter succinogenes. Second, a possible improvement in the digestibility of rice straw diet by husk supplementation was monitored in vivo. Four dietary treatments comprising RS (rice straw and concentrate), CHM (RS supplemented with Myanmar chickpea husk), CHE (RS with Egyptian chickpea husk) and LH (RS with lablab bean husk) were allocated to four wethers. The digestibility of acid detergent fiber was 3.1–5.5% greater in CHM and LH than RS. Total volatile fatty acid concentration was higher in LH than other treatments. Acetate proportion was higher in LH than RS. Ruminal abundance of F. succinogenes was 1.3–1.5 times greater in CHM and LH than RS. These results suggest that bean husk supplementation, especially lablab bean husk, might improve the nutritive value of rice straw diet by stimulating fibrolytic bacteria.     

Interaction of rumen bacteria as assumed by colonization patterns on untreated and alkali‐treated rice straw

Colonization patterns of representative rumen bacteria were compared between untreated rice straw (UTS) and sodium hydroxide‐treated rice straw (SHTS). UTS and SHTS were incubated in the rumen of sheep for 10 min, 1, 2, 6, 12, 24, 48 and 96 h using the nylon bag method. The population sizes of 13 representative bacterial species or groups were quantified by real‐time PCR. The total bacterial population size (abundance) was similar in both UTS and SHTS. Fibrobacter succinogenes showed a higher population size compared to other fibrolytic species and was detected at a higher level in SHTS (3.7%) than in UTS (2.6%). Ruminococcus albus and Ruminococcus flavefaciens were also detected at higher levels in SHTS (0.15% and 0.29%) than in UTS (0.03% and 0.18%). Population sizes of non‐fibrolytic species, such as Selenomonas ruminantium, Anaerovibrio lipolytica and Succinivibrio dextrinosolvens were higher in UTS than in SHTS. Coefficient of determination (r²) on population changes between bacterial species or groups were higher in UTS than in SHTS, suggesting the necessity of stronger bacterial interactions for UTS digestion. Therefore, not only colonization of fibrolytic species, but also synergistic interactions between different bacterial species may be key to the ruminal digestion of rice straw.     

Inclusion of novel bacteria in rumen microbiology: Need for basic and applied science

Rumen microbiology has made a significant contribution to the understanding of ruminant nutrition. However, further progress in research has been hindered by the incomplete analysis of the rumen microbiota comprised of bacteria, protozoa and fungi, most of which remain uncharacterized due to the difficulties in their isolation and cultivation. In order to maximize rumen fiber digestion, it is necessary to understand the community structure of rumen microbes, especially bacteria, and the factors that influence their composition. Recent advances in molecular biology techniques allow the analysis of such bacteria without cultivation, thereby identifying many functional, but uncultured, bacteria as new targets for basic and applied research. Specific uncultured bacterial groups are being considered as important members of a fibrolytic consortium in the rumen, judging by their ecologic distribution. The inclusion of such uncharacterized bacteria in analyses is crucial for understanding the rumen microbial community and its manipulation. In addition, these bacteria could potentially be candidates as probiotics and sources of enzymes for animal feed and other industrial uses.     

Effects of grass hay proportion in a corn silage‐based diet on rumen digesta kinetics and digestibility in dairy cows

In this study, we aimed to evaluate the effects of six levels of orchardgrass hay (GH) proportion (0%, 10%, 20%, 30%, 40% or 50% of dry matter) in finely chopped corn silage (CS)‐based diets on digesta kinetics of CS and GH in the rumen. Six non‐lactating, rumen‐cannulated Holstein cows were used in a 6 × 6 Latin square design. Ruminal digesta kinetics was measured by ruminal dosing of feed particle markers (dysprosium for CS, erbium for GH) followed by fecal sampling. The increase of GH proportion had a quadratic effect (P < 0.01) on total tract digestibility of neutral detergent fiber (NDF) and acid detergent fiber. The proportion of GH did not affect the particle size distribution of rumen digesta, total weight of dry matter or NDF in the rumen. The rates of large particle size reduction in the rumen for CS tended to increase linearly with increasing GH proportion (P = 0.077). A quadratic effect (P < 0.05) was found with increasing the GH proportion for the ruminal passage rate of small GH particles, but not for CS particles. The results suggested that associative effects between CS and GH could be generated on rumen digesta kinetics when cows were fed a CS‐based diet with an increased proportion of GH.     

Partial characterization of phylogeny,ecology and function of the fibrolytic bacterium Ruminococcus flavefaciens OS14, newly isolated from the rumen of swamp buffalo

The fibrolytic rumen bacterium Ruminococcus flavefaciensOS14 was isolated from swamp buffalo and its phylogenetic, ecological and digestive properties were partially characterized. Isolates from rumen contents of four swamp buffalo were screened for fibrolytic bacteria; one of the 40 isolates showed a distinctive feature of solubilizing cellulose powder in liquid culture and was identified as R. flavefaciens based on its 16S ribosomal DNA sequence. This isolate, OS14, was employed for detection and digestion studies, for which a quantitative PCR assay was developed and defined cultures were tested with representative forages in Thailand. OS14 was phylogenetically distant from other isolated and uncultured R. flavefaciens and showed limited distribution among Thai ruminants but was absent in Japanese cattle. OS14 digested rice straw and other tropical forage to a greater extent than the type strain C94 of R. flavefaciens. OS14 produced more lactate than C94, and digested para grass to produce propionate more extensively in co‐culture with lactate‐utilizing Selenomonas ruminantium S137 than a co‐culture of C94 with S137. These results indicate that phylogenetically distinct OS14 could digest Thai local forage more efficiently than the type strain, possibly forming a symbiotic cross‐feeding relationship with lactate‐utilizing bacteria. This strain might be useful for future animal and other industrial applications.     

The relationships between odd‐ and branched‐chain fatty acids to ruminal fermentation parameters and bacterial populations with different dietary ratios of forage and concentrate

The objectives of this study were to investigate the effect of different dietary ratios of forage and concentrate (F:C) on ruminal odd‐ and branched‐chain fatty acids (OBCFAs) contents and to evaluate the relationships between OBCFA and ruminal fermentation parameters as well as bacterial populations tested by real‐time PCR technique. The experimental design was a 3 × 3 Latin square. Three rumen‐fistulated dry Holstein cows were fed three rations with different dietary F:C ratios (F:C; 30:70, 50:50 and 70:30). The rumen samples were collected every two hours (0600, 0800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 0200 and 0400 h) over three consecutive days in each sampling period. The results showed that rumen OBCFA profiles are significantly (p < 0.05) affected by the dietary F:C ratios. The concentrations of C11:0, C13:0, iso‐C15:0, iso‐C16:0, iso‐C17:0 and C17:0 were higher in the cows fed dietary F:C ratio of 70:30 than those fed with other two rations. However, the concentrations of anteiso‐C15:0, C15:0 and total OBCFA were on the lowest level in the high forage diet. Correlation and regression analysis showed that ruminal OBCFAs had strong relationships with ruminal fermentation parameters and bacterial populations. In particular, the iso‐fatty acids had potential power to predict butyrate and isoacids metabolized in the rumen, whereas the fatty acids with 17 carbon atoms correlated with ruminal NH₃‐N content. The OBCFA contents have different relationships with fibrolytic and starch bacteria in the rumen. C17:0 and its isomers might be used to predict populations of fibrolytic bacteria.     

海子水牛瘤胃微生物的宏基因组学分析

为系统探讨海子水牛瘤胃内的微生物组成及木质纤维素降解酶系,本试验利用基于高通量测序的宏基因组学技术对海子水牛(2.5岁左右,平均体重为493 kg)瘤胃液样本进行组学分析。结果表明,共获得了77 283 638条reads,并拼接出744 712个scaffold。经prodigal分析后,共预测出827 044个基因。通过基因注释发现海子水牛瘤胃中含有多种木质纤维素降解微生物,如生黄瘤胃球菌(Ruminococcus flavefaciens)、白色瘤胃球菌(Ruminococcus albus)、产琥珀酸丝状杆菌(Fibrobacter succinogenes)及栖瘤胃普雷沃氏菌(Prevotella ruminicola)。此外,还发现有38 011个基因编码蛋白质具有木质纤维素降解酶活性,其中糖苷水解酶(GH)基因数量最多(17 877个),糖基转移酶(GT)(8 637个)、碳水化合物结合模块(CBM)(4 693个)和碳水化合物酯酶(CE)基因(4 214个)数量次之,多糖裂合酶(PL)(1 296个)和辅助氧化还原酶(AA)基因(934个)数量较少。在GH基因中,归属于GH2、GH43、GH97、GH3家族的基因较多,且编码蛋白质具有寡糖降解酶活性的基因数量最多。此外还发现了少量的纤维小体组分蛋白基因。结合其他物种肠胃宏基因组中GH基因比对分析,发现海子水牛瘤胃中的纤维素酶、半纤维素酶和分支降解酶比例与奶牛瘤胃较为接近。由此可见,海子水牛瘤胃内含有丰富的木质纤维素降解微生物及酶系,这将为筛选具有工业应用潜力的酶基因奠定理论基础。     

Ground transport stress affects bacteria in the rumen of beef cattle: A real‐time PCR analysis

Transport stress syndrome often appears in beef cattle during ground transportation, leading to changes in their capacity to digest food due to changes in rumen microbiota. The present study aimed to analyze bacteria before and after cattle transport. Eight Xianan beef cattle were transported over 1000 km. Rumen fluid and blood were sampled before and after transport. Real‐time PCR was used to quantify rumen bacteria. Cortisol and adrenocorticotrophic hormone (ACTH) were measured. Cortisol and ACTH were increased on day 1 after transportation and decreased by day 3. Cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus flavefaciens), Ruminococcus amylophilus and Prevotella albensis were increased at 6 h and declined by 15 days after transport. There was a significant reduction in Succinivibrio dextrinosolvens, Prevotella bryantii, Prevotella ruminicola and Anaerovibrio lipolytica after transport. Rumen concentration of acetic acid increased after transport, while rumen pH and concentrations of propionic and butyric acids were decreased. Body weight decreased by 3 days and increased by 15 days after transportation. Using real‐time PCR analysis, we detected changes in bacteria in the rumen of beef cattle after transport, which might affect the growth of cattle after transport.     

Effect of non‐starch‐polysaccharide‐degrading enzymes as feed additive on the rumen bacterial population in non‐lactating cows quantified by real‐time PCR

The effects of non‐starch‐polysaccharide‐degrading enzymes, added to a maize silage‐ and grass silage‐based total mixed ration (TMR) at least 14 h before feeding, on the rumen bacterial population were investigated. Six non‐lactating Holstein Friesian cows were allocated to three treatment groups using a duplicate 3 × 3 Latin square design with three 31‐day periods (29 days of adaptation and 2 days of sampling). Treatments were control TMR [69% forage and 31% concentrates on a dry matter (DM) basis] or TMR with 13.8 or 27.7 ml/kg of feed DM of Roxazyme G2 liquid with activities (U/ml enzyme preparation) of xylanase 260 000, β‐glucanase 180 000 and cellulase 8000 (DSM Nutritional Products, Basel, Switzerland). The concentrations of 16S rDNA of Anaerovibrio lipolytica, Fibrobacter succinogenes, Prevotella ruminicola, Ruminococcus flavefaciens, Selenomonas ruminantium and Treponema bryantii, and their relative percentage of total bacteria in rumen samples obtained before feeding and 3 and 7 h after feeding and from two rumen fractions were determined using real‐time PCR. Sampling time had only little influence, but bacterial numbers and the composition of the population differed between the transition layer between rumen fluid and the fibre mat (fraction A) and the rumen fluid (fraction B) highlighting the importance to standardize sampling. The 16S rDNA copies of total bacteria and the six bacterial species as well as the population composition were mainly unaffected by the high levels of exogenous enzymes supplemented at all sampling times and in both rumen fractions. Occasionally, the percentages of the non‐fibrolytic species P. ruminicola and A. lipolytica changed in response to enzyme supplementation. Some increases in the potential degradability of the diet and decreases in lag time which occurred collaterally indicate that other factors than changes in numbers of non‐particle‐associated bacteria are mainly responsible for the effects of exogenous enzymes.     

Effects of supplementing an active dry yeast product on rumen microbial community composition and on subsequent rumen fermentation of lactating cows in the mid‐to‐late lactation period

The effects of supplementing feed of cows in mid‐to‐late lactation with an active yeast product (Actisaf Sc 47) were evaluated using 15 Holstein cows in a replicated 3 × 3 Latin square design. The animals were fed a mixed ration with 33% neutral detergent fiber, consisting of timothy hay (29.8%), a commercial concentrate (70.0%) and commercial calcium triphosphate (0.2%), twice daily to meet 105% of their energy requirement. Yeast supplement was set at 0, 5 and 10 g per day over 21‐day periods, each of which consisted of 14 days for adaptation followed by 7 days of data collection. Milking performance, plasma metabolite parameters, rumen volatile fatty acids, lipopolysaccharide and microbial properties were measured. Although there were no significant differences in feeding and milking performance or blood parameters associated with supplementation, the acetate to propionate ratio in the rumen fluid tended to decrease (P = 0.08). The population of Bacteroidetes tended to be less prominent (P = 0.07) and the fibrolytic bacterium Fibrobacter significantly increased (P < 0.05) in the rumen fluid of the yeast 10 g group compared with that of the control. These data suggest that effects of supplementing live yeast to cows in mid‐to‐late lactation may be limited to microbial composition and fermentation characteristics in the rumen.     

Influence of fibrolytic enzymes in total mixed ration containing urea-molasses-treated sugarcane bagasse on the performance of lactating Holstein–Friesian crossbred cows

The aim of this study was to determine the effect of different levels of fibrolytic enzyme on nutrient utilization and milk production in dairy cows. Four multiparous early-to-mid-lactation Holstein–Friesian crossbred cows were randomly allocated in a 4 × 4 Latin square design. Cows were fed a balanced total mixed ration (TMR) on a dry matter (DM) basis containing 0, 1.2, 2.4, and 3.6 g/kg DM of fibrolytic enzyme in TMR, where the TMR comprises 60% concentrate supplemented with a fibrolytic enzyme at 0, 2, 4, and 6 g/kg DM of concentrate, and 40% urea-molasses-treated sugarcane bagasse (UMSB) was used as a roughage source. Fibrolytic enzyme supplementation in TMR containing UMSB did not affect dry matter intake (DMI) of dairy cows (p > 0.05). There was a quadratic effect of fibrolytic enzyme levels on the digestibility of DM, organic matter (OM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) (p < 0.05), and the maximal response was reached at 1.2–2.4 g/kg DM of fibrolytic enzyme added in the TMR. Furthermore, 3.5% fat-corrected milk, milk fat, total volatile fatty acids, and propionic acid were greater in a cow fed with 1.2–2.4 g/kg DM of fibrolytic enzyme, resulting in a lower ratio of acetic acid to propionic acid (p < 0.05). In conclusion, adding a fibrolytic enzyme in TMR containing UMSB improved nutrient utilization, rumen fermentation, and milk production of lactating dairy cows.     

Metagenomic profiles of the rumen microbiota during the transition period in low‐yield and high‐yield dairy cows

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low‐yield (LY) or high‐yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA‐based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.     

Provision of beta‐glucan prebiotics (cellooligosaccharides and kraft pulp) to calves from pre‐ to post‐weaning period on pasture

We conducted two feeding experiments to evaluate the effects of supplementation with either cellooligosaccharide or kraft pulp on growth performance in grazing beef calves (Japanese Black) from 4 weeks pre‐weaning to 12 to 16 weeks post‐weaning. In Experiment 1 (20‐week duration), nine calves (2.9‐month‐old females) were assigned to either a control group (CON) or an experimental group (CEL) fed cellooligosaccharide at a rate of 10 g/day mixed with concentrate. Average daily weight gain tended to be greater in CEL than in CON, especially after 1 month of weaning. In Experiment 2 (16‐week duration), 10 calves (2.0‐month‐old females) were assigned to either a control group or an experimental group (KRA) fed kraft pulp at a rate of 10% replacement of total digestible nutrients with concentrate. The proportion of fibrolytic bacteria increased and that of methanogenic Archaea decreased in the rumen microbial community composition of KRA calves in Experiment 2, whereas the decrease in Fibrobacter and Archaea was observed in CEL calves at first 4 weeks in Experiment 1. We conclude that beta‐glucan prebiotic supplementation to grazing calves at pre‐weaning would affect rumen microbial composition and modified rumen fermentation characteristics, leading to a better rumen environment via different means.     

The bio‐surfactant mannosylerythritol lipid acts as a selective antibacterial agent to modulate rumen fermentation

Methyl‐mannosylerythritol lipid (MEL), a new sugar esterified lipid synthesized by Pseudozyma aphidis, was assessed for its functionality in modulating rumen fermentation and microbiota toward more propionate and less methane production. A pure culture study using rumen representatives showed that MEL selectively inhibited the growth of most Gram‐positive bacteria including Streptococcus bovis, ruminococci, and Fibrobacter succinogenes, but not Gram‐negative bacteria such as Megasphaera elsdenii, Succinivibrio dextrinosolvens, and Selenomonas ruminantium. A batch culture study revealed that MEL significantly decreased methane production in a dose‐dependent manner with accumulation of hydrogen, while propionate production was enhanced. A continuous culture (Rusitec) study confirmed all of these changes. A feeding study revealed that sheep fed a MEL diet showed an increased proportion of propionate, while proportions of acetate and butyrate were decreased without affecting total VFA level. These changes disappeared after cessation of MEL feeding. Based on these results, dietary application of MEL can favorably modify rumen fermentation in terms of the efficiency of dietary energy utilization.     

Partial characterization of structure and function of a xylanase gene from the rumen hemicellulolytic bacterium Eubacterium ruminantium

A gene encoding for xylanase activity in the rumen hemicellulolytic bacterium Eubacterium ruminantium was cloned into pBR322 in Escherichia coli (E. coli ). The primary clone had a 5.7 kb insert produced by Eco RI partial digestion. Subcloning followed by sequencing allowed for the discovery that this enzyme has a glycosyl‐hydrolase family 10 catalytic domain with a family 9 carbohydrate binding module at C‐terminus and a region partially homologous to a family 22 carbohydrate binding module at N‐terminus. Cloned xylanase is specifically active against xylan and oligoxyloside to produce xylobiose and xylotriose, showing optimal pH and temperature at 7.0 and 50°C, respectively. Molecular size of the xylanase (91 kDa) was confirmed by zymogram analysis of the E. coli clone, which agreed with the predicted size from the DNA sequence. Functions of the two modules at C‐ and N‐termini were evaluated by using xylanase variants with and without the respective module and the C‐terminal module was found to be functional in binding to acid‐swollen cellulose and insoluble oat‐spelt xylan, whereas the N‐terminal module was inactive for binding them.     

Effect of Bacillus subtilis C-3102 supplementation in milk replacer on growth and rumen microbiota in preweaned calves

We aimed to assess the effect of feeding Bacillus subtilis C-3102 on the growth and rumen microbiota in the preweaned calves. Twelve newborn Japanese Black calves were randomly allocated to either the control (n = 6) or the treatment (n = 6) groups in the present study. Calves in the treatment group were offered B. subtilis C-3102 supplemented milk replacer throughout the preweaning period. Rumen fermentation during the first 21 days of life seemed to be slightly suppressed by feeding B. subtilis C-3102. This fermentation shift was probably attributed to the lower abundance of the core members of rumen microbiota until 21 days of age in the calves fed B. subtilis C-3102. However, feeding B. subtilis C-3102 did not influence the abundance of the core members of rumen microbiota at 90 days of age. Distribution of Sharpea spp. and Megasphaera spp., which potentially contribute to low methane production and are regarded as beneficial rumen bacteria, was higher in the rumen of calves fed B. subtilis C-3102 at 90 days of age. These results suggest that B. subtilis C-3102 supplementation in milk replacer could potentially contribute to the improvement of feed efficiency after weaning via the establishment of beneficial rumen bacteria.