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Article

The Effect of Ruminal Fluid Adaptation to a Direct Fed Microbial: In Vitro Methane Production and Fermentation Characteristics

by
Sreemol Suthan Nair
1,2,*,
S. Richard O. Williams
1,*,
Aodán S. ó Neachtain
1,
Renata Tognelli
1,2,
Subhash Chandra
3,
Pablo S. Alvarez-Hess
1,2,
Long Cheng
4,
Khageswor Giri
5 and
Joe L. Jacobs
1,3,6
1
Agriculture Victoria Research, Ellinbank, VIC 3821, Australia
2
School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
3
Agriculture Victoria, Tatura, VIC 3616, Australia
4
School of Agriculture, Food and Ecosystem Sciences, Faculty of Science, Dookie College, The University of Melbourne, Dookie, VIC 3647, Australia
5
Agriculture Victoria Research, Bundoora, VIC 3083, Australia
6
School of Applied Systems Biology, La Trobe University, Bundoora, VIC 3086, Australia
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(8), 473; https://doi.org/10.3390/fermentation11080473
Submission received: 15 July 2025 / Revised: 11 August 2025 / Accepted: 14 August 2025 / Published: 19 August 2025

Abstract

Direct-fed microbials (DFM) have emerged as a promising dietary strategy for enteric methane abatement. However, it is unclear whether in vitro studies trialing DFM should use ruminal fluid previously adapted to the DFM of interest or if the DFM can be directly added to an unadapted inoculum. Ten lactating, multiparous, rumen cannulated Holstein-Friesian cows were randomly allocated to one of two groups: 1) adapted, basal diet plus 4 g/d of a blend of Bacillus subtilis and Bacillus licheniformis (1.6 × 109 CFU/g each), delivered via the rumen canula; and 2) naive, basal diet only. Ruminal fluid from both groups was incubated in an in vitro 24-h batch culture system with two rates of Bacillus spp. and three feed substrates (hay, pasture, cereal grain), resulting in 12 treatments. Methane production was 16% greater, and total volatile fatty acid concentration was 7% greater in incubations using adapted ruminal fluid compared to those using naive ruminal fluid; however, neither parameter was affected when Bacillus spp. was added to the batch incubation system. Future in vitro studies evaluating DFM should consider including a period of in vivo adaptation to mimic their potential impact under in vivo feeding conditions.

1. Introduction

Methane is the second most significant greenhouse gas, contributing approximately 0.5 °C to observed global warming (from 2010 to 2019) [1]. The primary contributor of anthropogenic methane is the livestock sector, accounting for around 90% of global methane emissions [2,3]. Within livestock, ruminants are the main contributors towards methane emissions at a global scale [4,5]. Enteric methane production is a dietary inefficiency, with up to 10% of an animal’s gross energy intake lost as methane [6]. Addressing this challenge involves implementing a range of potential strategies, including management, nutritional, genetic, and advanced molecular technologies [7]. Dietary manipulation is a pragmatic nutritional strategy that can improve animal productivity while lowering methane emissions [8]. Amongst nutritional interventions, direct-fed microbials (DFM) have emerged as a promising methane mitigation strategy as they are sustainable and widely accepted by consumers and producers [9]. The US Food and Drug Administration defines DFM as feed products that contain live, naturally existing microbes [10]. These have been used in different livestock diets to enhance fiber digestion and lactate utilization, thereby stabilizing rumen pH [11,12]. Additionally, DFM have shown potential in mitigating enteric methane emissions by redirecting metabolic hydrogen from methanogenesis to alternative pathways, such as lactate, succinate, or propionate production, reductive acetogenesis, or anaerobic respiration [9].
Among DFM, several species of bacteria from the genus Bacillus (e.g., B. licheniformis and B. subtilis) have been identified as non-pathogenic additives and are widely used in animal feeds [13]. Bacillus spp. are classified as gram-positive, catalase-positive, spore-forming, aerobic, and facultative anaerobic bacteria [14,15], with over 2700 species of Bacillus identified to date [16]. These bacteria can adapt to adverse conditions, such as lower gastric pH, by producing spores that germinate in the intestinal lumen [17,18,19] of pigs, cattle, and poultry [20,21]. Feeding Bacillus spp. has been associated with improved dry matter intake (DMI), fiber digestibility, and average daily gain, while also showing potential to enhance nitrogen utilization and milk yield, without negatively affecting blood metabolites in dairy cattle [22,23]. The inclusion of Bacillus subtilis, Bacillus licheniformis, or their blend in the diets of ewes (at 2.56 × 109 CFU/day) [24] and dairy cows (at 2 to 6 × 1011 CFU/day) [25,26] has been associated with beneficial outcomes in terms of milk yield, milk fat concentration, and milk protein concentration. Beyond these productivity benefits, both in vitro [27,28] and in vivo [29,30] studies have demonstrated that Bacillus spp. supplementation can potentially reduce enteric methane emissions. The methane mitigation potential of Bacillus spp. without affecting DMI in dairy cows is associated with an increase in ruminal propionate concentration [31,32], a process that competes with methanogenesis for available hydrogen [33].
In vitro fermentation offers a rapid screening method to evaluate and test potential methane mitigants. The accuracy of this method may depend on the microbial composition of the ruminal fluid used. This is particularly relevant for additives such as DFM, where microbial adaptation can significantly alter rumen fermentation patterns and methane production [34]. However, it is unclear whether in vitro fermentation studies should use ruminal fluid inoculum from animals previously adapted to Bacillus spp., or if Bacillus spp. should be directly added to the incubation system. Moreover, there is no information available on the effect of using ruminal fluid from animals previously adapted or unadapted (naive) to Bacillus spp. on subsequent in vitro fermentation of substrate on methane production.
Our objective was to evaluate the effects of a) the addition of a Bacillus spp.-based DFM on in vitro total gas and methane production, and fermentation parameters when the ruminal fluid from animals previously adapted to Bacillus spp. was used, and b) direct addition of the DFM to a 24-h batch incubation system using different substrates.
We hypothesized that 1) incubations using ruminal fluid sourced from cows previously adapted to Bacillus spp. would result in a lower in vitro methane production and acetate to propionate ratio than those using ruminal fluid from naïve cows irrespective of substrate used, 2) adding Bacillus spp. to a substrate incubated in vitro will result in lower methane production and acetate to propionate ratio than incubations without added Bacillus spp. irrespective of substrate used, and 3) there would be no interaction between ruminal fluid source and the direct addition of the DFM irrespective of substrate used.

2. Materials and Methods

The research was conducted at the Ellinbank SmartFarm, Victoria, Australia (38°14′ S, 145°56′ E). All cows used in the experiment were familiarized with the experimental facilities prior to the study. The experiment was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes [35]. Approval to conduct the experiment was obtained from the DEECA Agricultural Research and Extension Animal Ethics Committee (Approval code: 2022-13, approved date: 13 December 2022).

2.1. Animals

Ten lactating, rumen cannulated (www.rumencannula.com), Holstein-Friesian cows producing 18.9 ± 3.35 kg milk/d (mean ± standard deviation), 269 ± 23.2 days in milk, consuming a grain mix of 8.9 kg DM and with a body weight of 630 ± 37.2 kg were randomly assigned one of two groups: 1) an adapted group (5 cows), receiving a DFM at a rate of 4 g/cow per day, and 2) a naive group (5 cows), that did not receive additional DFM. The dose of 4 g/cow per day was determined based on previous studies [36] and recommendations from the product manufacturer (Neoculi Pty Ltd., Burwood, VIC, Australia). Both groups were offered the same basal diet per cow, consisting of a perennial ryegrass (Lolium perenne L.)-dominant pasture (8 kg DM/d), pasture silage (4 kg DM/d), vetch (Vicia sativa L.) hay (3.9 kg DM/d), corn (Zea mays L.) silage (3.0 kg DM/d), and a grain mix (8.9 kg DM/d). The grain mix contained rolled barley (Hordeum vulgare L.) grain, 296 g/kg DM; rolled corn grain, 280 g/kg DM; rolled wheat (Triticum aestivum L.) grain, 148 g/kg DM; solvent extracted canola (Brassica napus L.) meal, 206 g/kg DM; limestone, 10.0 g/kg DM; molasses, 15.0 g/kg DM; sodium bicarbonate, 15.0 g/kg DM; and minerals, 28.6 g/kg DM. Most of the grain mix (7.0 kg DM/d) was offered in the dairy during milking; the remaining grain, vetch hay, and maize silage were mixed and offered on a feed pad; and the pasture and pasture silage were available in a paddock. Cows were milked and fed twice per day.
The DFM was a blend of Bacillus subtilis (DSM 5750) (1.6 × 109 CFU/g) and Bacillus licheniformis (DSM 5749) (1.6 × 109 CFU/g). Two grams of the DFM were weighed into gelatin capsules (Size #10 clear veterinary capsule; Torpac Inc., Fairfield, NJ, USA). One capsule was inserted directly into a cow’s rumen via the fistula after each milking (twice per day), providing 1.28 × 1010 CFU/day as per the manufacturer’s recommendation. Direct insertion of the DFM into the rumen via the fistula eliminated potential variability of dosing that could arise due to potential differences in daily animal dry matter intake.
Ruminal fluid samples were collected from both groups after a 14-day adaptation period [37] and used for subsequent in vitro analysis. Although only six cows were required as donors of ruminal fluid, three adapted and three naive, two additional spares were included for each group to account for potential illness or the need for any treatment (such as antibiotic administration) that could affect the composition of ruminal fluid.

2.2. In Vitro Treatments

Following the 14-day adaptation period, all animals continued on their feeding regime, and an in vitro experiment was performed using ruminal fluid sourced from both adapted and naive cows. The in vitro experiment included two Bacillus spp. rates (0 and the in vitro equivalent of 4 g/head per day (54.0 mg/bottle)) incubated in two sources of ruminal fluid (adapted or naive) and combined with three types of feed substrate (hay, pasture, grain), forming a 2 × 2 × 3 factorial treatment structure (Table 1). An incubation bottle with its associated ANKOM GP module was treated as the experimental unit. All treatments were present equally in both ANKOM systems (2) in each run. There were four water baths, two for each ANKOM system, and each water bath contained the complete set of treatments. ANKOM system 1 contained water baths 1 and 3, and ANKOM system 2 contained water baths 2 and 4. This procedure was replicated in three identical runs, conducted in consecutive weeks, over eight days total. The combination of three runs and four water baths was treated as twelve blocks, with each block having 12 treatment bottles. The 12 treatments were randomly assigned to the bottles in each block, with present treatments replicated only once in a block.
The calculation of in vitro Bacillus spp. dosage for each incubation bottle was scaled based on a rumen capacity of 70 L (approximately 16% of body weight) [38,39] and a daily intake of 4 g of the Bacillus spp. mixture per cow. The experiment was conducted over three identical in vitro runs, all completed within an 8-day span, with each run including four replications of each treatment, resulting in a total of 12 replications per treatment across the entire experiment. A power analysis-based determination of the number of replications per treatment could not be performed because the required prior information about residual variance was not available. We therefore determined the number of replications using the resource equation approach of Mead et al. [40] to ensure that residual degrees of freedom were at least 10 to 15. With 12 replications for each treatment, this resulted in 121 residual degrees of freedom.
In each in vitro run, a wheat–barley grain mix, vetch hay, and perennial ryegrass pasture were used as the in vitro feed substrates, with 1 g DM of individual substrate added per incubation bottle (4 bottles per substrate within run, Table 1). A subsample of each feed substrate incubated was analyzed for nutritive characteristics by wet chemistry [41] at Dairy One, NY, USA via Feed Central, Queensland (Table 2). Feeds were analyzed for concentrations of crude protein (CP) ([41]; method 990.03), acid detergent fiber (ADF) ([41]; method 7.074), neutral detergent fiber (NDF) ([41]; method 2002.04), lignin ([41]; method 949.04), non-fiber carbohydrates (NFC) ([41]; method 992.09), starch ([41]; method 996.11), ash ([41]; method 942.05), ether extract (EE) ([41]; method 2003.05). Concentrations of estimated ME were calculated using Equation (1) [42], as follows:
ME (MJ/kg DM) = [(1.01 × DE) − 1.88 + 0.019 × (EE − 3)],
where DE is digestible energy in MJ/kg DM (calculated according to NRC, 2001 [42]) and EE is ether extract as a percentage of DM.

2.3. Ruminal Fluid Collection and Preparation

For each run (days 14, 16, and 21 of feeding Bacillus spp.), ~400 mL of ruminal fluid was collected from each of the six (three naive and three adapted) cows. The same six animals were used across all three runs, ensuring consistency. The ruminal fluid was collected from multiple sites in the rumen using a copper pipe and a 100 mL syringe, with fluid pooled within groups. The pooled ruminal fluids were placed in 2 L glass bottles prewarmed to 39 °C. Samples were transported to the laboratory in an incubator (Thermoline laboratory incubator; Thermo Fisher Scientific Inc., Scoresby, Melbourne, VIC, Australia) set at 39 °C [43]. Before the in vitro run, the ruminal fluid was filtered under a constant flow of carbon dioxide [43,44], through a 500 µm mesh copper filter to remove suspended particles.

2.4. In Vitro Incubation

The in vitro experiment measured gas production using the automated ANKOM RF gas production system (ANKOM GP, ANKOM Technology, Macedon, NY, USA) over a 24-h period. This system comprised 310 mL ANKOM incubation bottles with associated radio frequency modules, which sat in oscillating water baths maintained at 39 °C (RATEK SWB20D, Ratek Instruments Pty Ltd., Boronia, Melbourne, VIC, Australia). Each ANKOM GP system was limited to communicating with a maximum of 50 linked radio frequency modules, and thus, two systems with their own individual modules were used in parallel.
Immediately prior to an in vitro run, 310 mL incubation bottles were filled with 25 mL of ruminal fluid and 75 mL of Kansas State buffer solution (pH 6.8), leaving 210 mL of headspace volume. The Kansas State buffer solution consisted of solution A containing (per liter of distilled water) 10 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, and 0.1 g of CaCl2H2O. Solution A was then brought to a pH of 6.8 by adding solution B, containing (per 100 mL of distilled water) 15 g of Na2CO3 and 1 g of Na2S·9H2O [45]. After the addition of ruminal fluid and buffer solution, each bottle was capped with an ANKOM GP module, and the headspace was flushed with carbon dioxide. Immediately after 24 h of fermentation, the incubation bottles were placed on ice to cease fermentation. The pH of the ruminal fluid in each bottle was measured using a Mettler-Toledo FG2 pH meter (Schwerzenbach, Switzerland), and samples were collected for gas and fermentation parameter analyses.

2.5. Sample Collection and Analysis

2.5.1. Gas Sampling and Analysis

At the completion of each 24 h incubation, separate gas samples were collected from the headspace of each bottle using an 18G needle and gas-tight glass syringe (SGE International Pty Ltd., Ringwood, VIC, Australia) and transferred into separate Exetainers® (12 mL soda glass vial, Labco Ltd., Buckinghamshire, UK) that had previously been evacuated. Methane proportions in the samples were determined by gas chromatography (GC). Samples were analyzed using an Agilent 7890A equipped with 3 detectors (TCD, µECD, FID), and a GILSON GX-271 autosampler for transferring the pressurized sample from Labco Exetainers® to the GC loops (1 mL × 2). The GC loops and sample inlet were flushed using helium between samples to avoid carryover. The columns used were in accordance with Hess et al., [44]. Methane results were reported in ppm and then converted into percentages.

2.5.2. Gas Production Calculation

Total gas production was measured continuously for 24 h in accordance with Russo et al. [43]. Gas pressure was measured every 5 s, and cumulative pressure was recorded at 5-min intervals. Settings were identical in each in vitro incubation run. Cumulative pressure measurements of gas vented were converted to moles using the ideal gas law (Equation (2)) and subsequently to mL of gas produced using Avogadro’s law (Equation (3)).
n = p(V/RT),
where n = gas produced in moles, p = pressure in kPa, V = headspace volume in L, T = temperature in Kelvin and R = universal gas constant, 8.314472 L kPa K−1 mol−1
Gas production (mL) = n × 22.4 × 1000,
To correct for gas production resulting from residual feed particle fermentation in the ruminal fluid, blank formulations were used consisting of ruminal fluid and buffer solution alone, with no feed substrate. Four blank formulations (two with adapted and two with naive ruminal fluid) were used in each run, two in each ANKOM GP system, with the gas production data from the blanks subtracted from the treatment bottles’ data. The resulting data was averaged for each treatment over the course of 3 runs and plotted against time to produce cumulative gas production curves for each treatment. The final sum of cumulative gas production was taken as total gas production (TGP). Methane proportions from gas sample analysis were then used to calculate methane production (MP) from TGP (Equation (4))
MP (mL) = TGP (mL) × methane% in headspace gas sample at 24 h,

2.5.3. In Vitro Fermentation Parameters

Volatile fatty acids (VFA) were determined from a 10 mL subsample transferred to a 15 mL plastic test tube and then frozen at 18 °C with no additional preservatives. Analysis was performed by capillary GC in accordance with Alvarez-Hess et al. [46].
Ammonia-N was determined from a 4.8 mL subsample transferred to a 10 mL plastic test tube to which 0.2 mL of 0.1 M HCl had been added, then frozen at −18 °C. This sample was analyzed for ammonia nitrogen (NH3-N) concentration by flow injection analysis (QuickChem 8500 Series 2 Flow Injection Analyzer; Lachat Instruments, Milwaukee, WI, USA) with reference to NH4Cl dissolved in 0.1 M HCl standards.

2.5.4. In Vitro Dry Matter Disappearance

Apparent in vitro dry matter disappearance of substrate dry matter (IVDDM) was calculated as per Kinley et al. [47], being the difference in substrate DM added before fermentation and substrate DM remaining after fermentation. Briefly, prior to fermentation, Duran #1 (DWK Life Sciences; Mainz, Germany) 50 mL borosilicate Gooch crucibles with a 5 mm dried sand filtration aid layer were labelled, and each crucible was dried overnight at 100 °C and weighed. Following incubation, the contents of an incubation bottle were gradually poured through a labelled crucible under vacuum, filtering out the buffered ruminal fluid and leaving only the residual substrate. The incubation bottles were rinsed with distilled water, and the washout was filtered to ensure all material was collected in the crucible. This was repeated for all bottles, with the contents of each bottle being filtered through a fresh crucible. The crucibles plus filtrate were then oven dried at 100 °C until constant weight, and the difference between pre- and post-weight of the crucibles was used to determine the IVDDM during the fermentation process.

2.6. Statistical Analyses

Data on gas production and VFAs from each incubation bottle were analyzed as per a randomized complete block design-based ANOVA, which included main effects of ruminal fluid source, rates of Bacillus spp. added in vitro, feed substrate types and their respective two-way and three-way interactions as treatment structure. The fitted ANOVA model can be written in the following equation form (Equation (5)):
y = μ + R + B + S + RB + RS + BS + RBS + b + ε
where y is the response variable of interest, μ is the overall mean, R is the main effect of ruminal fluid source, B is the main effect of direct addition of Bacillus spp., S is the main effect of feed substrate types, RB is the interaction between ruminal fluid source and Bacillus spp., RS is the interaction between ruminal fluid source and feed substrate type, BS is the interaction between Bacillus spp. and feed substrate type and RBS is the three-way interaction amongst ruminal fluid source, Bacillus spp. and feed substrate type, b with the effect of the block and ε the residual error term. The residuals were assumed to be normally distributed with zero mean and constant variance. The histogram of residuals and residuals versus fitted values plots were examined for normality of distribution with constant variance. Statistical significance of differences among treatments was assessed at 5% level of significance. Duncan’s letters indicating significant differences among treatment means were derived using Fisher’s unprotected Least Significant Difference (LSD) test.
All statistical analyses were performed in Genstat (version 22; VSN International, Hemel Hempstead, Hertfordshire, UK).

3. Results

3.1. Interactions

There were no significant two-way or three-way interactions between the different sources of ruminal fluid, two rates of Bacillus spp., or the three substrates for dry matter disappearance, gas production, NH3N, and total VFA at the end of the fermentation period (p ≥ 0.103, Table 3, Table 4 and Table 5 and Table S1). However, there were significant two-way interactions between ruminal fluid and substrate for pH, propionate, iso butyrate, butyrate, iso valerate, valerate, ratio of acetate to propionate (A:P), and (A+Bu:P) to propionate. Within most individual VFA, the effect of substrate type was influenced by the ruminal fluid source (p ≤ 0.028, Table 5). For pH, the extent of effect within a substrate type was influenced by ruminal fluid source (p = 0.003). There were also significant interactions between the addition of Bacillus spp. and ruminal fluid source for acetate, propionate, A:P, and A+B:P (p ≤ 0.037, Table 4 and Table 5). In the proportions of acetate and propionate, A:P and A+Bu:P, there was no effect of Bacillus spp. addition in adapted ruminal fluid, but there was an effect in naive ruminal fluid (Table 5). The other significant interaction was between the addition of Bacillus spp. and substrate type in the proportion of acetate, where the addition of Bacillus spp. varied with substrate type (p = 0.013, Table 5).

3.2. Ruminal Fluid Source Main Effect

In vitro total gas production per dry matter disappearance (TGP/IVDDM) was unaffected (p = 0.206, Table 3), and methane production per dry matter disappearance (MP/IVDDM) was 17% greater (p < 0.001) in incubations using adapted ruminal fluid compared to naive ruminal fluid.
The pH values were lower in incubations using adapted ruminal fluid when compared to those using naive ruminal fluid (p < 0.001, Table 4). Ammonia nitrogen (NH3-N) concentration (mg/L) from incubations using adapted ruminal fluid was 18% greater compared to incubations using naïve ruminal fluid (p < 0.001). Total VFA concentration was greater (p < 0.001) from incubations using adapted ruminal fluid, compared to incubations using naïve ruminal fluid. Acetate as a proportion of total VFA (mM/100 mM) was lower in incubations using adapted ruminal fluid compared to incubations using naïve ruminal fluid (p = 0.042), whereas there was no effect of the different sources of ruminal fluid on propionate concentrations (mM/100 mM, p = 0.760), A:P ratio (p = 0.205) or A+Bu:P ratio (p = 0.483).

3.3. In Vitro Addition of Bacillus spp. Main Effect

The TGP/IVDDM (p = 0.614) and MP/IVDDM values (p = 0.179) were unaffected when two rates of Bacillus spp. were directly incubated in the batch culture.
The pH (p = 0.073) and NH3-N (p = 0.800) concentrations were not influenced by the in vitro introduction of Bacillus spp. Acetate proportion was lower (p = 0.047), and propionate was greater (p = 0.005), resulting in a lower A:P ratio (p = 0.009) in these incubations compared to the incubations without added Bacillus spp.

3.4. Substrate Type Main Effect

The TGP/IVDDM (p < 0.001) and MP/IVDDM values (p < 0.001) were greater when grain was utilized as a substrate, when compared to other substrates.
The pH (p < 0.001) and NH3-N (p < 0.001) concentrations were lower in incubations that utilized grain as the substrate compared to those using hay or pasture as substrates. Total VFA concentration was greatest in incubations that used grain as the substrate (p < 0.001), while acetate (p = 0.673) and propionate (p = 0.817) proportions, A:P (p = 0.744), and A+Bu: P (p = 0.863) ratios were not affected by feed substrate type.

4. Discussion

4.1. Ruminal Fluid Source

In our study, incubations using ruminal fluid sourced from cows previously adapted to Bacillus spp. did not result in a lower in vitro methane production and acetate to propionate ratio than those using ruminal fluid from naïve cows, irrespective of substrate used. These findings do not support our first hypothesis. A greater methane production when adapted ruminal fluid was used did not correspond to a difference in TGP but did result in an expected alteration in the methane to total gas ratio. To our knowledge, this is the first report in which the ruminal fluid from cows adapted to DFM has been used for evaluating methane mitigation potential, rather than directly adding the DFM to a batch fermentation culture. Our in vitro findings after DFM supplementation and adaptation align with the in vivo results of Cappellozza et al. [36], where dairy cows that were supplemented with 3 g/day of the same Bacillus spp. based DFM product used in our study had greater acetate and lower propionate concentrations in ruminal fluid than the cows that were not supplemented. This difference in VFA could explain the greater methane production seen in Bacillus spp. fed cows, as methanogenesis directly competes with propionate formation for available hydrogen [33]. However, other in vivo studies incorporating Bacillus spp. demonstrate contrasting results. For example, methane yield was 10% lower in dairy cows supplemented with 50 g of B. subtilis/day for 15 days, containing 2 × 1010 CFU/g [30], compared to the unsupplemented group. This dosage was over 78 times higher than the total CFU/day used in our study. When B. licheniformis was supplemented to sheep at a similar dose rate as our study, 2.5 × 109 CFU/day, methane production was 6% lower compared to the unsupplemented groups [29]. If this dose rate showed efficacy in smaller ruminants, it suggests that dairy cows with a larger rumen volume may require a greater dose of Bacillus spp. to achieve a similar reduction in methane production. This could explain why we did not observe a reduction in methane in our study. Another interesting finding from our study was the significant effect of the ruminal fluid source on methane production. The donor cows’ diet, specifically the Bacillus spp. fed, played a crucial role in influencing methane production. Our findings do align with the broader literature on the influence of donor cows’ diets on in vitro methane production, as demonstrated by Alvarez-Hess et al. [48], who suggested that ruminal fluid from cows adapted to a treatment could have a different response in vitro compared to when ruminal fluid from naive cows is used. If we had used only one source of ruminal fluid, our results measuring in vitro fermentation would likely have drawn different conclusions from what we observed using two different sources. The microbial population in the rumen has been reported to adapt to the diet fed, thereby affecting subsequent in vitro degradation of substrate and influencing fermentation parameters [49]. Moreover, any changes resulting from this adaptation may not be of equivalent magnitude to those occurring in vivo when considering factors such as stage of lactation, basal diet, and passage rate [50]. However, a limitation of this study that should be acknowledged is that the delivery of the DFM via rumen cannula does not represent typical farm conditions, where the bacterial blend would be incorporated into the daily feed, and individual variations in dry matter intake could also play a significant role. Thus, we suggest that further research is required to validate these results under more typical feeding conditions.
Fermentation parameters from in vivo experiments are more analogous to our results using adapted ruminal fluid. Kawauchi et al. [51] investigated the impact of feeding Bacillus subtilis diets at a rate of 1.0 × 1010 CFU/day on rumen fermentation in non-lactating crossbred cows, revealing no difference in the A:P ratio between the control and Bacillus spp.-fed groups. Similar patterns were observed for different Bacillus spp. strains. For example, when concentrate mixed with Bacillus toyonensis (2.0 × 1011 CFU/ton of concentrate) was fed at the rate of 2% body weight to Nellore bulls, the A:P ratio remained unchanged after 133 days of supplementation [52]. In contrast, Dias et al. [53] reported that cattle supplemented with 2 g/day of the same blend of Bacillus spp. used in our study (6.4 × 109 CFU/day) had a lower A:P ratio after 28 days of adaptation than the control animals, indicating a potential beneficial effect of these strains on rumen fermentation traits. Although the source of ruminal fluid did not affect the A:P ratio in our study, the acetate proportions were lower when adapted ruminal fluid was used, while propionate concentrations remained similar between adapted and naive groups. We also observed a lower final in vitro pH when using ruminal fluid from adapted animals compared to naive cows, which is consistent with an increase in total VFA [54]. This finding aligns with the in vivo results of Qiao et al. [25], who supplemented Chinese Holstein cows with the same Bacillus spp. blend used in our study, at the rate of 2 × 1011 CFU/day—over 15 times higher than the inclusion rate in our trial. Consistent with our results, Miguel et al. [55] reported that total VFA concentration was 9 to 19% greater when different roughages in a total mixed ration (TMR) were inoculated with a coculture of Bacillus subtilis and L. acidophilus in vitro, compared to TMR without the coculture inoculation. An adaptation period of 14 days has been shown to be sufficient for adapting cattle to forage-based diets [56], and an adaptation period of 15 days has been shown to be sufficient when adapting cows to Bacillus spp. [30,57]. We collected ruminal fluid on days 14, 16, and 21, so based on these references [30,56,57], our cows should have been adapted to the introduced Bacillus spp., which contributed to the observed results.
Incubations that used ruminal fluid from adapted animals showed a 20% greater NH3 -N concentration compared to those that used ruminal fluid from naive animals. In line with these results, Sun et al. (2013) reported that ruminal NH3-N concentration increased by 38% with supplementation of a B. subtilis DFM to dairy cows for 70 days, with the levels remaining elevated by 11% until 1 week after the cessation of feeding. Bacillus spp. have been shown to secrete a variety of enzymes, including proteases and amylases [26]. For example, B. subtilis secretes subtilisin, a proteolytic enzyme [58], that facilitates the breakdown of dietary protein within the rumen, thus providing peptides and amino acids for microbial protein synthesis. The elevated NH3-N concentration in our experiment could be due to enhanced dietary protein degradation by the increased proteolytic Bacillus spp. populations in the ruminal fluid [25,26]. Ghorbani et al. [59] observed an increase in amylolytic and proteolytic bacteria, along with a reduction in protozoal counts, following adaptation to Bacillus spp. This shift likely enhanced the ruminal recycling of bacterial nitrogen, resulting in elevated levels of ruminal NH3-N. A similar mechanism may have affected the outcomes observed in our study. Under in vivo conditions, the elevated NH3-N concentrations observed are advantageous for the cows, as the synthesis of microbial protein, which relies on NH3-N, peptides, and amino acids, results in the production of rumen bypass protein, thereby enhancing protein availability for dairy cows [60]. The findings reported in this research provide further evidence of the potential productive benefits of adding these DFM to the diet of dairy cows to enhance performance parameters of the animal.

4.2. In Vitro Addition of Bacillus spp.

Adding Bacillus spp. to a substrate incubated in vitro did not result in lower methane production than incubations without added Bacillus spp. However, there was a lower relative proportion of acetate, accompanied by a greater propionate proportion and a subsequent 7% lower A:P ratio when Bacillus spp. was added to the 24-h batch culture, thus we partially accept our second hypothesis.
In agreement with our results, the incorporation of a blend of B. subtilis and B. licheniformis with grass silage and corn silage showed no significant differences in methane yield (mL/g organic matter) for both silages compared to the control silages after 48 h of in vitro fermentation [61]. Similarly, the addition of Bacillus coagulans in vitro did not impact methane production when different fibrous agricultural by-products were used as substrates [62]. In contrast, in vitro methane production following the direct addition of B. subtilis was 12.5% greater, while B. licheniformis addition reduced methane production regardless of the different dose rates, ranging from 2.5 to 7.5 × 106 CFU [27]. In a separate experiment, in vitro gas production was 14% greater than the control group when 1 × 105 CFU/g B. licheniformis was incubated with hybrid Pennisetum silage in the first 24 h, with no significant difference in gas production when the time was extended to 48 h [63]. The lack of in vitro response to the addition of Bacillus spp. in our study may be linked to the short duration of the incubation period, suggesting the possibility of treatment differences with longer incubation periods. A 24-h batch system is likely insufficient for the DFM to colonize the medium and produce an effect, whereas feeding the DFM to cows over an extended period and then collecting the ruminal fluid did show a response. The TGP/IVDDM was not different when Bacillus spp. was incorporated in vitro. Cappellozza et al. [64] used the same blend of Bacillus spp. as in our study and found that the mean TGP/IVDDM ranged from 76.5 to 79.7 mL/g after 24 h when different fiber-based feedstuffs and commercial dairy TMR were used. The TGP/IVDDM in our study is double the amount reported by Cappellozza et al. [64]. One possible explanation is that the microbial biomass was greater for the substrate incubated in those treatments that used adapted rumen fluid along with an equivalent of 4 g of Bacillus spp. This increased microbial activity may have led to greater competition for nutrients [65], resulting in the degradation of some bacteria and consequently greater gas production. Furthermore, the increased microbial activity and limited amount of substrate (1 g of substrate per bottle) limited our ability to measure fiber digestibility parameters, as limited residual material precluded further chemical analysis.
The direct addition of Bacillus spp. to in vitro batch cultures yielded promising outcomes in terms of the A:P ratio. Consistent with our findings, prior research with in vitro supplementation of the same combination of Bacillus spp. has demonstrated a similar shift in VFA proportions, favoring propionate while decreasing acetate proportions [66]. Furthermore, an in vitro study conducted by Sun et al. [67] demonstrated that the addition of Bacillus subtilis natto, either live or autoclaved at a dosage of 1 × 1011 CFU, resulted in an increase in propionate production. A change in the ratio of acetate to propionate has previously been associated with a change in methane yield (g CH4/kg DMI) in dairy cows [68,69]. Our finding that the direct in vitro addition of Bacillus spp. increased propionate production without affecting methane production is inconsistent with these viewpoints. It is possible that the stress response of resident bacteria in the ruminal fluid, from the sudden introduction of Bacillus spp. to in vitro batch cultures, could have caused this discrepancy [29,70]. Other underlying mechanisms may also be involved, given ruminal methanogenesis is a complex biochemical process influenced by numerous factors, such as ruminal formate production as a substrate for methanogenesis and ruminal abundance of H2-producing and H2-consuming bacteria, proteobacteria, fungi, and ciliate protozoa [71]. Although notable differences in fermentation parameters were observed, the underlying microbial mechanisms remain unknown. Future studies incorporating microbiome profiling would provide valuable insight into how DFM supplementation influences rumen microbial structure and function.

4.3. Interaction Between Bacillus spp. and Substrates

Each of the three incubated substrates displayed distinct methane production levels, with grain yielding the greatest, but there were no interactive effects observed between either the ruminal fluid source or the in vitro Bacillus spp. Addition, across all three substrates, thus, we accept our third hypothesis.
The TGP/IVDDM and MP/IVDDM were notably greater for the grain substrate, regardless of the ruminal fluid source and the addition of Bacillus spp. in vitro. Studies have shown that different substrates ferment differently [72,73]. Gas production results from the microbial degradation of feed and the buffering of acids generated during in vitro ruminal fermentation and can serve as an indirect measurement of carbohydrate degradability [74]. In our study, the maximum total TGP/IVDDM was positively related to readily fermentable substrates [75,76] and negatively related to the ADF and NDF concentrations. Consistent with this finding, Cappellozza et al. [64] found that the mean total TGP/IVDDM after 24 and 48 h was greater when the same blend of Bacillus spp. used in our study was incubated with different TMR compared to incubation with various forages. Supporting our methane results, Sarmikasoglou et al. [28] found that B. subtilis supplementation in vitro increased methane production when incubated with a substrate formulated to reflect an early lactation diet (19% CP, 31% starch), but decreased methane production with a substrate representing a mid-lactation diet (15% CP, 25% starch). Similarly, Martínez et al. [77] observed that in vitro methane production per gram of substrate was 17.5% greater with a high concentrate substrate (forage: concentrate (F:C) ratio- 30:70), when compared to a high forage substrate (F:C ratio- 70:30) after 24 h of in vitro incubation. Grains rich in soluble carbohydrates increase the population of ciliate protozoa and stimulate hydrogen transfer to methanogens, resulting in greater methane production [78].
We did not observe any variations in individual VFA concentration when different substrates were used, and the A:P ratios were similar among all three substrates. When grain was used as the substrate, it produced more total VFA. Similarly, Zicarelli et al. [79] observed a substantial increase in total VFA concentration during in vitro rumen fermentation as concentrate proportions rose from 0 to 50% when combined with forage. The NH3-N concentrations in our study were greater when ryegrass was used as the substrate and lowest for grain. This is likely due to the greater crude protein concentration in the ryegrass and the highly degradable nature of protein, as observed in pastures by Repetto et al. [80].
Despite the variations attributed to the fermentable properties of each feed type, there were no interactive effects on gas production and fermentation parameters between the rumen fluid source, direct in vitro addition of Bacillus spp., and the substrate incubated. This consistency indicates that the effect of Bacillus spp. was uniform across all three substrates. However, there was an interaction between the substrate and ruminal fluid source. For instance, no difference was observed in A:P between the substrates, which is contrary to previous reports [81]. However, the interaction between substrate and ruminal fluid appears to have muddied the effect of substrate, leading to our experiment not detecting any main effect of substrate on A:P. The interaction between substrate and ruminal fluid appeared to be driven by the response of hay to the ruminal fluid source. Hay produced the lowest amount of acetate and the greatest amount of propionate in adapted ruminal fluid, but the greatest acetate and lowest propionate in naïve ruminal fluid. It is not clear why hay behaved differently in different types of ruminal fluid.
Similar to our findings, previous in vitro studies showed no interactive effects on gas production when different forage compositions in a TMR were combined with the presence of Bacillus spp. based DFM [64]. Our results suggest that Bacillus spp. based DFM can potentially be incorporated into various production systems with similar effects. However, an in vivo validation is recommended for further confirmation. It is important to note that individual animal variation could influence the observed effects, particularly if different animals were used for each treatment. To minimize this, we selected animals from the same herd, breed, lactation stage, and basal diet to ensure similar rumen function. The same six animals were used across all three runs for consistency. Additionally, multiple animals were included in rumen sampling, and samples were pooled to reduce individual variation, strengthening the reliability of our results.

5. Conclusions

Incubations using ruminal fluid collected from cows adapted to Bacillus spp. had greater methane production in vitro than incubations using ruminal fluid from naive cows, whereas adding Bacillus spp. to in vitro batch cultures had no effect on methane production. This discrepancy indicates that a 24-h in vitro fermentation period is not adequate for the DFM to sufficiently colonize the media. Both the supplementation of Bacillus spp. to cows and the in vitro addition of Bacillus spp. did change the VFA concentration but did not mitigate methane production. The consistent effect of Bacillus spp. across all substrates suggests its potential efficacy across diverse production systems. Future in vitro studies should consider incorporating ruminal fluid from animals previously adapted to DFM to better replicate the microbial environment and improve the predictive value of in vivo methane mitigation assessments.
However, we note that there were some limitations to this study. For example, the use of a small amount of feed in the incubation bottles limited our ability to undertake detailed assessments on fiber digestibility, which is known to be closely related to methane production. Future studies should look to undertake in sacco measurements on parameters including organic matter, ADF, and NDF digestibility of feeds when DFM are offered. Further studies may also consider dose responses to optimize rates of DFM feeding for different classes of livestock.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11080473/s1, Table S1: Interactive effect of ruminal fluid, Bacillus rate, and substrate on dry matter disappearance and gas production at the end of the fermentation period.

Author Contributions

Conceptualization, S.S.N., S.R.O.W., P.S.A.-H. and J.L.J.; methodology, S.S.N., S.R.O.W., S.C., P.S.A.-H. and J.L.J.; formal analysis, S.C. and K.G.; investigation, S.S.N., A.S.ó.N., R.T. and P.S.A.-H.; resources, P.S.A.-H. and J.L.J.; data curation, S.S.N., S.R.O.W. and A.S.ó.N.; writing—original draft preparation, S.S.N.; writing—review and editing, S.S.N., S.R.O.W., A.S.ó.N., R.T., P.S.A.-H., L.C., K.G. and J.L.J.; visualization, S.R.O.W. and S.C.; supervision, S.R.O.W., P.S.A.-H., L.C. and J.L.J.; project administration, P.S.A.-H. and J.L.J.; funding acquisition, J.L.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Neoculi Pty Ltd. (Unit 4, 25-37 Huntingdale Rd, Burwood VIC 3125), grant number AGVR221. The funding organization had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Institutional Review Board Statement

The study was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Animal use was approved by the DEECA Agricultural Research and Extension Animal Ethics Committee (Approval 2022-13, approved 13 December 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from the corresponding author upon reasonable request.

Acknowledgments

This work would not have been possible without the contributions of the science and technical staff at Agriculture Victoria Research, Ellinbank SmartFarm, Victoria, Australia.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
A:PRatio of acetate to propionate
A+Bu:PRatio of acetate plus butyrate to propionate
ADFAcid detergent fiber
CFUColony Forming Units
DFMDirect-fed microbials
DMDry matter
DMIDry matter intake
IVDDMIn vitro dry matter disappearance
NDFNeutral detergent fiber
TGPTotal gas production
TMRTotal mixed ration
VFAVolatile fatty acids

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Table 1. In vitro experiment design of each run.
Table 1. In vitro experiment design of each run.
Ruminal Fluid SourceBacillus Rate (Equivalence)Substrate (1 g)Treatment
Naive0 g/head/dayHayNH0
PastureNP0
GrainNG0
4 g/head/dayHayNH4
PastureNP4
GrainNG4
Adapted0 g/head/dayHayAH0
PastureAP0
GrainAG0
4 g/head/dayHayAH4
PastureAP4
GrainAG4
Table 2. Nutrient composition of substrates incubated (g/kg DM unless otherwise stated).
Table 2. Nutrient composition of substrates incubated (g/kg DM unless otherwise stated).
ItemWheat-Barley Grain MixVetch Hay Perennial Ryegrass
Crude protein175150253
Acid detergent fiber75448236
Neutral detergent fiber135539401
Lignin339022
Non-fiber carbohydrates592182156
Starch4281211
Ash71110134
Ether extract26.218.757.2
Metabolizable energy (MJ/kg DM)12.48.0211.5
Table 3. Effect of ruminal fluid, Bacillus rate, and substrate on dry matter disappearance and gas production at the end of the fermentation period.
Table 3. Effect of ruminal fluid, Bacillus rate, and substrate on dry matter disappearance and gas production at the end of the fermentation period.
IVDDM 1 (g/g DM Substrate)TGP
(mL)
TGP/IVDDM (mL/g IVDDM)MP
(mL)
MP/TGP (mL/mL of TGP)MP/IVDDM (mL/g IVDDM)
Ruminal fluid (R)Adapted0.5797.717313.70.1223.6
Naive0.5996.016411.80.1119.7
SED0.0054.507.090.4790.3050.933
Bacillus probiotic (B)00.5997.616612.50.1221.0
40.5896.217013.00.1222.3
SED0.0084.507.090.4790.3050.933
Substrate
(S)
Grain0.67 c135 c206 b20.2 c0.14 b30.4 b
Hay0.46 a71.9 a158 a8.0 a0.10 a17.8 a
Pasture0.61 b83.0 b141 a10.0 b0.11 a16.7 a
SED0.01045.518.680.5870.3731.143
p-valueR0.0050.7030.206<0.001<0.001<0.001
B0.3020.7650.6140.2440.2780.179
S<0.001<0.001<0.001<0.001<0.001<0.001
B × S0.6010.6940.4810.7810.9830.592
R × B0.5180.1030.2950.7360.4480.563
R × S0.1240.8210.9360.4530.4120.330
R × B × S0.8730.1930.4130.7670.7050.970
1 IVDDM = In vitro dry matter disappearance, TGP = total gas production, TGP/IVDDM = total gas production per dry matter disappearance, MP = methane production, MP/TGP = methane production as a proportion of total gas production, MP/IVDDM = methane production per dry matter disappearance. a, b, c Within column within main effect, superscripts indicate difference at p < 0.05.
Table 4. Effect of ruminal fluid, Bacillus rate, and substrate on pH, ammonia (mg/L), total volatile fatty acids (mM), individual volatile fatty acids (mM/100 mM total fatty acids), and selected volatile fatty acid ratios at the end of the fermentation period.
Table 4. Effect of ruminal fluid, Bacillus rate, and substrate on pH, ammonia (mg/L), total volatile fatty acids (mM), individual volatile fatty acids (mM/100 mM total fatty acids), and selected volatile fatty acid ratios at the end of the fermentation period.
pHNH3-N (mg/L)Total VFA (mM)A 1
(mM/100 mM)
P (mM/100 mM)iBu
(mM/100 mM)
Bu
(mM/100 mM)
iV
(mM/100 mM)
V
(mM/100 mM)
A:PA+Bu:P
Ruminal fluid (R)Adapted6.0910668.961.422.20.7013.31.131.052.833.45
Naive6.1486.763.962.622.10.6412.50.971.012.933.51
SED0.0154.891.090.5860.4510.0120.4380.0410.0650.0720.081
Bacillus probiotic (B)06.1095.866.562.621.50.6712.91.071.062.983.59
46.1397.066.261.422.80.6712.91.031.012.783.37
SED0.0154.891.090.5860.4510.0120.4380.0410.0650.0720.081
Substrate
(S)
Grain5.87 a70.5 a75.7 c61.722.20.70 b13.11.101.042.843.45
Hay6.30 c97.9 b58.4 a62.122.30.65 a12.81.011.012.893.49
Pasture6.19 b121 c65.0 b62.322.00.67 ab12.81.051.062.913.51
SED0.0195.991.340.7180.5530.0150.5370.0510.0800.0880.099
p-valueR<0.001<0.001<0.0010.0420.760<0.0010.068<0.0010.5550.2050.483
B0.0730.8000.8020.0470.0050.6430.9960.3360.4790.0090.007
S<0.001<0.001<0.0010.6730.8170.0100.7890.1720.7870.7440.863
B × S0.7250.9640.7770.0130.1180.2940.0800.3820.1730.0500.118
R × B0.9370.8050.6630.0080.0130.5610.1770.4700.0880.0150.037
R × S0.0030.4610.5330.308<0.001<0.001<0.001<0.0010.027<0.001<0.001
R × B × S0.4290.3540.2750.4240.2570.1780.8310.1910.3310.3160.363
1 A = acetate, P = propionate, iBu = iso butyrate, Bu = butyrate, iV = iso valerate, V = valerate A:P = ratio of acetate to propionate, A+Bu:P = ratio of (acetate + butyrate) to propionate. a, b, c Within column within main effect, superscripts indicate difference at p < 0.05.
Table 5. Interactive effect of ruminal fluid, Bacillus rate, and substrate on pH, ammonia (mg/L), total volatile fatty acids (mM), individual volatile fatty acids (mM/100 mM total fatty acids), and selected volatile fatty acid ratios at the end of the fermentation period.
Table 5. Interactive effect of ruminal fluid, Bacillus rate, and substrate on pH, ammonia (mg/L), total volatile fatty acids (mM), individual volatile fatty acids (mM/100 mM total fatty acids), and selected volatile fatty acid ratios at the end of the fermentation period.
Interactions 1,2pHNH3-N (mg/L)Total VFA (mM)A 3
(mM/100 mM)
P (mM/100 mM)iBu
(mM/100 mM)
Bu
(mM/100 mM)
iV
(mM/100 mM)
V
(mM/100 mM)
A:PA+Bu:P
B × S0G5.86 a69.3 a76.2 c62.8 bc21.2 a0.68 bc13.1 a1.08 ab1.06 a3.01 b3.65 b
3G5.87 a71.6 a75.2 c60.6 a23.3 b0.71 c13.2 a1.12 b1.02 a2.68 a3.26 a
0H6.28 c98.2 b58.8 c61.4 ab22.3 ab0.66 ab13.5 a1.05 ab0.96 a2.86 ab3.48 ab
3H6.32 c97.6 b58.0 c62.7 bc22.3 ab0.63 a12.2 a0.96 a1.06 a2.92 ab3.49 ab
0P6.18 b119.7 c64.6 b63.7 c21.1 a0.67 abc12.2 a1.08 ab1.16 a3.06 b3.65 b
3P6.20 b121.8 c65.5 b61.0 ab22.8 b0.66 ab13.4 a1.01 ab0.96 a2.76 a3.36 a
p Value0.7250.9640.7770.0130.1180.2940.0800.3820.1730.0500.118
SED0.0278.481.901.010.780.0220.760.0720.1130.1250.141
R × BA06.08 a106.1 b68.7 b61.2 a22.1 b0.70 b13.6 b1.14 b1.02 a2.84 a3.48 a
A36.11 ab106.1 b69.0 b61.6 a22.3 b0.70 b13.0 ab1.13 b1.09 a2.83 a3.43 a
N06.13 bc85.4 a64.3 a64.3 b20.9 a0.65 a12.2 b1.00 a1.09 a3.11 b3.71 b
N36.16 c87.9 a63.5 a61.3 a23.3 b0.63 a12.8 ab0.94 a0.94 a2.74 a3.31 a
p Value0.9370.8050.6630.0080.0130.5610.1770.4700.0880.0150.037
SED0.0226.921.550.820.630.0180.620.0600.0930.1020.115
R × SAG5.81 a77.1 ab78.0 e61.5 ab21.0 b0.71 c14.43 b1.22 cd1.01 ab2.96 bc3.66 c
AH6.31 e106.6 c60.2 ab60.8 a25.2 d0.61 b11.43 a0.87 ab0.94 a2.47 a2.93 a
AP6.15 c134.6 d68.3 c62.0 ab20.4 ab0.78 d14.13 b1.31 d1.20 b3.07 cd3.76 c
NG5.92 b63.9 a73.5 d61.9 ab23.4 c0.68 c11.85 a0.99 b1.06 ab2.72 b3.24 b
NH6.29 e89.2 b56.6 a63.3 b19.4 a0.69 c14.24 b1.15 c1.07 ab3.30 d4.05 d
NP6.22 d106.8 c61.7 b62.7 ab23.5 c0.55 a11.48 a0.78 a0.92 a2.75 b3.24 b
p Value0.0030.4610.5330.308<0.001<0.001<0.001<0.0010.027<0.001<0.001
SED0.0278.481.901.020.780.0220.7590.0720.1130.1250.141
R × B × SA0G5.81 a76.8 ab77.2 e61.4 abc20.8 ab0.68 cde14.90 c1.13 bcd0.92 ab3.01 bc3.75 bc
A3G5.81 a77.3 ab78.7 e61.5 abc21.3 ab0.74 ef13.95 bc1.31 de1.11 bc2.92 b3.58 b
A0H6.29 ef111.7 de61.5 b60.0 a25.3 c0.63 bc12.22 ab0.93 ab0.90 ab2.43 a2.92 a
A3H6.33 f101.5 cd59.0 ab61.7 abc25.2 c0.59 ab10.64 a0.80 a0.99 abc2.52 a2.94 a
A0P6.13 c129.7 ef67.6 c62.3 abcd20.4 ab0.79 f13.76 bc1.35 e1.25 c3.09 bc3.77 bc
A3P6.18 cd139.5 f69.1 c61.1 abc20.4 ab0.78 f14.50 c1.27 de1.16 bc3.05 bc3.76 bc
N0G5.90 b61.8 a75.2 de64.1 cd21.5 ab0.69 cde11.36 a1.03 bc1.20 bc3.01 bc3.54 b
N3G5.94 b65.9 a71.7 cd59.8 a25.2 c0.67 cd12.34 ab0.94 ab0.92 ab2.44 a2.94 a
N0H6.26 ef84.8 abc56.0 a62.9 bcd19.3 a0.69 de14.68 c1.17 cde1.01 abc3.28 c4.05 c
N3H6.32 f93.7 bcd57.1 ab63.7 cd19.4 a0.68 cde13.80 bc1.12 bcd1.12 bc3.32 c4.04 c
N0P6.22 de109.8 de61.7 b65.0 d21.8 b0.56 a10.64 a0.81 a1.07 abc3.04 bc3.54 b
N3P6.22 de104.0 cd61.8 b60.3 ab25.2 c0.55 a12.32 ab0.745 a0.76 a2.46 a2.95 a
p Value0.4290.3540.2750.4240.2570.1780.8310.1910.3310.3160.363
SED0.03911.992.691.431.110.0311.0740.1020.1600.1770.200
1 B = Bacillus spp. addition, S = substrate type and R = ruminal fluid type. 2 Treatment combinations of Bacillus spp. (0 = without, 4 = with), substrate (G = grain, H = hay, P = pasture), and ruminal fluid type (A = adapted, N = naive). 3 A = acetate, P = propionate, iBu = iso butyrate, Bu = butyrate, iV = iso valerate, V = valerate A:P = ratio of acetate to propionate, A+Bu:P = ratio of (acetate + butyrate) to propionate, a, b, c, d, e, f Within column within main effect, superscripts indicate difference at p < 0.05.
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Nair, S.S.; Williams, S.R.O.; ó Neachtain, A.S.; Tognelli, R.; Chandra, S.; Alvarez-Hess, P.S.; Cheng, L.; Giri, K.; Jacobs, J.L. The Effect of Ruminal Fluid Adaptation to a Direct Fed Microbial: In Vitro Methane Production and Fermentation Characteristics. Fermentation 2025, 11, 473. https://doi.org/10.3390/fermentation11080473

AMA Style

Nair SS, Williams SRO, ó Neachtain AS, Tognelli R, Chandra S, Alvarez-Hess PS, Cheng L, Giri K, Jacobs JL. The Effect of Ruminal Fluid Adaptation to a Direct Fed Microbial: In Vitro Methane Production and Fermentation Characteristics. Fermentation. 2025; 11(8):473. https://doi.org/10.3390/fermentation11080473

Chicago/Turabian Style

Nair, Sreemol Suthan, S. Richard O. Williams, Aodán S. ó Neachtain, Renata Tognelli, Subhash Chandra, Pablo S. Alvarez-Hess, Long Cheng, Khageswor Giri, and Joe L. Jacobs. 2025. "The Effect of Ruminal Fluid Adaptation to a Direct Fed Microbial: In Vitro Methane Production and Fermentation Characteristics" Fermentation 11, no. 8: 473. https://doi.org/10.3390/fermentation11080473

APA Style

Nair, S. S., Williams, S. R. O., ó Neachtain, A. S., Tognelli, R., Chandra, S., Alvarez-Hess, P. S., Cheng, L., Giri, K., & Jacobs, J. L. (2025). The Effect of Ruminal Fluid Adaptation to a Direct Fed Microbial: In Vitro Methane Production and Fermentation Characteristics. Fermentation, 11(8), 473. https://doi.org/10.3390/fermentation11080473

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