The Effect of Ruminal Fluid Adaptation to a Direct Fed Microbial: In Vitro Methane Production and Fermentation Characteristics
Abstract
1. Introduction
2. Materials and Methods
2.1. Animals
2.2. In Vitro Treatments
2.3. Ruminal Fluid Collection and Preparation
2.4. In Vitro Incubation
2.5. Sample Collection and Analysis
2.5.1. Gas Sampling and Analysis
2.5.2. Gas Production Calculation
2.5.3. In Vitro Fermentation Parameters
2.5.4. In Vitro Dry Matter Disappearance
2.6. Statistical Analyses
3. Results
3.1. Interactions
3.2. Ruminal Fluid Source Main Effect
3.3. In Vitro Addition of Bacillus spp. Main Effect
3.4. Substrate Type Main Effect
4. Discussion
4.1. Ruminal Fluid Source
4.2. In Vitro Addition of Bacillus spp.
4.3. Interaction Between Bacillus spp. and Substrates
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
A:P | Ratio of acetate to propionate |
A+Bu:P | Ratio of acetate plus butyrate to propionate |
ADF | Acid detergent fiber |
CFU | Colony Forming Units |
DFM | Direct-fed microbials |
DM | Dry matter |
DMI | Dry matter intake |
IVDDM | In vitro dry matter disappearance |
NDF | Neutral detergent fiber |
TGP | Total gas production |
TMR | Total mixed ration |
VFA | Volatile fatty acids |
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Ruminal Fluid Source | Bacillus Rate (Equivalence) | Substrate (1 g) | Treatment |
---|---|---|---|
Naive | 0 g/head/day | Hay | NH0 |
Pasture | NP0 | ||
Grain | NG0 | ||
4 g/head/day | Hay | NH4 | |
Pasture | NP4 | ||
Grain | NG4 | ||
Adapted | 0 g/head/day | Hay | AH0 |
Pasture | AP0 | ||
Grain | AG0 | ||
4 g/head/day | Hay | AH4 | |
Pasture | AP4 | ||
Grain | AG4 |
Item | Wheat-Barley Grain Mix | Vetch Hay | Perennial Ryegrass |
---|---|---|---|
Crude protein | 175 | 150 | 253 |
Acid detergent fiber | 75 | 448 | 236 |
Neutral detergent fiber | 135 | 539 | 401 |
Lignin | 33 | 90 | 22 |
Non-fiber carbohydrates | 592 | 182 | 156 |
Starch | 428 | 12 | 11 |
Ash | 71 | 110 | 134 |
Ether extract | 26.2 | 18.7 | 57.2 |
Metabolizable energy (MJ/kg DM) | 12.4 | 8.02 | 11.5 |
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) | Adapted | 0.57 | 97.7 | 173 | 13.7 | 0.12 | 23.6 |
Naive | 0.59 | 96.0 | 164 | 11.8 | 0.11 | 19.7 | |
SED | 0.005 | 4.50 | 7.09 | 0.479 | 0.305 | 0.933 | |
Bacillus probiotic (B) | 0 | 0.59 | 97.6 | 166 | 12.5 | 0.12 | 21.0 |
4 | 0.58 | 96.2 | 170 | 13.0 | 0.12 | 22.3 | |
SED | 0.008 | 4.50 | 7.09 | 0.479 | 0.305 | 0.933 | |
Substrate (S) | Grain | 0.67 c | 135 c | 206 b | 20.2 c | 0.14 b | 30.4 b |
Hay | 0.46 a | 71.9 a | 158 a | 8.0 a | 0.10 a | 17.8 a | |
Pasture | 0.61 b | 83.0 b | 141 a | 10.0 b | 0.11 a | 16.7 a | |
SED | 0.0104 | 5.51 | 8.68 | 0.587 | 0.373 | 1.143 | |
p-value | R | 0.005 | 0.703 | 0.206 | <0.001 | <0.001 | <0.001 |
B | 0.302 | 0.765 | 0.614 | 0.244 | 0.278 | 0.179 | |
S | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 | |
B × S | 0.601 | 0.694 | 0.481 | 0.781 | 0.983 | 0.592 | |
R × B | 0.518 | 0.103 | 0.295 | 0.736 | 0.448 | 0.563 | |
R × S | 0.124 | 0.821 | 0.936 | 0.453 | 0.412 | 0.330 | |
R × B × S | 0.873 | 0.193 | 0.413 | 0.767 | 0.705 | 0.970 |
pH | NH3-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:P | A+Bu:P | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Ruminal fluid (R) | Adapted | 6.09 | 106 | 68.9 | 61.4 | 22.2 | 0.70 | 13.3 | 1.13 | 1.05 | 2.83 | 3.45 |
Naive | 6.14 | 86.7 | 63.9 | 62.6 | 22.1 | 0.64 | 12.5 | 0.97 | 1.01 | 2.93 | 3.51 | |
SED | 0.015 | 4.89 | 1.09 | 0.586 | 0.451 | 0.012 | 0.438 | 0.041 | 0.065 | 0.072 | 0.081 | |
Bacillus probiotic (B) | 0 | 6.10 | 95.8 | 66.5 | 62.6 | 21.5 | 0.67 | 12.9 | 1.07 | 1.06 | 2.98 | 3.59 |
4 | 6.13 | 97.0 | 66.2 | 61.4 | 22.8 | 0.67 | 12.9 | 1.03 | 1.01 | 2.78 | 3.37 | |
SED | 0.015 | 4.89 | 1.09 | 0.586 | 0.451 | 0.012 | 0.438 | 0.041 | 0.065 | 0.072 | 0.081 | |
Substrate (S) | Grain | 5.87 a | 70.5 a | 75.7 c | 61.7 | 22.2 | 0.70 b | 13.1 | 1.10 | 1.04 | 2.84 | 3.45 |
Hay | 6.30 c | 97.9 b | 58.4 a | 62.1 | 22.3 | 0.65 a | 12.8 | 1.01 | 1.01 | 2.89 | 3.49 | |
Pasture | 6.19 b | 121 c | 65.0 b | 62.3 | 22.0 | 0.67 ab | 12.8 | 1.05 | 1.06 | 2.91 | 3.51 | |
SED | 0.019 | 5.99 | 1.34 | 0.718 | 0.553 | 0.015 | 0.537 | 0.051 | 0.080 | 0.088 | 0.099 | |
p-value | R | <0.001 | <0.001 | <0.001 | 0.042 | 0.760 | <0.001 | 0.068 | <0.001 | 0.555 | 0.205 | 0.483 |
B | 0.073 | 0.800 | 0.802 | 0.047 | 0.005 | 0.643 | 0.996 | 0.336 | 0.479 | 0.009 | 0.007 | |
S | <0.001 | <0.001 | <0.001 | 0.673 | 0.817 | 0.010 | 0.789 | 0.172 | 0.787 | 0.744 | 0.863 | |
B × S | 0.725 | 0.964 | 0.777 | 0.013 | 0.118 | 0.294 | 0.080 | 0.382 | 0.173 | 0.050 | 0.118 | |
R × B | 0.937 | 0.805 | 0.663 | 0.008 | 0.013 | 0.561 | 0.177 | 0.470 | 0.088 | 0.015 | 0.037 | |
R × S | 0.003 | 0.461 | 0.533 | 0.308 | <0.001 | <0.001 | <0.001 | <0.001 | 0.027 | <0.001 | <0.001 | |
R × B × S | 0.429 | 0.354 | 0.275 | 0.424 | 0.257 | 0.178 | 0.831 | 0.191 | 0.331 | 0.316 | 0.363 |
Interactions 1,2 | pH | NH3-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:P | A+Bu:P | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
B × S | 0G | 5.86 a | 69.3 a | 76.2 c | 62.8 bc | 21.2 a | 0.68 bc | 13.1 a | 1.08 ab | 1.06 a | 3.01 b | 3.65 b |
3G | 5.87 a | 71.6 a | 75.2 c | 60.6 a | 23.3 b | 0.71 c | 13.2 a | 1.12 b | 1.02 a | 2.68 a | 3.26 a | |
0H | 6.28 c | 98.2 b | 58.8 c | 61.4 ab | 22.3 ab | 0.66 ab | 13.5 a | 1.05 ab | 0.96 a | 2.86 ab | 3.48 ab | |
3H | 6.32 c | 97.6 b | 58.0 c | 62.7 bc | 22.3 ab | 0.63 a | 12.2 a | 0.96 a | 1.06 a | 2.92 ab | 3.49 ab | |
0P | 6.18 b | 119.7 c | 64.6 b | 63.7 c | 21.1 a | 0.67 abc | 12.2 a | 1.08 ab | 1.16 a | 3.06 b | 3.65 b | |
3P | 6.20 b | 121.8 c | 65.5 b | 61.0 ab | 22.8 b | 0.66 ab | 13.4 a | 1.01 ab | 0.96 a | 2.76 a | 3.36 a | |
p Value | 0.725 | 0.964 | 0.777 | 0.013 | 0.118 | 0.294 | 0.080 | 0.382 | 0.173 | 0.050 | 0.118 | |
SED | 0.027 | 8.48 | 1.90 | 1.01 | 0.78 | 0.022 | 0.76 | 0.072 | 0.113 | 0.125 | 0.141 | |
R × B | A0 | 6.08 a | 106.1 b | 68.7 b | 61.2 a | 22.1 b | 0.70 b | 13.6 b | 1.14 b | 1.02 a | 2.84 a | 3.48 a |
A3 | 6.11 ab | 106.1 b | 69.0 b | 61.6 a | 22.3 b | 0.70 b | 13.0 ab | 1.13 b | 1.09 a | 2.83 a | 3.43 a | |
N0 | 6.13 bc | 85.4 a | 64.3 a | 64.3 b | 20.9 a | 0.65 a | 12.2 b | 1.00 a | 1.09 a | 3.11 b | 3.71 b | |
N3 | 6.16 c | 87.9 a | 63.5 a | 61.3 a | 23.3 b | 0.63 a | 12.8 ab | 0.94 a | 0.94 a | 2.74 a | 3.31 a | |
p Value | 0.937 | 0.805 | 0.663 | 0.008 | 0.013 | 0.561 | 0.177 | 0.470 | 0.088 | 0.015 | 0.037 | |
SED | 0.022 | 6.92 | 1.55 | 0.82 | 0.63 | 0.018 | 0.62 | 0.060 | 0.093 | 0.102 | 0.115 | |
R × S | AG | 5.81 a | 77.1 ab | 78.0 e | 61.5 ab | 21.0 b | 0.71 c | 14.43 b | 1.22 cd | 1.01 ab | 2.96 bc | 3.66 c |
AH | 6.31 e | 106.6 c | 60.2 ab | 60.8 a | 25.2 d | 0.61 b | 11.43 a | 0.87 ab | 0.94 a | 2.47 a | 2.93 a | |
AP | 6.15 c | 134.6 d | 68.3 c | 62.0 ab | 20.4 ab | 0.78 d | 14.13 b | 1.31 d | 1.20 b | 3.07 cd | 3.76 c | |
NG | 5.92 b | 63.9 a | 73.5 d | 61.9 ab | 23.4 c | 0.68 c | 11.85 a | 0.99 b | 1.06 ab | 2.72 b | 3.24 b | |
NH | 6.29 e | 89.2 b | 56.6 a | 63.3 b | 19.4 a | 0.69 c | 14.24 b | 1.15 c | 1.07 ab | 3.30 d | 4.05 d | |
NP | 6.22 d | 106.8 c | 61.7 b | 62.7 ab | 23.5 c | 0.55 a | 11.48 a | 0.78 a | 0.92 a | 2.75 b | 3.24 b | |
p Value | 0.003 | 0.461 | 0.533 | 0.308 | <0.001 | <0.001 | <0.001 | <0.001 | 0.027 | <0.001 | <0.001 | |
SED | 0.027 | 8.48 | 1.90 | 1.02 | 0.78 | 0.022 | 0.759 | 0.072 | 0.113 | 0.125 | 0.141 | |
R × B × S | A0G | 5.81 a | 76.8 ab | 77.2 e | 61.4 abc | 20.8 ab | 0.68 cde | 14.90 c | 1.13 bcd | 0.92 ab | 3.01 bc | 3.75 bc |
A3G | 5.81 a | 77.3 ab | 78.7 e | 61.5 abc | 21.3 ab | 0.74 ef | 13.95 bc | 1.31 de | 1.11 bc | 2.92 b | 3.58 b | |
A0H | 6.29 ef | 111.7 de | 61.5 b | 60.0 a | 25.3 c | 0.63 bc | 12.22 ab | 0.93 ab | 0.90 ab | 2.43 a | 2.92 a | |
A3H | 6.33 f | 101.5 cd | 59.0 ab | 61.7 abc | 25.2 c | 0.59 ab | 10.64 a | 0.80 a | 0.99 abc | 2.52 a | 2.94 a | |
A0P | 6.13 c | 129.7 ef | 67.6 c | 62.3 abcd | 20.4 ab | 0.79 f | 13.76 bc | 1.35 e | 1.25 c | 3.09 bc | 3.77 bc | |
A3P | 6.18 cd | 139.5 f | 69.1 c | 61.1 abc | 20.4 ab | 0.78 f | 14.50 c | 1.27 de | 1.16 bc | 3.05 bc | 3.76 bc | |
N0G | 5.90 b | 61.8 a | 75.2 de | 64.1 cd | 21.5 ab | 0.69 cde | 11.36 a | 1.03 bc | 1.20 bc | 3.01 bc | 3.54 b | |
N3G | 5.94 b | 65.9 a | 71.7 cd | 59.8 a | 25.2 c | 0.67 cd | 12.34 ab | 0.94 ab | 0.92 ab | 2.44 a | 2.94 a | |
N0H | 6.26 ef | 84.8 abc | 56.0 a | 62.9 bcd | 19.3 a | 0.69 de | 14.68 c | 1.17 cde | 1.01 abc | 3.28 c | 4.05 c | |
N3H | 6.32 f | 93.7 bcd | 57.1 ab | 63.7 cd | 19.4 a | 0.68 cde | 13.80 bc | 1.12 bcd | 1.12 bc | 3.32 c | 4.04 c | |
N0P | 6.22 de | 109.8 de | 61.7 b | 65.0 d | 21.8 b | 0.56 a | 10.64 a | 0.81 a | 1.07 abc | 3.04 bc | 3.54 b | |
N3P | 6.22 de | 104.0 cd | 61.8 b | 60.3 ab | 25.2 c | 0.55 a | 12.32 ab | 0.745 a | 0.76 a | 2.46 a | 2.95 a | |
p Value | 0.429 | 0.354 | 0.275 | 0.424 | 0.257 | 0.178 | 0.831 | 0.191 | 0.331 | 0.316 | 0.363 | |
SED | 0.039 | 11.99 | 2.69 | 1.43 | 1.11 | 0.031 | 1.074 | 0.102 | 0.160 | 0.177 | 0.200 |
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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
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 StyleNair, 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 StyleNair, 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