Avian (IgY) anti-methanogen antibodies for reducing ruminal methane production: in vitro assessment of their effects

2008 ◽  
Vol 48 (2) ◽  
pp. 260 ◽  
Author(s):  
S. R. Cook ◽  
P. K. Maiti ◽  
A. V. Chaves ◽  
C. Benchaar ◽  
K. A. Beauchemin ◽  
...  
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Antibody Activity ◽  
Early Lactation ◽  
Ch4 Production ◽  
Transient Nature ◽  
Methanobrevibacter Ruminantium ◽  
Freeze Dried ◽  
Egg Powder

In vitro dry matter disappearance (IVDMD) and production of methane, volatile fatty acids (VFA) and ammonia from an early lactation diet or from freeze-dried alfalfa were assessed in the presence of anti-methanogen antibody treatments in two in vitro ruminal incubations (experiments 1 and 2). In experiment 1, hens were immunised with crude cell preparations of Methanobrevibacter smithii, Methanobrevibacter ruminantium or Methanosphaera stadtmanae and complete Freund’s adjuvant (CFA). Semipurified egg antibodies (IgY) prepared from the hens’ eggs (α-SMICFA, α-RUMCFA, or α-STADCFA, respectively) were dispensed into 24 replicate vials (400 μL per vial) containing 500 mg of an early lactation total mixed ration (18% crude protein; 33% neutral detergent fibre; DM basis). Vials containing an equal volume of semipurified antibodies from eggs of non-immunised hens were included as a control. In experiment 2, hens were immunised with one of the three antigenic preparations combined with Montanide ISA 70 adjuvant. Triplicate vials per time point included 0.6 g of freeze-dried egg powder (α-SMIMon, α-RUMMon, α-STADMon; 19.0 ± 2.6 mg IgY/g) or a mixture of all three (ComboMon) and 500 mg of freeze-dried alfalfa. Total gas, methane production and pH were measured at intervals over 24 h. After 24 h, samples were analysed for VFA, ammonia and IVDMD. In experiment 1, cumulative CH4 production was similar (P > 0.05) among treatments at each sampling time. At 24 h, average CH4 production across treatments was 27.03 ± 0.205 mg/g DM. In experiment 2, α-SMIMon, α-STADMon and ComboMon reduced methane production at 12 h (P ≤ 0.05) compared with the control, but by 24 h, CH4 levels in all treatments were similar (P > 0.05) to the control. At 24 h, total VFA concentrations were lower (P < 0.05) in α-RUMMon and α-SMIMon than in the control. The transient nature of the inhibition of methane production by the antibodies may have arisen from instability of the antibodies in ruminal fluid, or to the presence of non-culturable methanogens unaffected by the antibody activity that was administered.

scholarly journals Pomegranate seed oil rich in conjugated linolenic acids reduces in vitro methane production

1970 ◽  
Vol 46 (3) ◽  
pp. 325-335
Author(s):  
E. Maleki ◽  
G.Y. Meng ◽  
M. Faseleh Jahromi ◽  
R. Jorfi ◽  
A. Khoddami ◽  
...  
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Seed Oil ◽  
Control Diet ◽  
Nutrient Utilization ◽  
Gas Production ◽  
Metabolizable Energy ◽  
Ruminal Fermentation ◽  
Ch4 Production

The objective of this study was to determine the effect of pomegranate (Punica granatum L.) seed oil (PSO) on gas and methane (CH4) production, ruminal fermentation and microbial populations under in vitro conditions. Three treatments consisting of a control diet containing 10 mg tallow (CON); the control diet with 5 mg PSO + 5 mg tallow (MPSO) and the control diet containing 10 mg PSO (HPSO) were compared. Ten mg of the experimental fat/oil samples were inserted into a gas-tight 100 mL plastic syringe containing 30 mL of an incubation inoculum and 250 mg of a basic substrate of a hay/concentrate (1/1, w/w) mixture. In vitro gas production was recorded over 0, 2, 4, 6, 8, 10, 12 and 24 h of incubation. After 24 hours, incubation was stopped, and methane production, pH, volatile fatty acids (VFAs) and microbial counts were measured in the inoculant. Gas production at 4, 6, 8, 10, 12 and 24 h incubation, metabolizable energy and in vitro organic matter disappearance increased linearly and quadratically as level of PSO increased. Furthermore, the 10 mg PSO (HPSO) decreased CH4 production by 21.0% compared with the control (CON) group. There were no significant differences in total and individual VFA concentrations between different levels of PSO, except for butyric acid. After 24 h of incubation, methanogenesis decreased in the HPSO compared with the MPSO and CON treatments. In addition, total bacteria and protozoa counts increased with rising PSO levels, while population methanogenesis declined significantly. These results suggested that PSO could reduce methane emissions, which might be beneficial to nutrient utilization and growth in ruminants.

scholarly journals Gas Production, Digestibility and Efficacy of Stored or Fresh Plant Extracts to Reduce Methane Production on Different Substrates

Animals ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 146 ◽  
Author(s):  
Abiodun Mayowa Akanmu ◽  
Abubeker Hassen ◽  
Festus Adeyemi Adejoro
Keyword(s):  
Plant Extracts ◽  
Crude Extract ◽  
Methane Production ◽  
Plant Material ◽  
Volatile Fatty Acids ◽  
Plant Secondary Metabolites ◽  
Gas Production ◽  
Growth Promoters ◽  
Freeze Dried

Natural compounds such as plant secondary metabolites (PSM) can be used to replace antibiotic growth promoters as rumen modifiers. In this study, the effectiveness of stored and freshly extracted Aloe vera (AV), Azadirachta indica (AZ), Moringa oleifera (MO), Jatropha curcas (JA), Tithonia diversifolia (TD) and Carica papaya (CP) crude extract and monensin on in vitro gas and methane production, organic matter digestibility (IVOMD) and volatile fatty acids (VFA) were evaluated using a total mixed ration (TMR), lucerne or Eragrostis curvula substrates. Fresh extracts were processed from the same batch of frozen (−20 °C) plant material a few days before the trial while the stored extracts were extracted and stored at 4 °C for 12 months prior to the study. Extraction was done by solubilising 50 g freeze-dried plant material in 500 mL 100% methanol. Four mL of reconstituted 50 mg crude extract per 1000 mL distilled water was added per incubation vial, which already contained 400 mg substrate and in vitro fermentation, and gas production and IVOMD evaluation were carried out using standard procedures. Results showed that storing plant extracts for 12 months did not affect the activity or stability of metabolites present in the crude extracts, as shown by the lack of differences in total gas production (TGP) and methane produced between fresh or stored extracts across the substrates. In the TMR substrate, plant extracts increased IVOMD but did not affect TGP and methane production, whereas monensin did not have any effect. Plant extracts increased IVOMD of Eragrostis substrate and supressed methane production to a greater extent than monensin (p < 0.05). It can be concluded that storing plant extracts for up to 12 months did not compromise their efficacy. In addition, the use of 50 mg/kg of AV, AZ, MO, JA, TD and CP extract to a forage-based diet will reduce methane production while improving feed digestibility.

Asparagopsis taxiformis decreases enteric methane production from sheep

2018 ◽  
Vol 58 (4) ◽  
pp. 681 ◽  
Author(s):  
Xixi Li ◽  
Hayley C. Norman ◽  
Robert D. Kinley ◽  
Michael Laurence ◽  
Matt Wilmot ◽  
...  
Keyword(s):  
Organic Matter ◽  
Methane Production ◽  
Volatile Fatty Acids ◽  
Animal Health ◽  
Long Term Effects ◽  
Asparagopsis Taxiformis ◽  
Ch4 Production ◽  
Enteric Methane ◽  
High Fibre

Asparagopsis taxiformis concentrates halogenated compounds that are known to inhibit cobamide-dependent methanogenesis in vitro and, therefore, has potential to mitigate enteric methane production. The present study investigated the effect of Asparagopsis on methane (CH4) production from sheep offered a high-fibre pelleted diet (offered at 1.2 × maintenance) at five inclusion levels of Asparagopsis for 72 days (0% (control), 0.5%, 1%, 2% and 3% organic matter basis as offered). Individual animal CH4 measurements were conducted at 21-day intervals using open-circuit respiration chambers. Asparagopsis inclusion resulted in a consistent and dose-dependent reduction in enteric CH4 production over time, with up to 80% CH4 mitigation at the 3% offered rate compared with the group fed no Asparagopsis (P < 0.05). Sheep fed Asparagopsis had a significantly lower concentration of total volatile fatty acids and acetate, but a higher propionate concentration. No changes in liveweight gain were identified. Supplementing Asparagopsis in a high-fibre diet (<2% organic matter) resulted in significant and persistent decreases in enteric methanogenesis over a 72-day period. Granulomatous and keratotic ruminal mucosa changes were identified in several sheep with Asparagopsis supplementation. While the outcomes of the present study may be extrapolated to feedlot to achieve the antimethanogenic effect associated with Asparagopsis, further work is required to define the long-term effects on productivity and animal health.

scholarly journals Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria inIn VitroRuminal Cultures

2011 ◽  
Vol 77 (8) ◽  
pp. 2634-2639 ◽  
Author(s):  
Zhenming Zhou ◽  
Qingxiang Meng ◽  
Zhongtang Yu
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Cellulolytic Bacteria ◽  
Bacterial Populations ◽  
Content Type ◽  
Specific Pcr ◽  
Methanogen Population ◽  
Propynoic Acid

ABSTRACTThe objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens usingin vitrocultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyltrans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce theFibrobacter succinogenespopulation. SN reduced theRuminococcus albuspopulation, while PA and 2NEOH increased the populations of bothR. albusandRuminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide futureinvivostudies to develop effective mitigation of methane emission from ruminants.

PSXIII-2 Effect of varying proportions of cereal rye and turnip on ruminal fermentation and methane output through in vitro batch culture

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 461-461
Author(s):  
Jordan L Cox-O’Neill ◽  
Vivek Fellner ◽  
Alan J Franluebbers ◽  
Deidre D Harmon ◽  
Matt H Poore ◽  
...  
Keyword(s):  
Methane Production ◽  
Dry Matter ◽  
Ruminal Fermentation ◽  
Variable Response ◽  
Cool Season ◽  
Cereal Rye ◽  
Freeze Dried ◽  
Main Effect ◽  
Cool Season Grass

Abstract Ruminant animal performance has been variable in studies grazing annual cool-season grass and brassica monocultures and mixtures. There is little understanding of the fermentation mechanisms causing variation. The aim of this study was to determine apparent dry matter (DM) digestibility, methane, and volatile fatty acid (VFA) concentration from different proportions of cereal rye (Secale cereal; R) and turnip (Brassica rapa L.; T) (0R:100T, 40R:60T, 60R:40T, and 100R:0T) via in vitro batch fermentation. Freeze-dried forage samples from an integrated crop-livestock study was assembled into the four treatments with a 50:50 leaf to root ratio for turnip. Measurements were made following a 48 hr fermentation with 2:1 buffer and ruminal fluid inoculum. Data were analyzed using Mixed Procedure of SAS with batch (replicate) and treatment (main effect) in the model; differences were declared at P ≤ 0.05, with tendencies declared at &gt; 0.05 but &lt; 0.10. Rumen apparent DM digestibility (26.8%; overall mean) was not different among treatments. Methane production was less (P &lt; 0.01) with inclusion of turnip ranging from 774 nmol/ml for 0R:100T to 1416 nmol/ml for 100R:0T. Total VFA production, acetate to propionate ratio, acetate, and valerate were not affected by forage treatments (117 mM, 1.45, 39.84 mol/100 mol, and 7.86 mol/100 mol, respectively; overall mean). Propionate, isobutyrate, and isovalerate concentrations were greater and butyrate concentration less with greater (P &lt; 0.01) proportions of rye in the mixture. No effect of R:T ratio on digestibility or total VFA production along with the observed differences in individual VFA concentration do not explain variable response in grazing animals. Additionally, methane production results indicate that grazing turnips could potentially reduce methane production and thus reduce ruminant livestock’s contribution to greenhouse gas emissions.

scholarly journals Modelling the impact of the macroalgae Asparagopsis taxiformis on rumen microbial fermentation

2020 ◽  
Author(s):  
Rafael Muñoz-Tamayo ◽  
Juana C. Chagas ◽  
Mohammad Ramin ◽  
Sophie J. Krizsan
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Mean Squared Error ◽  
Microbial Fermentation ◽  
Model Structure ◽  
Red Macroalgae ◽  
Asparagopsis Taxiformis ◽  
The Impact

AbstractBackgroundThe red macroalgae Asparagopsis taxiformis is a potent natural supplement for reducing methane production from cattle. A. taxiformis contains several anti-methanogenic compounds including bromoform that inhibits directly methanogenesis. The positive and adverse effects of A. taxiformis on the rumen microbiota are dose-dependent and operate in a dynamic fashion. It is therefore key to characterize the dynamic response of the rumen microbial fermentation for identifying optimal conditions on the use of A. taxiformis as a dietary supplement for methane mitigation. Accordingly, the objective of this work was to model the effect of A. taxiformis supplementation on the rumen microbial fermentation under in vitro conditions. We adapted a published mathematical model of rumen microbial fermentation to account for A. taxiformis supplementation. We modelled the impact of A. taxiformis on the fermentation and methane production by two mechanisms, namely (i) direct inhibition of the growth rate of methanogenesis by bromoform and (ii) hydrogen control on sugars utilization and on the flux distribution towards volatile fatty acids production. We calibrated our model using a multi-experiment estimation approach that integrated experimental data with six macroalgae supplementation levels from a published in vitro study assessing the dose-response impact of A. taxiformis on rumen fermentation.Resultsour model captured satisfactorily the effect of A. taxiformis on the dynamic profile of rumen microbial fermentation for the six supplementation levels of A. taxiformis with an average determination coefficient of 0.88 and an average coefficient of variation of the root mean squared error of 15.2% for acetate, butyrate, propionate, ammonia and methane.Conclusionsour results indicated the potential of our model as prediction tool for assessing the impact of additives such as seaweeds on the rumen microbial fermentation and methane production in vitro. Additional dynamic data on hydrogen and bromoform are required to validate our model structure and look for model structure improvements. We are working on model extensions to account for in vivo conditions. We expect this model development can be useful to help the design of sustainable nutritional strategies promoting healthy rumen function and low environmental footprint.

The effects of essential oil and condensed tannin on fermentation and methane production under in vitro conditions

2016 ◽  
Vol 56 (10) ◽  
pp. 1707 ◽  
Author(s):  
Brittany Pinski ◽  
Mevlüt Günal ◽  
Amer A. AbuGhazaleh
Keyword(s):  
Fatty Acid ◽  
Methane Production ◽  
Volatile Fatty Acid ◽  
Condensed Tannin ◽  
Total Volatile ◽  
Tannin Extract ◽  
Ch4 Production ◽  
N Concentration ◽  
Total Volatile Fatty Acid

The potential of five different essential oils (EO) and quebracho condensed tannin extract (QCT) as antimethanogenic additives in ruminant feeds were investigated. The first experiment was conducted to screen the effects rosemary oil, sage oil, cinnamon oil (CNO), eucalyptus oil and myrrh oil at 500 mg/L of culture fluid on methane (CH4) production under in vitro conditions. Rumen contents were collected from a cannulated Holstein dairy cow and used for a 24-h batch-culture experiment. Treatments were a control (CON) or CON plus EO at 500 mg/L. Results showed that CNO decreased CH4 production and, therefore, was selected for Experiment 2. The second experiment was designed to test the effects of CNO at three different dose levels on CH4 production and fermentation in 24-h batch-culture experiments. Treatments were CON or CON plus CNO supplemented at 125, 250 and 500 mg/L. Relative to CON, CNO decreased total gas production at the 250 and 500 mg/L doses. All doses of CNO decreased CH4 production. Total volatile fatty acid production was lower in cultures incubated with CNO at the 500 mg/L. Ammonia-N concentration decreased in cultures incubated with CNO at the 500 mg/L. The third experiment was designed to test the effects of QCT on CH4 production and fermentation in 24-h batch cultures. Treatments were CON or CON plus QCT at 25, 50 and 75 g/kg of diet DM. Relative to CON, total volatile fatty acid concentration increased with the 50 g/kg QCT, but was similar to the 25 and 75 g/kg QCT. The proportions of acetate decreased, while the proportions of propionate increased with the 25 g/kg QCT compared with CON. Methane production was not affected in cultures incubated with QCT. Relative to CON, all doses of QCT decreased ammonia-N concentration. In conclusion, results from the present study showed that except for CNO, EO tested in the study had no effects on rumen CH4 production. Addition of CNO to rumen cultures at 125 and 250 mg/L reduced CH4 production without negative effects on rumen fermentation. Quebracho condensed tannin-extract supplementation had no effects on CH4 production and fermentation parameters except for ammonia-N concentration.

Investigating Eremophila glabra as a bioactive agent for preventing lactic acidosis in sheep

2010 ◽  
Vol 50 (6) ◽  
pp. 449 ◽  
Author(s):  
P. G. Hutton ◽  
Z. Durmic ◽  
P. E. Vercoe
Keyword(s):  
Lactic Acidosis ◽  
Volatile Fatty Acids ◽  
Native Plant ◽  
Rumen Ph ◽  
Freeze Dried ◽  
Australian Native Plant ◽  
And Control ◽  
Higher Doses

The Australian native plant Eremophila glabra was tested as a potential agent for preventing lactic acidosis in sheep after it was observed to be effective against acidosis in vitro. Ruminally fistulated wethers were infused via rumen cannula with single doses of kibbled wheat (14 g/kg bodyweight) and either virginiamycin (Eskalin500; AB, 80 mg/kg of wheat plus 100 g milled oaten hay/kg of wheat, n = 6), E. glabra (EG, 100 g freeze-dried and milled leaf material per kg of wheat, n = 10) or milled oaten hay (Control, 100 g milled oaten hay/kg of wheat, n = 16). Rumen samples were collected immediately before infusion and then 2, 4, 6, 8, 12, 16 and 24 h after the infusion. The samples were analysed for pH, D-lactate, volatile fatty acids (VFA) and osmolality. Rumen pH and D-lactate values indicative of acidosis were detected in the Control and EG groups. The pH nadir of the rumen was 12 h after the wheat infusion, at which time the values in the EG (pH = 4.87) and Control (pH = 5.09) groups were lower (P < 0.05) than in the AB group (pH = 5.63) and the D-lactate concentrations were higher (P < 0.05) in the EG and Control groups (24 mmol/L and 15 mmol/L, respectively) than in the AB group (0.9 mmol/L). At the same time, total VFA concentration was higher (P < 0.05) in the AB group (102 mmol/L) than in the Control (65 mmol/L) and the EG (14 mmol/L) groups. Rumen osmolality did not differ between groups. Virginiamycin was effective at preventing lactic acidosis. However, the inclusion of dried leaves from E. glabra at a similar level that was effective in vitro did not prevent lactic acidosis in vivo, and the reasons behind this remain unclear. The study demonstrates the difficulty in converting in vitro results to in vivo and highlights the need to test the plant at higher doses in vivo.

scholarly journals Effects of Geraniol and Camphene on in Vitro Rumen Fermentation and Methane Production

2017 ◽  
Vol 48 (2) ◽  
pp. 63-69
Author(s):  
M. Joch ◽  
V. Kudrna ◽  
B. Hučko
Keyword(s):  
Methane Emission ◽  
Methane Production ◽  
Dry Matter ◽  
Volatile Fatty Acids ◽  
Rumen Fluid ◽  
Positive Control ◽  
Rumen Fermentation ◽  
Dry Matter Digestibility

AbstractThe objective of this study was to determine the effects of geraniol and camphene at three dosages (300, 600, and 900 mg l-1) on rumen microbial fermentation and methane emission in in vitro batch culture of rumen fluid supplied with a 60 : 40 forage : concentrate substrate (16.2% crude protein, 33.1% neutral detergent fibre). The ionophore antibiotic monensin (8 mg/l) was used as positive control. Compared to control, geraniol significantly (P < 0.05) reduced methane production with increasing doses, with reductions by 10.2, 66.9, and 97.9%. However, total volatile fatty acids (VFA) production and in vitro dry matter digestibility were also reduced (P < 0.05) by all doses of geraniol. Camphene demonstrated weak and unpromising effects on rumen fermentation. Camphene did not decrease (P > 0.05) methane production and slightly decreased (P < 0.05) VFA production. Due to the strong antimethanogenic effect of geraniol a careful selection of dose and combination with other antimethanogenic compounds may be effective in mitigating methane emission from ruminants. However, if a reduction in total VFA production and dry matter digestibility persisted in vivo, geraniol would have a negative effect on animal productivity.

Effects of gallic acid on in vitro rumen fermentation and methane production using rumen simulation (Rusitec) and batch-culture techniques

2019 ◽  
Vol 59 (2) ◽  
pp. 277 ◽  
Author(s):  
C. Wei ◽  
J. Guyader ◽  
L. Collazos ◽  
K. A. Beauchemin ◽  
G. Y. Zhao
Keyword(s):  
Gallic Acid ◽  
Batch Culture ◽  
Methane Production ◽  
Gas Production ◽  
Short Term ◽  
Ch4 Production ◽  
N Metabolism ◽  
In Vitro Rumen Fermentation

Two experiments were conducted to investigate the effects of adding gallic acid (GA) to ruminant diets on long- and short-term in vitro rumen fermentation and methane (CH4) production, and to test possible interactions between GA and ethanol on fermentation. The first experiment was conducted using the rumen simulation technique (Rusitec), as a completely randomised block design with four replications and the following four doses of GA: 0, 5, 10 and 20 mg GA/g dry matter (DM). Ethanol was used in all treatments to increase the solubilisation of GA in rumen fluid. The experimental period lasted 16 days, of which the first 7 days were for adaptation and the subsequent 9 days were for sampling. The second experiment was a 48-h batch-culture incubation conducted as a completely randomised design with a 4 (GA dose; 0, 10, 20, and 40 mg GA/g DM) × 2 (with or without ethanol) arrangement of treatments. In the Rusitec experiment, addition of GA up to 20 mg/g DM did not affect DM disappearance (DMD), organic matter (OM) disappearance, neutral detergent-fibre disappearance (NDFD), acid detergent-fibre disappearance (ADFD) or starch disappearance (P &gt; 0.05), but crude protein disappearance was linearly decreased (P = 0.04) from 78.3% to 72.0%. Daily gas production and CH4 production expressed as mL/g DM and mL/g DMD were not affected by addition of GA (P &gt; 0.05). Addition of GA up to 20 mg/g DM increased butyrate and isovalerate production (P &lt; 0.05) and tended to increase isobutyrate (P = 0.09) and decrease heptanoate production (P = 0.07). In the batch-culture experiment, adding GA up to 40 mg/g DM linearly increased 48-h DMD, NDFD and ADFD (P &lt; 0.05) and decreased (P &lt; 0.05) CH4 expressed as mL/g DMD, mL/g NDFD and mL/g ADFD. Methane production was decreased after 24 h and 48 h only when GA was added at 10 mg/g DM without ethanol. Fermentation liquid pH and concentration of ammonia-nitrogen (ammonia-N) were also reduced (P &lt; 0.05) with an increasing concentration of GA. Treatments with ethanol notably enhanced 48-h DMD, NDFD, ADFD, gas production (mL/g DM, mL/g OM or mL/g DMD), CH4 production (mL/g DM, mL/g DMD or mL/g NDFD), total volatile fatty acid concentration, the acetate:propionate ratio, acetate, valerate, isovalerate and caproate molar proportions (P &lt; 0.01) and decreased propionate, butyrate and isobutyrate molar proportions (P &lt; 0.01). Significant dose of GA × ethanol interaction was observed only for acetate molar proportion (P = 0.03). In conclusion, our study suggests that the beneficial effects of GA on feed digestion and CH4 production may be short term, while improvements in N metabolism may be sustained over the long term. It may be useful to conduct long-term in vivo studies using a range of diets and doses to verify whether GA can be used as a feed additive to mitigate enteric CH4 production and improve N metabolism of ruminants.

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