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The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Barley grain sources with variable kernel sizes makes adequate and consistent processing of kernels challenging. This study evaluated how the severity of processing for reconstituted high-moisture (65% on DM basis) barley (RHB) affects ensiling characteristics and in vitro ruminal fermentation. Three independent sources of light (<630 g/500 mL) and heavy (>670 g/500 mL) barley were blended to create 4 sources of variable kernel sized barley (646 g/500 mL). Reconstituted high-moisture barley rolled using a 1.40-mm gap width for finely processed RHB (RHBF), a 1.86-mm gap width for medium processed RHB (RHBM), or a 2.31-mm gap width for coarsely processed RHB (RHBC) was ensiled for 1 or 5 mo with dry rolled barley (DRB; roller gap width 1.86 mm) used as a control. The 1-mo RHB and the DRB were further evaluated using the rumen in vitro simulation technique (RUSITEC) to investigate the effects of severity of processing for RHB on ruminal fermentation, and gas, methane, and microbial protein production. Using a randomized complete block design (n = 4), 16 fermenters from 2 RUSITEC apparatuses were used to assess the 4 sources and 4 processing treatments. The addition of water increased kernel width before rolling and resulted in increased kernel length, width, and thickness for RHB relative to DRB. Increasing processing severity for RHB linearly increased kernel width. The percentage of fine particles (<1.18 mm) was greater for DRB than RHBF, but did not differ by processing severity for RHB. Dry matter, OM, and starch disappearance were not different between DRB and RHBF, but linearly increased with increasing processing severity for RHB. Fermenter pH tended to be less for DRB relative to RHBF. In conclusion, the reduction in fine particles with the addition of water for RHB may prevent a decline in fermenter pH and when processed to achieve the same processing index using a smaller roller gap width, yielded similar DM and OM disappearance suggesting a lesser risk for low ruminal pH without compromising digestibility. [ABSTRACT FROM AUTHOR]

ABSTRACT: Barley grain sources with variable kernel sizes makes adequate and consistent processing of kernels challenging. This study evaluated how the severity of processing for reconstituted high-moisture (65% on DM basis) barley (RHB) affects ensiling characteristics and in vitro ruminal fermentation. Three independent sources of light (670 g/500 mL) barley were blended to create 4 sources of variable kernel sized barley (646 g/500 mL). Reconstituted high-moisture barley rolled using a 1.40-mm gap width for finely processed RHB (RHBF), a 1.86-mm gap width for medium processed RHB (RHBM), or a 2.31-mm gap width for coarsely processed RHB (RHBC) was ensiled for 1 or 5 mo with dry rolled barley (DRB; roller gap width 1.86 mm) used as a control. The 1-mo RHB and the DRB were further evaluated using the rumen in vitro simulation technique (RUSITEC) to investigate the effects of severity of processing for RHB on ruminal fermentation, and gas, methane, and microbial protein production. Using a randomized complete block design (n = 4), 16 fermenters from 2 RUSITEC apparatuses were used to assess the 4 sources and 4 processing treatments. The addition of water increased kernel width before rolling and resulted in increased kernel length, width, and thickness for RHB relative to DRB. Increasing processing severity for RHB linearly increased kernel width. The percentage of fine particles (

ABSTRACT The ruminant-microorganism symbiosis is unique by providing high-quality food from fibrous materials but also contributes to the production of one of the most potent greenhouse gases—methane. Mitigating methanogenesis in ruminants has been a focus of interest in the past decades. One of the promising strategies to combat methane production is the use of feed supplements, such as seaweeds, that might mitigate methanogenesis via microbiome modulation and direct chemical inhibition. We conducted in vitro investigations of the effect of three seaweeds (Ascophyllum nodosum, Asparagopsis taxiformis, and Fucus vesiculosus) harvested at different locations (Iceland, Scotland, and Portugal) on methane production. We applied metataxonomics (16S rRNA gene amplicons) and metagenomics (shotgun) methods to uncover the interplay between the microbiome’s taxonomical and functional states, methanogenesis rates, and seaweed supplementations. Methane concentration was reduced by A. nodosum and F. vesiculosus, both harvested in Scotland and A. taxiformis, with the greatest effect of the latter. A. taxiformis acted through the reduction of archaea-to-bacteria ratios but not eukaryotes-to-bacteria. Moreover, A. taxiformis application was accompanied by shifts in both taxonomic and functional profiles of the microbial communities, decreasing not only archaeal ratios but also abundances of methanogenesis-associated functions. Methanobrevibacter “SGMT” (M. smithii, M. gottschalkii, M. millerae or M. thaueri; high methane yield) to “RO” (M. ruminantium and M. olleyae; low methane yield) clades ratios were also decreased, indicating that A. taxiformis application favored Methanobrevibacter species that produce less methane. Most of the functions directly involved in methanogenesis were less abundant, while the abundances of the small subset of functions that participate in methane assimilation were increased.IMPORTANCEThe application of A. taxiformis significantly reduced methane production in vitro. We showed that this reduction was linked to changes in microbial function profiles, the decline in the overall archaeal community counts, and shifts in ratios of Methanobrevibacter “SGMT” and “RO” clades. A. nodosum and F. vesiculosus, obtained from Scotland, also decreased methane concentration in the total gas, while the same seaweed species from Iceland did not.

Simple Summary: Garlic oil and cinnamaldehyde are plant-derived bioactive compounds with antimicrobial activity, which might contribute to improving the efficiency of rumen fermentation. However, an apparent adaptation of microbial populations to these compounds is usually observed over time. In this study, the effects of garlic oil and cinnamaldehyde on rumen fermentation and microbial populations were assessed in two periods of time using an in vitro system (Rusitec fermenters). Despite this possible adaptation, garlic oil supplementation reduced methane production and enhanced the energy profile of fermentation end products over time, whereas cinnamaldehyde appeared to increase the microbial protein supply to the animal. Thus, the use of these compounds as feed additives could contribute to increasing the efficiency and sustainability of ruminant production systems, bringing both economic and environmental benefits. Garlic oil (GO) and cinnamaldehyde (CIN) have shown potential to modify rumen fermentation. The aim of this study was to assess the effects of GO and CIN on rumen fermentation, microbial protein synthesis (MPS), and microbial populations in Rusitec fermenters fed a mixed diet (50:50 forage/concentrate), as well as whether these effects were maintained over time. Six fermenters were used in two 15-day incubation runs. Within each run, two fermenters received no additive, 180 mg/L of GO, or 180 mg/L of CIN. Rumen fermentation parameters were assessed in two periods (P1 and P2), and microbial populations were studied after each of these periods. Garlic oil reduced the acetate/propionate ratio and methane production (p < 0.001) in P1 and P2 and decreased protozoal DNA concentration and the relative abundance of fungi and archaea after P1 (p < 0.05). Cinnamaldehyde increased bacterial diversity (p < 0.01) and modified the structure of bacterial communities after P1, decreased bacterial DNA concentration after P2 (p < 0.05), and increased MPS (p < 0.001). The results of this study indicate that 180 mg/L of GO and CIN promoted a more efficient rumen fermentation and increased the protein supply to the animal, respectively, although an apparent adaptive response of microbial populations to GO was observed. [ABSTRACT FROM AUTHOR]

This study investigated the combined impact of essential oil blends (EOBs) and fumaric acid (FA) on ruminal fermentation in dairy cows using the rumen simulation technique (RUSITEC) system. Three rumen-cannulated, non-lactating Holstein Friesian cows served as inoculum donors. The substrate, a total mixed ration (TMR), comprised corn silage, alfalfa hay, and a concentrate mix in a 3:1:1 ratio. The four treatments evaluated were Control (TMR without additives), EFA1 (TMR + EOB1 + FA), EFA2 (TMR + EOB2 + FA), and EFA3 (TMR + EOB3 + FA). Sixteen fermentation chambers were randomly assigned to the treatments, each with four replicates, following a completely randomized design during a 9-day experimental period. EOBs and FA were added at 10 µL/g feed and 3% of TMR, respectively. After a 4-day adaptation, samples were collected for 5 days. Results revealed that EFA1 significantly reduced (p = 0.0351) CH4 emissions by 60.2% without negatively impacting dry matter disappearance, fiber fraction digestibility, pH, or gas volume. All EFAs increased (p < 0.001) the propionate molar proportion and decreased (p < 0.001) the acetate-to-propionate ratio. EFA2 decreased (p < 0.05) the acetate proportion by 3.3% compared to the control. In conclusion, EFA1 is recommended as an effective nutritional intervention to mitigate CH4 emissions and optimize ruminal fermentation in dairy cows. [ABSTRACT FROM AUTHOR]

The effect of cicer milkvetch (Astragalus cicer L.) and sainfoin (Onobrychis viciifolia Scop.) on ruminal fermentation, methane production, and microbial nitrogen synthesis was assessed in two experiments. Experiment 1 analyzed two legumes, cicer milkvetch and sainfoin at two stages (vegetative and late flower) incubated with alfalfa (Medicago sativa) at five inclusion rates 0:100; 25:75, 50:50, 75:25, and 100:0 (as DM) in batch culture. Experiment 2 analyzed vegetative cicer milkvetch and alfalfa incubated in ratios of 25:75, 50:50, 75:25, and 100:0 (as DM) in continuous culture systems (RUSITEC). In batch culture, increased dry matter disappearance (DMD), and propionate percentage (%total), and reduced methane (mg·g−1 DMD) occurred with vegetative cicer milkvetch inclusion. In RUSITEC, DMD linearly increased (P < 0.01), acetate:propionate ratio quadratically decreased, while ammonia (NH3-N) concentration (P < 0.01) and butyrate percentage (%total) linearly decreased (P < 0.05) with increasing inclusion of cicer milkvetch. No differences were observed for methane (CH4) production (mg·g−1 DMD), or short chain fatty acid (SCFA) production (mmol·day−1). Microbial nitrogen synthesis and efficiency of protein synthesis linearly increased (P < 0.05) with increased inclusion of cicer milkvetch. Results suggest cicer milkvetch may result in synchronicity of energy and nitrogen during rumen fermentation, which could enhance cattle production. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to investigate the in vitro dose-dependent effects of sigla storax (Styrax liquidus) on rumen microbiota and rumen microbial fermentation in comparison to monensin as a positive control. Methods and Results: This study was carried out using a rumen simulation model (Rusitec). Treatments consisted of no additive (control), 10 mg l−1 of monensin sodium salt, 100 mg l−1 (Low-Sigla), and 500 mg l−1 (High-Sigla) of sigla storax (n = 6/treatment). In addition to rumen fermentation characteristics, rumen microbial composition was investigated using 16S rRNA sequencing. The methane variables and the acetate to propionate ratio decreased in the both High-Sigla and monensin groups (P < 0.05). High-Sigla had no effect on ammonia, total SCFA and nutrition degradation, while monensin decreased these parameters (P < 0.05). Unlike monensin, the sigla storax treatments did not affect the alpha or beta diversity indexes of the microbiota. The relative abundance of Methanomethylophilaceae and Ruminococcaceae decreased with High-Sigla and monensin (P < 0.05), and Atopobiaceae and Eggerthellaceae decreased with the both doses of sigla storax as well as monensin treatments (P < 0.05). Syntrophococcus, DNF00809, and Kandleria were among the genera that most decreased with High-Sigla and monensin (Q < 0.07) and were strongly positively correlated with methane production (r = 0.52–0.56). Conclusions: The high dose of sigla storax (500 mg l−1) decreased methane in the rumen ecosystem without adverse effects on nutrient degradation and SCFA production, and without dramatically impacting the microbial composition. Sigla storax might be a novel feed additive to mitigate methane in cattle. [ABSTRACT FROM AUTHOR]

Garlic oil (GO) and cinnamaldehyde (CIN) have shown potential to modify rumen fermentation. The aim of this study was to assess the effects of GO and CIN on rumen fermentation, microbial protein synthesis (MPS), and microbial populations in Rusitec fermenters fed a mixed diet (50:50 forage/concentrate), as well as whether these effects were maintained over time. Six fermenters were used in two 15-day incubation runs. Within each run, two fermenters received no additive, 180 mg/L of GO, or 180 mg/L of CIN. Rumen fermentation parameters were assessed in two periods (P1 and P2), and microbial populations were studied after each of these periods. Garlic oil reduced the acetate/propionate ratio and methane production (p < 0.001) in P1 and P2 and decreased protozoal DNA concentration and the relative abundance of fungi and archaea after P1 (p < 0.05). Cinnamaldehyde increased bacterial diversity (p < 0.01) and modified the structure of bacterial communities after P1, decreased bacterial DNA concentration after P2 (p < 0.05), and increased MPS (p < 0.001). The results of this study indicate that 180 mg/L of GO and CIN promoted a more efficient rumen fermentation and increased the protein supply to the animal, respectively, although an apparent adaptive response of microbial populations to GO was observed.

This study investigated the combined impact of essential oil blends (EOBs) and fumaric acid (FA) on ruminal fermentation in dairy cows using the rumen simulation technique (RUSITEC) system. Three rumen-cannulated, non-lactating Holstein Friesian cows served as inoculum donors. The substrate, a total mixed ration (TMR), comprised corn silage, alfalfa hay, and a concentrate mix in a 3:1:1 ratio. The four treatments evaluated were Control (TMR without additives), EFA1 (TMR + EOB1 + FA), EFA2 (TMR + EOB2 + FA), and EFA3 (TMR + EOB3 + FA). Sixteen fermentation chambers were randomly assigned to the treatments, each with four replicates, following a completely randomized design during a 9-day experimental period. EOBs and FA were added at 10 µL/g feed and 3% of TMR, respectively. After a 4-day adaptation, samples were collected for 5 days. Results revealed that EFA1 significantly reduced (p = 0.0351) CH4 emissions by 60.2% without negatively impacting dry matter disappearance, fiber fraction digestibility, pH, or gas volume. All EFAs increased (p < 0.001) the propionate molar proportion and decreased (p < 0.001) the acetate-to-propionate ratio. EFA2 decreased (p < 0.05) the acetate proportion by 3.3% compared to the control. In conclusion, EFA1 is recommended as an effective nutritional intervention to mitigate CH4 emissions and optimize ruminal fermentation in dairy cows.

The aim of this study was to distinguish effects due to diet composition from those triggered by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system (RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined. Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal grain, 32% hay) diets were more digestible (p < 0.05) than high forage (10% cereal grain, 78% hay) diets, resulting in a greater (p < 0.05) formation of most fermentation end-products and microbial protein in the rumen. However, there were no significant (p > 0.05) differences between diets in methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of oleic acid, and relative proportion of conjugated linoleic acid were increased (p < 0.05) with high forage diets. A decline in rumen pH from 6.8 to 6.4 decreased (p < 0.05) feed digestibility, protein degradability, and the daily outputs of some fermentation end-products (gas, VFA, acetate, ammonia) but had no effect (p > 0.05) on the synthesis of microbial protein, and on the output of methane, propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid relative proportions were observed within this pH range. The overall multivariate analysis revealed a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined pH effects on ruminal fermentation and microbial communities. [ABSTRACT FROM AUTHOR]

• Reducing nitrogen fertiliser application decreased grass crude protein content. • Nitrogen fertiliser reduction induced subtle changes in rumen microbiome. • Reducing nitrogen fertiliser decreased daily nitrogen excretion by beef cattle. • Fertiliser nitrogen application rate had no effect on RUSITEC methane output. Nitrogen (N) excretion by cattle, particularly urinary N, can have detrimental environmental impacts on air and water quality, and contributes to greenhouse gas emissions. The effects of increasing the application rate of inorganic N fertiliser from 15 (N15) to 80 (N80) kg/ha per cut to Lolium perenne dominant swards in summer, on intake, rumen fermentation, rumen microbial populations, apparent total-tract digestibility and N-balance in beef cattle, and in vitro fermentation and methane output, were studied. Sixteen suckler-bred Charolais steers, used in a randomised block design experiment, were offered fresh grass mechanically harvested 21-d after N fertiliser application. Similar grass was incubated in an eight-vessel in vitro RUSITEC system. Grass crude protein concentration was 50 g/kg dry matter (DM) higher for N80 compared to N15. There was no difference in grass DM intake between treatments. Rumen fermentation variables did not differ between treatments, except for the molar proportion of propionate, which was greater for N80 than N15. Gross microbial community structure in the rumen was not significantly altered by inorganic N fertiliser application rate. The relative abundance of individual genera Lachnospiraceae_NK3A320_group and Ruminococcaceae_NK4A214_group, and Acetitomaculum were significantly lower at the higher N application rate. Mean plasma urea concentration was greater for N80 compared to N15. In vivo digestibility of DM, organic matter, neutral detergent fibre and acid detergent fibre was unaffected by fertiliser N application rate. Nitrogen intake was 75 g/d greater, and urinary and faecal N excretion were 20 and 5 g/d greater, respectively, for N80 than N15. The quantity of N retained and N use efficiency was greater for N80 compared to N15. In vitro NH 3 concentration was greater for N80 than N15, whereas other rumen fermentation variables, and in vitro methane and total gas output, did not differ between treatments. Reducing the inorganic N fertiliser rate applied to mid-season temperate grass reduced N excretion from beef cattle, which is environmentally beneficial, with no effect on in vitro methane production. [ABSTRACT FROM AUTHOR]

Dietary tannins can affect rumen microbiota and enteric fermentation to mitigate methane emissions, although such effects have not yet been fully elucidated. We tested two subunits of hydrolyzable tannins named gallic acid (GA) and ellagic acid (EA), alone (75 mg/g DM each) or combined (150 mg/g DM in total), using the Rusitec system. EA and EA+GA treatments decreased methane production, volatile fatty acids, nutrient degradation, relative abundance of Butyrivibrio fibrisolvens , Fibrobacter succinogenes , Ruminococcus flavefaciens but increased Selenomonas ruminantium . EA and EA+GA increased urolithins A and B. Also, EA and EA+GA reduced bacterial richness, with limited effects on archaeal richness. For bacteria, Megasphaera elsdenii was more abundant after EA and EA+GA, while Methanomethylophilaceae dominated archaea in all treatments. EA was more effective than GA in altering rumen microbiota and fermentation but GA did not reduce VFA and nutrient degradation. Thus, dietary supplementation of EA-plant extracts for ruminants may be considered to mitigate enteric methane, although a suitable dosage must be ensured to minimize the negative effects on fermentation.

Ruminant livestock contribute significantly to global methane production and mitigation of which is of utmost importance. Feed additives represent a cost-effective means of achieving this. A potential target for such additives is rumen Oxidative Reduction Potential (ORP), a parameter which influences CH 4 production rates, with methanogenesis occurring optimally at ORPs below -300 mV. Thus, a controlled elevation of rumen ORP represents a potentially benign means of methanogen suppression. This research involved assessing a range of oxygen-releasing compounds for their ability to increase rumen ORP and inhibit methanogenesis, using the in-vitro rumen simulation technique (RUSITEC). Seven potential CH 4 inhibitors were tested in a 21-day trial monitoring biogas volume, CH 4 content, ORP, digestibility, ammonia, and volatile fatty acids concentration. The additives evaluated included: liquid peroxide (H 2 O 2 ) and urea hydrogen peroxide (UHP), as well as slower reacting species (calcium and magnesium peroxide), in addition to encapsulated liquid H 2 O 2 for controlled, slow release. Consistent CH 4 reductions of >50 % were observed from all additives. Reduced neutral detergent fibre (NDF) digestibility and a reduction in total volatile fatty acids (VFAs) was observed for some treatments, but MgO 2 and encapsulated H 2 O 2 reduced CH 4 volume by 62 % and 58 %, respectively, and had no detrimental effects on digestibility (p > 0.05) or on VFA production. Ex-situ ORP measurements demonstrated significant increases in ORP upon addition of the additives, with MgO 2 and encapsulated H 2 O 2 inducing a more moderate effect suggesting a controlled additive release was achieved with the slow-release format of encapsulated liquid H 2 O 2 . Thus, potential slow-release forms deemed suitable to progress to bolus or pellet format in-vivo were identified and could enable a longer-lasting suppression of methanogens within the rumen, facilitating application in both intensive and pasture-based production systems., (Copyright © 2024. Published by Elsevier B.V.)

The ruminant-microorganism symbiosis is unique by providing high-quality food from fibrous materials but also contributes to the production of one of the most potent greenhouse gases-methane. Mitigating methanogenesis in ruminants has been a focus of interest in the past decades. One of the promising strategies to combat methane production is the use of feed supplements, such as seaweeds, that might mitigate methanogenesis via microbiome modulation and direct chemical inhibition. We conducted in vitro investigations of the effect of three seaweeds ( Ascophyllum nodosum , Asparagopsis taxiformis , and Fucus vesiculosus ) harvested at different locations (Iceland, Scotland, and Portugal) on methane production. We applied metataxonomics (16S rRNA gene amplicons) and metagenomics (shotgun) methods to uncover the interplay between the microbiome's taxonomical and functional states, methanogenesis rates, and seaweed supplementations. Methane concentration was reduced by A. nodosum and F. vesiculosus , both harvested in Scotland and A. taxiformis , with the greatest effect of the latter. A. taxiformis acted through the reduction of archaea-to-bacteria ratios but not eukaryotes-to-bacteria. Moreover, A. taxiformis application was accompanied by shifts in both taxonomic and functional profiles of the microbial communities, decreasing not only archaeal ratios but also abundances of methanogenesis-associated functions. Methanobrevibacter "SGMT" ( M. smithii, M. gottschalkii, M. millerae or M. thaueri ; high methane yield) to "RO" ( M. ruminantium and M. olleyae ; low methane yield) clades ratios were also decreased, indicating that A. taxiformis application favored Methanobrevibacter species that produce less methane. Most of the functions directly involved in methanogenesis were less abundant, while the abundances of the small subset of functions that participate in methane assimilation were increased., Importance: The application of A. taxiformis significantly reduced methane production in vitro . We showed that this reduction was linked to changes in microbial function profiles, the decline in the overall archaeal community counts, and shifts in ratios of Methanobrevibacter "SGMT" and "RO" clades. A. nodosum and F. vesiculosus , obtained from Scotland, also decreased methane concentration in the total gas, while the same seaweed species from Iceland did not., Competing Interests: The authors declare no conflict of interest.

ABSTRACT: The physiological function of the reticulorumen plays an essential role in ruminant nutrition, and detailed knowledge of rumen motility can further advance understanding of ruminant nutrition and physiology. Rumen motility was simulated by setting different stirrer rotation speeds in a rumen simulation technique (RUSITEC) system. The aim of this study was to investigate the effects of rotation speeds on rumen fermentation, saturation factor of dissolved gases, hydrogen (H2) and methane (CH4) emissions, microbial protein synthesis, and selected microbial population using RUSITEC. The experiment was performed according to a balanced 3 × 3 Latin square design, and each period included 7 d for adaptation and 3 d for sampling. Three motility treatments included 5, 15, and 25 rpm rotation speeds. Daily total gas and H2 and CH4 emissions had quadratic responses to the increasing rotation speed and were highest at 15 rpm. Quadratic and linear responses (highest at 5 rpm) to increasing rotation speed were observed for saturation factors of H2 and CH4, liquid-dissolved H2 and CH4 concentrations, and headspace concentration of H2 in the gas phase, whereas increasing rotation speed linearly decreased saturation factors of CO2 and liquid-dissolved CO2 concentration. Quadratic and linear responses to increasing rotation speed were observed for molar percentages of acetate, ammonia, and microbial protein concentration, whereas increasing rotation speed quadratically increased pH and decreased total volatile fatty acid concentration and acetate-to-propionate ratio. The 15-rpm rotation speed had the highest values of total volatile fatty acids, acetate molar percentage, and microbial protein concentration. Quadratic and linear responses to increasing rotation speed were observed for copy numbers of solid-associated fungi and fluid-associated bacteria, fungi, and protozoa, while increasing rotation speed linearly increased copy numbers of solid-associated protozoa. Rotation at 15 rpm increased populations of fungi and protozoa in the solid rumen contents and the population of bacteria and fungi in the liquid rumen contents. In summary, this study provides insights on the biofunction of proper rumen motility (i.e., at a rotation speed of 15 rpm), such as improving feed fermentation, increasing gas emissions with decreased dissolved gas concentrations and saturation factors, and promoting microbial colonization and microbial protein synthesis, although further increase in rotation speed (i.e., to 25 rpm) decreases feed fermentation and microbial protein synthesis.

Inclusion of the red seaweed Asparagopsis taxiformis as a feed additive, has led to significant reductions in methane (CH4) production from ruminants. However, dietary supplementation with this seaweed is negatively associated with health and environmental concerns mainly due to its bromoform content, a compound with potential carcinogenic properties. Thus, there is renewed focus on ascertaining the anti-methanogenic potential of locally grown brown and green seaweeds, which typically do not contain bromoform. The objective of this study was to investigate the effects of selected brown and green seaweeds on diet digestibility, ruminal fermentation patterns, total gas (TGP) and CH4 production in vitro, using the rumen simulation technique system. In experiment 1, Pelvetia canaliculata (PEC) was examined. In experiment 2, Cystoseira tamariscifolia (CYT), Bifurcaria bifurcata (BIB), Fucus vesiculosus (FUV), Himanthalia elongata (HIM) and Ulva intestinalis (ULI) were analysed. Ascophyllum nodosum (ASC) was included in both experiments. A diet containing A. taxiformis (ASP1; ASP2) and an unsupplemented diet (CON) were included as positive and negative controls, respectively in both experiments. All seaweeds were included at a rate of 10 g/kg dry matter (DM) into a control diet of 50:50 (w:w) forage:concentrate. The seven brown and green seaweeds assessed failed to affect absolute CH4 emissions or alter fermentation patterns. In experiment 1, seaweed treatment had no effect on diet digestibility, CH4%, CH4 mmol/d or CH4 L/d (P>0.1), however ASP1 reduced CH4 mmol/g DOM by 49% (P

Aim: This study aimed to characterize the critical points for determining the development of dysbiosis associated with feed intolerances and ruminal acidosis. Methods and Results: A metabologenomics approach was used to characterize dynamic microbial and metabolomics shifts using the rumen simulation technique (RUSITEC) by feeding native cornstarch (ST), chemically modified cornstarch (CMS), or sucrose (SU). SU and CMS elicited the most drastic changes as rapidly as 4 h after feeding. This was accompanied by a swift accumulation of d‐lactate, and the decline of benzoic and malonic acid. A consistent increase in Bifidobacterium and Lactobacillus as well as a decrease in fibrolytic bacteria was observed for both CMS and ST after 24 h, indicating intolerances within the fibre degrading populations. However, an increase in Lactobacillus was already evident in SU after 8 h. An inverse relationship between Fibrobacter and Bifidobacterium was observed in ST. In fact, Fibrobacter was positively correlated with several short‐chain fatty acids, while Lactobacillus was positively correlated with lactic acid, hexoses, hexose‐phosphates, pentose phosphate pathway (PENTOSE‐P‐PWY), and heterolactic fermentation (P122‐PWY). Conclusions: The feeding of sucrose and modified starches, followed by native cornstarch, had a strong disruptive effect in the ruminal microbial community. Feed intolerances were shown to develop at different rates based on the availability of glucose for ruminal microorganisms. Significance and Impact of the study: These results can be used to establish patterns of early dysbiosis (biomarkers) and develop strategies for preventing undesirable shifts in the ruminal microbial ecosystem. [ABSTRACT FROM AUTHOR]

Feeding winery by-products (WBP) could affect the bovine microbiome because of their phenol compounds and a transfer of WBP-associated microbiota. This work examined changes in the underexplored solid-associated rumen microbiome following the inclusion of WBP. Using the rumen simulation technique, fermenters were inoculated with the inoculum of donor cows and were fed one of six dietary treatments including a control diet of 70 % hay +30 % concentrate (CON), control diet + 3.7 % commercial grapeseed extract (EXT), 65 % hay + 25 % concentrate + 10 % grape pomace (GP-low), 56 % hay + 24 % concentrate + 20 % grape pomace (GP-high), 70 % hay + 25 % concentrate + 5 % grapeseed meal (GS-low), and 65 % hay + 25 % concentrate + 10 % grapeseed meal (GS-high) (dry matter basis). The compositional changes of bacteria, archaea and fungi in the solid fractions were based on 16S and ITS2 rRNA sequencing. The alpha- and beta-diversity of the microbiota were unaffected. However, treatment modified the bacterial composition at low taxonomic levels. Butyrivibrio fibrisolvens , Treponema bryantii , and bacterium MC2010 decreased in EXT, while Treponema berlinense was increased in GP-high and GP-low compared to CON. Concerning fungi, GS-high increased Candida spp. , Lachancea spp. , Microdochium spp. , Mucor spp. , Pichia spp. , Saturnispora spp. , and Zygosaccharomyces spp. compared to CON. Many non- Saccharomyces yeasts were detected in WBP samples but absent in donor cows and CON samples. The genera affected by treatment were not the major contributors to the ruminal degradation of nutrients. The results indicate a sensitivity of rumen solid bacteria to grape phenols when delivered as an extract and a transfer of WBP-associated microbiota into the rumen. • Rumen bacteria and fungi were sensitive to grape phenols in an extract form. • Winery by-products introduced non- Saccharomyces yeasts into the rumen community. • Affected genera were not major contributors to the nutrient degradation. • Donor cows' microbiota data are essential when interpreting the in vitro effects. [ABSTRACT FROM AUTHOR]

The effects of supplementing grass with a high-energy, low-protein concentrate on intake, rumen fermentation, rumen microbial communities, apparent total tract digestibility and N-balance in beef cattle, and rumen fermentation and methane output in vitro, were studied. Sixteen Charolais steers (mean live weight (s.d.) 564 (24.3) kg)), were used in a completely randomised block design experiment, and offered either zero-grazed perennial ryegrass (Lolium perenne L.) ad libitum (GO), or the same grass with 2.25 kg citrus pulp dry matter (DM) daily (GCP). During the measurement phase, DM intake was restricted at 0.85 of their pre-determined intake. The same diets were incubated in an eight-vessel RUSITEC system. Supplementation decreased grass DM intake (6.59 vs. 7.54 kg/d) but increased total DM intake by 0.95 kg/d (P < 0.01). Rumen pH (P = 0.05) and NH 3 concentration (P < 0.05) decreased, and total volatile fatty acid (VFA) concentrations (P < 0.01), molar proportions of propionate (P = 0.08) and butyrate (P < 0.05) increased for GCP compared to GO. Supplementation significantly altered the overall rumen microbial community structure compared to GO, driven principally by changes in relative abundance of Prevotella , Methoanobrevibacter and Lachnospiraceae genera. Plasma urea concentration was lower (P < 0.05) for GCP than GO. There was no effect (P > 0.10) of supplementation on apparent total tract digestibility of DM, OM and ADFom, but aNDFom digestibility tended to be lower (P = 0.07) and crude protein (N × 6.25) digestibility was lower (P < 0.01) for GCP compared to GO. Nitrogen intake (200 g/d) did not differ (P > 0.05) between treatments. Faeces N loss was 9 g/d greater (P < 0.01), and urine (P < 0.01) and total (P < 0.05) N losses were 22 and 13 g/d lower, respectively, for GCP compared to GO. In terms of N-balance (g/kg N intake), faecal N excretion was greater and urinary N excretion was lower (P < 0.01) resulting in a greater N use efficiency (P = 0.06) for GCP than GO. In vitro rumen NH 3 concentrations were lower (P < 0.01) for GCP compared to GO, whereas in vitro rumen pH, lactic acid, VFA concentrations and molar proportions, and methane and total gas output did not differ (P > 0.05) between treatments. Supplementing grass with a low-protein, high-energy feed may be a strategy to reduce urinary and total N excretion in beef cattle grazing temperate grass pasture. • Supplementing grass with citrus pulp reduced rumen ammonia and plasma urea levels. • Overall rumen microbial community structure was altered by supplementation. • Supplementation reduced urinary and total nitrogen excretion. • Nitrogen use efficiency was enhanced by supplementation. • Supplementation had no effect on in vitro RUSITEC methane output. [ABSTRACT FROM AUTHOR]

Introduction: Sodium monensin, a common antibiotic in cattle feed, poses risks such as toxicity and reduced fertility, prompting regulatory scrutiny and interest in probiotic alternatives to improve gastrointestinal health and nutrient absorption. Microencapsulated probiotics offer potential as substitutes for antibiotics, aiming to enhance ruminal function and weight gain, explored through in vitro and in vivo models. Justification: In response to growing concerns about antibiotic resistance, the livestock industry, which continues to rely on antibiotics and growth-promoting agents, seeks solutions to improve rumen efficiency in systems based on tropical pastures. Objective: To evaluate the feed efficiency of cattle subjected to 2 isoenergetic and isoproteic diets: a commercial diet with monensin vs a diet of microencapsulated probiotics (FF) (Fortcell Feed, Bialtec S.A.S). Methods: A semi-continuous Rusitec fermenter with 8 vessels was used for a 17-day incubation period, evaluating treatments with sodium monensin and probiotics. Additionally, field tests were conducted on commercial cattle farms, involving 270 animals, to compare weight gain performance between a product containing antibiotics and microencapsulated probiotics. Results and Discussion: In vitro test results showed that the microencapsulated probiotic treatment had higher degradability (P<0.05) in various parameters, such as dry matter by 11%, and organic matter by 8%. The in vivo test evidenced higher daily weight gain (115 g/day/animal) with microencapsulated probiotics than with sodium monensin treatment. Significant differences in body weight gain between diets were found through Monte Carlo simulation, suggesting that diets with better feed conversion ratios offered a better gross margin. Conclusion: The use of microencapsulated probiotics in cattle feed salt shows promise as a potential alternative to sodium monensin. [ABSTRACT FROM AUTHOR]

Dairy products have a crucial role in the diets of over 6 billion people worldwide, providing essential nutrients such as energy, high-quality protein, and micronutrients. With dairy cows accounting for nearly 81% of global milk production, strategies to increase milk production sustainably are vital, especially with the U.S. being the second-largest producer. Essential oil blends (EOB), onion peel extract (OPE), and prebiotics such as mannan oligosaccharides (MOS) and galactooligosaccharides (GOS) offer promising avenues to enhance bovine milk production. These additives have the potential to modify rumen ecology and fermentation activity, while also mitigating greenhouse gas emissions in dairy cows. Therefore, this study investigated the effects of combining EOB, OPE, GOS, and/or MOS on specific rumen metabolites and metabolic pathways using the rumen simulation technique (RUSITEC) system. Rumen-cannulated non-lactating Holstein Friesian cows (n = 3) were used as rumen fluid donors. Total mixed ration (TMR) consisted of corn silage (60%), alfalfa hay (20%), and concentrate (20%). Using a completely randomized design, 16 fermentation vessels were divided into 4 groups with 4 replicates each over a 9-d period. The treatments included a control group (CON; TMR only), GEO (TMR+GOS+EOB+OPE), MEO (TMR+MOS+EOB+OPE), and OLEO (TMR+OLG+EOB+OPE). The EOB comprised of garlic, lemongrass, cumin, lavender, and nutmeg (4:2:2:1:1), while OLG contained GOS and MOS in equal proportions. EOB was added at 10 μL/g, and OPE, GOS, MOS, and OLG were included at 30 mg/g TMR. Rumen effluents were collected over 5 d for quantitative untargeted metabolome analysis. Analysis of rumen effluents using liquid chromatography-mass spectrometry and MetaboAnalyst 5.0 revealed 495 detected and identified metabolites. Volcano plot analysis showed no significant differences (P ≥ 0.05) in metabolite abundance between the CON and MEO, GEO, or OLEO treatments. Partial least squares-discriminant analysis (PLS-DA) also indicated no clear separation between the CON and treatment groups, suggesting no significant alteration in the metabolome. Compared with CON, the relative concentrations of dopamine 4-O-glucuronide, proline, and histamine were less while pyrocatechol was greater in MEO group. Isomer 1 of 2-hydroxyhepta-2,4-dienedioic acid, histamine, and dopamine 4-O-glucuronide were less while 2-aminomuconic acid and isomer 1 of hulupinic acid were greater in the GEO group. Histamine was less and hydroxypyridine was greater in the OLEO group. Phosphonate and phosphinate metabolism tended to be upregulated in OLEO. Meanwhile, metabolic pathways related to amino acids, vitamins, nitrogen metabolism, and primary bile acid biosynthesis remained unaltered by MEO, GEO and OLEO treatments. In summary, the study demonstrated that the inclusion of MEO, GEO, and OLEO had no adverse effects on rumen metabolites. Future research should focus on combinations of nutraceuticals that could positively impact nutrient metabolism and production performance of dairy cows. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of two brown Icelandic seaweed samples (Ascophyllum nodosum and Fucus vesiculosus) on in vitro methane production, nutrient degradation, and microbiota composition. A total mixed ration (TMR) was incubated alone as control or together with each seaweed at two inclusion levels (2.5 and 5.0% on a dry matter basis) in a long-term rumen simulation technique (Rusitec) experiment. The incubation period lasted 14 days, with 7 days of adaptation and sampling. The methane concentration of total gas produced was decreased at the 5% inclusion level of A. nodosum and F. vesiculosus by 8.9 and 3.6%, respectively (P < 0.001). The total gas production was reduced by all seaweeds, with a greater reduction for the 5% seaweed inclusion level (P < 0.001). Feed nutrient degradation and the production of volatile fatty acids and ammonia in the effluent were also reduced, mostly with a bigger effect for the 5% inclusion level of both seaweeds, indicating a reduced overall fermentation (all P ≤ 0.001). Microbiota composition was analyzed by sequencing 16S rRNA amplicons from the rumen content of the donor cows, fermenter liquid and effluent at days 7 and 13, and feed residues at day 13. Relative abundances of the most abundant methanogens varied between the rumen fluid used for the start of incubation and the samples taken at day 7, as well as between days 7 and 13 in both fermenter liquid and effluent (P < 0.05). According to the differential abundance analysis with q2-ALDEx2, in effluent and fermenter liquid samples, archaeal and bacterial amplicon sequence variants were separated into two groups (P < 0.05). One was more abundant in samples taken from the treatment without seaweed supplementation, while the other one prevailed in seaweed supplemented treatments. This group also showed a dose-dependent response to seaweed inclusion, with a greater number of differentially abundant members between a 5% inclusion level and unsupplemented samples than between a 2.5% inclusion level and TMR. Although supplementation of both seaweeds at a 5% inclusion level decreased methane concentration in the total gas due to the high iodine content in the seaweeds tested, the application of practical feeding should be done with caution. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of two brown Icelandic seaweed samples (Ascophyllum nodosum and Fucus vesiculosus) on in vitro methane production, nutrient degradation, and microbiota composition. A total mixed ration (TMR) was incubated alone as control or together with each seaweed at two inclusion levels (2.5 and 5.0% on a dry matter basis) in a long-term rumen simulation technique (Rusitec) experiment. The incubation period lasted 14 days, with 7 days of adaptation and sampling. The methane concentration of total gas produced was decreased at the 5% inclusion level of A. nodosum and F. vesiculosus by 8.9 and 3.6%, respectively (P < 0.001). The total gas production was reduced by all seaweeds, with a greater reduction for the 5% seaweed inclusion level (P < 0.001). Feed nutrient degradation and the production of volatile fatty acids and ammonia in the effluent were also reduced, mostly with a bigger effect for the 5% inclusion level of both seaweeds, indicating a reduced overall fermentation (all P ≤ 0.001). Microbiota composition was analyzed by sequencing 16S rRNA amplicons from the rumen content of the donor cows, fermenter liquid and effluent at days 7 and 13, and feed residues at day 13. Relative abundances of the most abundant methanogens varied between the rumen fluid used for the start of incubation and the samples taken at day 7, as well as between days 7 and 13 in both fermenter liquid and effluent (P < 0.05). According to the differential abundance analysis with q2-ALDEx2, in effluent and fermenter liquid samples, archaeal and bacterial amplicon sequence variants were separated into two groups (P < 0.05). One was more abundant in samples taken from the treatment without seaweed supplementation, while the other one prevailed in seaweed supplemented treatments. This group also showed a dose-dependent response to seaweed inclusion, with a greater number of differentially abundant members between a 5% inclusion level and unsupplemented samples than between a 2.5% inclusion level and TMR. Although supplementation of both seaweeds at a 5% inclusion level decreased methane concentration in the total gas due to the high iodine content in the seaweeds tested, the application of practical feeding should be done with caution.

Experimental data on the effects of cleavers (Galium aparine) and yarrow (Achillea millefolium) extracts on rumen microbial fermentation are scarce. The objective of this study was to determine the effects of cleavers and yarrow extracts on in vitro ruminal fermentation. Incubation trial was carried out using the long-term rumen simulation technique (RUSITEC). The experiment lasted 10 days. After an adaptation period of 5 days, the fermentation vessels divided into 3 groups; first three vessels received no additives (control), second three vessels received 500 mg/L cleavers extract daily, and third three vessels received 500 mg/L yarrow extract daily. Supplementations of cleavers and yarrow extracts had no significant effect on ruminal pH, total volatile fatty acids (VFA), acetate, propionate and methane production, NH3-N concentration and, total protozoa. However, both extracts decreased dry matter digestibility (DMD) (P

The aim of this study was to distinguish effects due to diet composition from those triggered by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system (RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined. Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal grain, 32% hay) diets were more digestible (p < 0.05) than high forage (10% cereal grain, 78% hay) diets, resulting in a greater (p < 0.05) formation of most fermentation end-products and microbial protein in the rumen. However, there were no significant (p > 0.05) differences between diets in methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of oleic acid, and relative proportion of conjugated linoleic acid were increased (p < 0.05) with high forage diets. A decline in rumen pH from 6.8 to 6.4 decreased (p < 0.05) feed digestibility, protein degradability, and the daily outputs of some fermentation end-products (gas, VFA, acetate, ammonia) but had no effect (p > 0.05) on the synthesis of microbial protein, and on the output of methane, propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid relative proportions were observed within this pH range. The overall multivariate analysis revealed a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined pH effects on ruminal fermentation and microbial communities.

The physiological function of the reticulorumen plays an essential role in ruminant nutrition, and detailed knowledge of rumen motility can further advance understanding of ruminant nutrition and physiology. Rumen motility was simulated by setting different stirrer rotation speeds in a rumen simulation technique (RUSITEC) system. The aim of this study was to investigate the effects of rotation speeds on rumen fermentation, saturation factor of dissolved gases, hydrogen (H 2) and methane (CH 4) emissions, microbial protein synthesis, and selected microbial population using RUSITEC. The experiment was performed according to a balanced 3 × 3 Latin square design, and each period included 7 d for adaptation and 3 d for sampling. Three motility treatments included 5, 15, and 25 rpm rotation speeds. Daily total gas and H 2 and CH 4 emissions had quadratic responses to the increasing rotation speed and were highest at 15 rpm. Quadratic and linear responses (highest at 5 rpm) to increasing rotation speed were observed for saturation factors of H 2 and CH 4 , liquid-dissolved H 2 and CH 4 concentrations, and headspace concentration of H 2 in the gas phase, whereas increasing rotation speed linearly decreased saturation factors of CO 2 and liquid-dissolved CO 2 concentration. Quadratic and linear responses to increasing rotation speed were observed for molar percentages of acetate, ammonia, and microbial protein concentration, whereas increasing rotation speed quadratically increased pH and decreased total volatile fatty acid concentration and acetate-to-propionate ratio. The 15-rpm rotation speed had the highest values of total volatile fatty acids, acetate molar percentage, and microbial protein concentration. Quadratic and linear responses to increasing rotation speed were observed for copy numbers of solid-associated fungi and fluid-associated bacteria, fungi, and protozoa, while increasing rotation speed linearly increased copy numbers of solid-associated protozoa. Rotation at 15 rpm increased populations of fungi and protozoa in the solid rumen contents and the population of bacteria and fungi in the liquid rumen contents. In summary, this study provides insights on the biofunction of proper rumen motility (i.e., at a rotation speed of 15 rpm), such as improving feed fermentation, increasing gas emissions with decreased dissolved gas concentrations and saturation factors, and promoting microbial colonization and microbial protein synthesis, although further increase in rotation speed (i.e., to 25 rpm) decreases feed fermentation and microbial protein synthesis. [ABSTRACT FROM AUTHOR]

Methane emissions by ruminants contribute to global warming and result in a loss of dietary energy for the animals. One possibility of reducing methane emissions is by dietary strategies. In the present trial, we investigated the long-term effects of Mootral, a feed additive consisting of garlic powder (Allium sativum) and bitter orange extracts (Citrus aurantium), on fermentation parameters and the microbial community in the rumen simulation technique (RUSITEC) system. The experiment lasted 38 days and was divided into three phases: an equilibration period of 7 days, a baseline period (BL) of 3 days, and experimental period (EP) of 28 days. Twelve fermentation vessels were divided into three groups (n = 4): control (CON), short-term (ST), and long-term (LT) application. From day 11 to day 27, 1.7 g of Mootral was added to the ST vessels; LT vessels received 1.7 g of Mootral daily for the entire EP. With the onset of Mootral application, methane production was significantly reduced in both groups until day 18. Thereafter, the production rate returned to the initial quantity. Furthermore, the short chain fatty acid fermentation profile was significantly altered by Mootral application; the molar proportion of acetate decreased, while the proportions of propionate and butyrate increased. Metabolomic analysis revealed further changes in metabolite concentrations associated with the Mootral supplementation period. The methyl coenzyme-M reductase gene copy number was reduced in the liquid and solid phase, whereas the treatment did not affect the abundance of bacteria. At the end of the BL, Methanomicrobia was the most abundant archaeal class. Mootral supplementation induced an increase in the relative abundance of Methanomassiliicoccales and a reduction in the relative abundance of Methanomicrobia, however, this effect was transient. Abundances of bacterial families were only marginally altered by the treatment. In conclusion, Mootral has the transient ability to reduce methane production significantly due to a selective effect on archaea numbers and archaeal community composition with little effect on the bacterial community.

Methane emissions by ruminants contribute to global warming and result in a loss of dietary energy for the animals. One possibility of reducing methane emissions is by dietary strategies. In the present trial, we investigated the long-term effects of Mootral, a feed additive consisting of garlic powder (Allium sativum) and bitter orange extracts (Citrus aurantium), on fermentation parameters and the microbial community in the rumen simulation technique (RUSITEC) system. The experiment lasted 38 days and was divided into three phases: an equilibration period of 7 days, a baseline period (BL) of 3 days, and experimental period (EP) of 28 days. Twelve fermentation vessels were divided into three groups (n = 4): control (CON), short-term (ST), and long-term (LT) application. From day 11 to day 27, 1.7 g of Mootral was added to the ST vessels; LT vessels received 1.7 g of Mootral daily for the entire EP. With the onset of Mootral application, methane production was significantly reduced in both groups until day 18. Thereafter, the production rate returned to the initial quantity. Furthermore, the short chain fatty acid fermentation profile was significantly altered by Mootral application; the molar proportion of acetate decreased, while the proportions of propionate and butyrate increased. Metabolomic analysis revealed further changes in metabolite concentrations associated with the Mootral supplementation period. The methyl coenzyme-M reductase gene copy number was reduced in the liquid and solid phase, whereas the treatment did not affect the abundance of bacteria. At the end of the BL, Methanomicrobia was the most abundant archaeal class. Mootral supplementation induced an increase in the relative abundance of Methanomassiliicoccales and a reduction in the relative abundance of Methanomicrobia, however, this effect was transient. Abundances of bacterial families were only marginally altered by the treatment. In conclusion, Mootral has the transient ability to reduce methane production significantly due to a selective effect on archaea numbers and archaeal community composition with little effect on the bacterial community. [ABSTRACT FROM AUTHOR]

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The objective of this study was to evaluate the effect of dried distillers grains with solubles (DDGS) and red-osier dogwood (ROD) extract on in vitro fermentation characteristics, nutrient disappearance, and microbial profiles using the rumen simulation technique. The experiment was a completely randomized design with a 2 × 2 factorial arrangement of treatments and four replicates per treatment. A basal diet [10% barley silage, 87% dry-rolled barley grain, and 3% vitamin and mineral supplement, dry matter (DM) basis] and a DDGS diet (as per basal diet with 25% of wheat DDGS replacing an equal portion of barley grain) were supplemented with ROD extract at 0 and 1% (DM basis), respectively. The experimental period was 17 d, consisting 10 days of adaptation and 7 days of data and sample collection. The substitution of wheat DDGS for barley grain did not affect gas production; disappearances of DM, organic matter, and crude protein; total volatile fatty acid (VFA) production; and microbial protein production. However, replacing barley grain with wheat DDGS increased (P = 0.01) fermenter pH and molar proportion of branched-chain VFA, switched (P = 0.06) the fermentation pattern to higher acetate production due to increased (P = 0.01) disappearance of neutral detergent fiber (NDF), and decreased (P = 0.08) methane (CH4) production. In the basal barley diet, the ROD extract increased the acetate to propionate (A:P) ratio (P = 0.08) and reduced the disappearance of starch (P = 0.06) with no effect on any other variables. No effects of ROD in the DDGS diet were observed. The number of operational taxonomic unit (OTUs) and the Shannon diversity index of the microbial community had little variation among treatments. Taxonomic analysis revealed no effect of adding the ROD extract on the relative abundance of bacteria at the phylum level with either the basal diet or DDGS diet, while at the genus level, the microbial community was affected by the addition of both DDGS and the ROD extract. Prevotella and Fibrobacter were the most abundant genera in the basal diet; however, Treponema became the most abundant genus with the addition of the ROD extract. These results indicated that the substitution of wheat DDGS for barley grain may mitigate enteric CH4 emissions. The trend of reduced starch fermentability and increased NDF disappearance with the addition of ROD extract suggests a reduced risk of rumen acidosis and an improvement in the utilization of fiber for cattle-fed high-grain diet.

This study aimed to determine the influence of sorghum ensiled with unsalable pumpkin at 20 or 40% dry matter (DM) basis on rumen fermentation characteristics and rumen microbial communities using the rumen simulation technique (RUSITEC). The experiment used a completely randomised design including silages comprising (1) 100% sorghum; (2) 80% sorghum + 20% DM pumpkin; or (3) 60% sorghum + 40% DM pumpkin. Each RUSITEC run (n = 2) was 15 d long, including 6 d of adaptation and 9 d of sampling. Dry matter digestibility (DMD) was measured on d 8 and 10–13. Gas production was measured daily, whereas methane and volatile fatty acids (VFA) production were measured from d 7–15. Solid-associated microbes (SAM) were collected on d 5, 10 and 15, whereas liquid-associated microbes (LAM) were collected after 15-d incubation. The V4 region of the 16S rRNA gene and the ITS1 region were sequenced to identify archaeal, bacterial and fungal communities. Ensiling 40% DM pumpkin with sorghum increased DMD and decreased the ratio of acetate to propionate (P ≤ 0.01). Both bacterial SAM and LAM communities were dominated by Megasphaera, and had the highest relative abundance (P = 0.03) with 40% DM pumpkin after 5 d incubation in the SAM community, while species of the Aspergillus genus dominated fungal SAM and LAM communities with 20 or 40% DM unsalable pumpkin. Therefore, ensiling up to 40% DM unsalable pumpkin with sorghum produces a high-quality ruminant feed with minimal influence on the rumen microbial population. Key points: • Including 40% DM unsalable pumpkin decreased acetate:propionate • Ensiling unsalable pumpkin with sorghum increases digestibility in a RUSITEC • Rumen microbial communities were slightly influenced by unsalable pumpkin inclusion [ABSTRACT FROM AUTHOR]

Plant based by-products (BP) produced from food and bioethanol industries are human inedible, but can be recycled into the global food chain by ruminant livestock. However, limited data is available on the methanogenesis potential associated with supplementing a solely BP formulated concentrate to a pastoral based diet. Therefore the objective of this in vitro study was to investigate the effects of BP inclusion rate (in a formulated concentrate) to a pasture based diet on dietary digestibility, rumen fermentation patterns, methane production and the prokaryotic microbial community composition. Diets consisted of perennial ryegrass and one of two supplementary concentrates, formulated to be isonitrogenous (16% CP) and isoenergetic (12.0 MJ/ME/kg), containing either 35% BP, barley and soybean meal (BP35) or 95% BP (BP95) offered on a 50:50 basis, however, starch, NDF and fat content varied. The BPs, included in equal proportions on a DM basis, were soyhulls, palm kernel expeller and maize dried distillers grains. The BP35 diet had greater (P < 0.05) digestibility of the chemical constituents DM, OM, CP, NDF, ADF. Greater total VFA production was seen in the BP35 diet (P < 0.05). Daily methane production (mmol/day; +22.7%) and methane output per unit of total organic matter digested (MPOMD; +20.8%) were greatest in the BP35 diet (P < 0.01). Dietary treatment influenced microbial composition (PERMANOVA; P = 0.023) with a greater relative abundance of Firmicutes (adj P < 0.01) observed in the BP35. The Firmicutes:Bacteroidetes ratio was significantly reduced in the BP95 diet (P < 0.01). The relative proportions of Proteobacteria (adj P < 0.01), Succinivibrionaceae (adj P < 0.03) and Succinivibrio (adj P = 0.053) increased in the BP95 diet. The abundance of Proteobacteria was found to be negatively associated with daily methane production (rs, −0.71; P < 0.01) and MPOMD (rs, −0.65; P < 0.01). Within Proteobacteria, the relationship of methane production was maintained with the mean abundance of Succinivibrio (rs, −0.69; P < 0.01). The abundance of the Firmicutes phyla was found to be positively correlated with both daily methane production (rs, 0.79; P < 0.001) and MPOMD (rs, 0.75; P < 0.01). Based on in vitro rumen simulation data, supplementation of an exclusively BP formulated concentrate was shown to reduce daily methane output by promoting a favorable alteration to the rumen prokaryotic community.

Abstract Microbial degradation in the rumen and dietary availability of methionine amino acid have been reported as limiting in dairy ruminants. The aim of the present study was to examine the post-ruminal effects of feeding ruminants different concentrations of rumen-protected methionine (RPM) in low crude protein diets using the long-term rumen simulation method (Rusitec) followed by in vitro abomasum and ileum digestibility technique. The experiment contained four treatment groups: (1) high protein, without RPM supplementation (HP); (2) low protein, without RPM supplementation (LP); (3) low protein supplementation with low RPM (LPLM); and (4) low protein supplementation with high RPM (LPHM) mixed per 20 ± 0.04 g basal diet in every fermenter. The results showed that the LPLM and LPHM groups had significantly higher disappearance of crude protein and neutral detergent fiber in the abomasum and ileum than the HP treatment (P 0.05). The proportions of short-chain fatty acids and total volatile fatty acids in the abomasum and ileum were the same between the LPHM and HP groups (P > 0.05); however, the LPLM group was found to be significantly (P < 0.05) lower than the HP group and similar to the LP group (P > 0.05). Rusitec pH before or after changing feed bags and daily ammonia nitrogen production in the abomasum and ileum were non significantly (P > 0.05) different among all groups. In conclusion, RPM supplementation with low crude protein diets promoted post-ruminal digestibility and production of volatile fatty acids.

Background: Brewers' spent grain (BSG) typically contains 20% – 29% crude protein (CP) with high concentrations of glutamine, proline and hydrophobic and non-polar amino acid, making it an ideal material for producing value-added products like bioactive peptides which have antioxidant properties. For this study, protein was extracted from BSG, hydrolyzed with 1% alcalase and flavourzyme, with the generated protein hydrolysates (AlcH and FlaH) showing antioxidant activities. This study evaluated the effects of AlcH and FlaH on gas production, ruminal fermentation characteristics, nutrient disappearance, microbial protein synthesis and microbial community using an artificial rumen system (RUSITEC) fed a high-grain diet. Results: As compared to the control of grain only, supplementation of FlaH decreased (P < 0.01) disappearances of dry matter (DM), organic matter (OM), CP and starch, without affecting fibre disappearances; while AlcH had no effect on nutrient disappearance. Neither AlcH nor FlaH affected gas production or VFA profiles, however they increased (P < 0.01) NH3-N and decreased (P < 0.01) H2 production. Supplementation of FlaH decreased (P < 0.01) the percentage of CH4 in total gas and dissolved-CH4 (dCH4) in dissolved gas. Addition of monensin reduced (P < 0.01) disappearance of nutrients, improved fermentation efficiency and reduced CH4 and H2 emissions. Total microbial nitrogen production was decreased (P < 0.05) but the proportion of feed particle associated (FPA) bacteria was increased with FlaH and monensin supplementation. Numbers of OTUs and Shannon diversity indices of FPA microbial community were unaffected by AlcH and FlaH; whereas both indices were reduced (P < 0.05) by monensin. Taxonomic analysis revealed no effect of AlcH and FlaH on the relative abundance (RA) of bacteria at phylum level, whereas monensin reduced (P < 0.05) the RA of Firmicutes and Bacteroidetes and enhanced Proteobacteria. Supplementation of FlaH enhanced (P < 0.05) the RA of genus Prevotella, reduced Selenomonas, Shuttleworthia, Bifidobacterium and Dialister as compared to control; monensin reduced (P < 0.05) RA of genus Prevotella but enhaced Succinivibrio. Conclusions: The supplementation of FlaH in high-grain diets may potentially protect CP and starch from ruminal degradation, without adversely affecting fibre degradation and VFA profiles. It also showed promising effects on reducing CH4 production by suppressing H2 production. Protein enzymatic hydrolysates from BSG using flavourzyme showed potential application to high value-added bio-products. [ABSTRACT FROM AUTHOR]

The effects of rice bran and corn on ensiling characteristics of potato vines were investigated. Ruminal fermentation of potato vine silage was measured using a rumen simulation technique (Rusitec). Potato vines were treated (fresh weight basis) for ensiling without additives (control), with 15% rice bran and with 15% rice bran plus 3% corn, 5% corn, or 7% corn, respectively. Silage with 5% corn was chosen for a ruminal fermentation trial, based on the results of ensiling quality. Comparison of rumen fermentation between two silage (silage with 5% corn and whole corn silage) diets was conducted using a Rusitec apparatus. Adding rice bran and corn improved the potato vine silage quality (P <.05). Compared with whole corn silage, potato vine silage diet had lower (P <.05) methane production, ruminal pH, acetate, and acetate to propionate ratio and higher (P <.05) total volatile fatty acids, total nitrogen (N), ammonia-N, and microbial-N. These findings indicated that rice bran and corn improved ensiling fermentation characteristics of potato vines, and that additive-treated potato vine silage had no adverse effects on rumen fermentation and could improve the energy and N utilisation and alleviate the methane pollution of the rumen compared with whole corn silage. [ABSTRACT FROM AUTHOR]

Experimental data on the effects of cleavers (Galium aparine) and yarrow (Achillea millefolium) extracts on rumen microbial fermentation are scarce. The objective of this study was to determine the effects of cleavers and yarrow extracts on in vitro ruminal fermentation. Incubation trial was carried out using the long-term rumen simulation technique (RUSITEC). The experiment lasted 10 days. After an adaptation period of 5 days, the fermentation vessels divided into 3 groups; first three vessels received no additives (control), second three vessels received 500 mg/L cleavers extract daily, and third three vessels received 500 mg/L yarrow extract daily. Supplementations of cleavers and yarrow extracts had no significant effect on ruminal pH, total volatile fatty acids (VFA), acetate, propionate and methane production, NH3-N concentration and, total protozoa. However, both extracts decreased dry matter digestibility (DMD) (P<0.05). Butyrate production, on the other hand, increased with cleavers extract (P<0.05). In conclusion, there were only small effects of cleavers and yarrow extracts on the investigated microbial fermentation characteristics. Nevertheless, it may be considered advantageous for feed conversion that plant extracts did not suppress ruminal fermentation in spite of decreasing DMD. [ABSTRACT FROM AUTHOR]

Alfalfa (Medicago sativa L.) silage (AS) is an important feedstuff in ruminant nutrition. However, its high non-protein nitrogen content often leads to poor ruminal nitrogen retention. Various pre-ensiling treatments differing with respect to dry matter concentrations, wilting intensities and sucrose addition have been previously shown to improve the quality and true protein preservation of AS, and have substantial effects on in vitro ruminal fermentation of the resulting silages. However, it is unknown how these pre-ensiling treatments affect the ruminal microbiota composition, and whether alterations in the microbiota explain previously observed differences in ruminal fermentation. Therefore, during AS incubation in a rumen simulation system, liquid and solid phases were sampled 2 and 7 days after first incubating AS, representing an early (ET) and late (LT) time point, respectively. Subsequently, DNA was extracted and qPCR (bacteria, archaea, and anaerobic fungi) and prokaryotic 16S rRNA gene amplicon sequence analyses were performed. At the ET, high dry matter concentration and sucrose addition increased concentrations of archaea in the liquid phase (P = 0.001) and anaerobic fungi in the solid phase (P < 0.001). At the LT, only sucrose addition increased archaeal concentration in the liquid phase (P = 0.014) and anaerobic fungal concentration in the solid phase (P < 0.001). Bacterial concentrations were not affected by pre-ensiling treatments. The prokaryotic phylogenetic diversity index decreased in the liquid phase from ET to LT (P = 0.034), whereas the solid phase was not affected (P = 0.060). This is suggestive of a general adaption of the microbiota to the soluble metabolites released from the incubated AS, particularly regarding the sucrose-treated AS. Redundancy analysis of the sequence data at the genus level indicated that sucrose addition (P = 0.001), time point (P = 0.001), and their interaction (P = 0.001) affected microbial community composition in both phases. In summary, of the pre-ensiling treatments tested sucrose addition had the largest effect on the microbiota, and together with sampling time point affected microbiota composition in both phases of the rumen simulation system. Thus, microbiota composition analysis helped to understand the ruminal fermentation patterns, but could not fully explain them.

Methane (CH4) from ruminal feed degradation is a major pollutant from ruminant livestock, which calls for mitigation strategies. The purpose of the present 4 × 2 factorial arrangement was to investigate the dose–response relationships between four doses of the CH4 inhibitor 3-nitrooxypropanol (3-NOP) and potential synergistic effects with low (LC) or high (HC) concentrate feed proportions (CFP) on CH4 reduction as both mitigation approaches differ in their mode of action (direct 3-NOP vs. indirect CFP effects). Diet substrates and 3-NOP were incubated in a rumen simulation technique to measure the concentration and production of volatile fatty acids (VFA), fermentation gases as well as substrate disappearance. Negative side effects on fermentation regarding total VFA and gas production as well as nutrient degradability were observed for neither CFP nor 3-NOP. CH4 production decreased from 10% up to 97% in a dose-dependent manner with increasing 3-NOP inclusion rate (dose: p < 0.001) but irrespective of CFP (CFP × dose: p = 0.094). Hydrogen gas accumulated correspondingly with increased 3-NOP dose (dose: p < 0.001). In vitro pH (p = 0.019) and redox potential (p = 0.066) varied by CFP, whereas the latter fluctuated with 3-NOP dose (p = 0.01). Acetate and iso-butyrate (mol %) decreased with 3-NOP dose, whereas iso-valerate increased (dose: p < 0.001). Propionate and valerate varied inconsistently due to 3-NOP supplementation. The feed additive 3-NOP was proven to be a dose-dependent yet effective CH4 inhibitor under conditions in vitro. The observed lack of additivity of increased CFP on the CH4 inhibition potential of 3-NOP needs to be verified in future research testing further diet types both in vitro and in vivo.

The aim of this study was to investigate the effects of dry extracts of Urtica dioica, Matricaria chamomilla, and Vitex agnus-castus with high phenolic contents on rumen microbial fermentation as compared with those of monensin, a common ionophore antibiotic, using Rumen Simulation Technique (RUSITEC) under normal and acidosis conditions. The treatments were as follows: negative control (no additive), positive control (5 mg/d monensin), and extracts of U. dioica (500 mg/d), M. chamomilla (500 mg/d), and V. agnus-castus (500 mg/d). Neither the plant extracts nor monensin altered the ruminal pH under normal or acidosis conditions. All the treatments affected total volatile fatty acid (VFA) production, propionate production, and dry matter digestibility (DMD), regardless of the fermentation conditions. All three extracts increased (P<0.05) total VFA production similar to that observed with monensin (P<0.05). M. chamomilla and V. agnus-castus increased propionate production and DMD similar to that obtained with monensin (P<0.05). In contrast to the monensin treatment, all three extracts increased acetate production under normal conditions (P<0.05). Under acidosis conditions, acetate production remained unchanged in the U. dioica and V. agnus-castus treatments, as well as in the monensin treatment. Under both conditions, the acetate-to-propionate (A:P) ratio decreased only in the monensin treatment (P<0.05). U. dioica and M. chamomilla had antiprotozoal effects (P<0.05) similar to those of monensin, regardless of the condition. The NH3-N concentration declined only in the V. agnus-castus treatment under acidosis conditions (P<0.05). Similar to the monensin treatment, lactate concentrations remained unchanged in the V. agnus-castus treatment under both conditions. In conclusion, plant extracts stimulated fermentative activity of rumen microorganisms under normal and acidosis conditions. Although they did not improve ruminal pH, U. dioica and V. agnus-castus extracts had more favorable effects on some fermentation parameters under acidosis conditions. [ABSTRACT FROM AUTHOR]

Beef cattle partition dietary nitrogen (N) into meat or excrete it mainly in faeces and urine, which can contribute significantly to water and air pollution. The effects of two inorganic nitrogen (N) fertilizer application rates—15 (LN) or 80 (HN) kg N/ha—to Lolium perenne‐dominant swards in autumn, on herbage chemical composition, intake, digestion and N balance in beef cattle, and in vitro fermentation and methane production were studied. Four growing beef steers used in a 2 × 2 crossover design experiment were offered zero‐grazed grass harvested 21 days post‐N application between July and October. The same grasses were incubated in an eight‐vessel rumen simulation technique in a randomized complete block experiment. Grass dry‐matter (DM) concentration was 26 g/kg lower and crude protein (CP) concentration was 35 g/kg DM higher for HN compared to LN. There was no difference in herbage DM intake or in vivo DM digestibility between treatments. Nitrogen intake and the digestibility of N were higher for HN compared to LN. Total and urine N loss were 41 and 45 g/day greater, respectively, for HN compared to LN, but faecal N loss did not differ between treatments. The quantity of N retained and therefore N‐use efficiency did not differ between LN and HN (25% vs. 22%). In vitro rumen pH and ammonia‐N concentrations were higher for HN compared to LN, whereas volatile fatty acid concentrations and molar proportions did not differ between treatments. In vitro methane and total gas production were 0.9 mmol/day and 280 ml/day lower for HN than LN respectively. Under the particular conditions of this experiment, reducing fertilizer N application rate reduced total and urinary N excretion, which has potential environmental benefits; however, methane output measured in vitro was increased. [ABSTRACT FROM AUTHOR]

This study investigated the effect of treatment of wheat straw using ammonia fiber expansion (AFEX) and exogenous fibrolytic enzymes (Viscozyme) on fiber digestibility, rumen fermentation, microbial protein synthesis, and microbial populations in an artificial rumen system [Rumen Simulation Technique (RUSITEC)]. Four treatments were assigned to 16 vessels (4 per treatment) in 2 RUSITEC apparatuses in a randomized block design. Treatments were arranged as a 2 × 2 factorial using untreated or AFEX-treated wheat straw with or without exogenous fibrolytic enzymes [0 or 500 μg of protein/g straw dry matter (DM)]. Fibrolytic enzymes were applied to straw, prior to sealing in nylon bags. The concentrate mixture was provided in a separate bag within each fermentation vessel. The RUSITECs were adapted for 8 d and disappearance of DM, neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) was measured after 48 h of incubation. Ammonia fiber expansion increased (P < 0.01) the disappearance of wheat straw DM (69.6 vs. 38.3%), NDF (65.6 vs. 36.8%), ADF (61.4 vs. 36.0%), and CP (68.3 vs. 24.0%). Total dietary DM, organic matter (OM), and NDF disappearance was also increased (P ≤ 0.05) by enzymes. Total microbial protein production was greater (P < 0.01) for AFEX-treated (72.9 mg/d) than untreated straw (63.1 mg/d). Total gas and methane (CH4) production (P < 0.01) were also greater for AFEX-treated wheat straw than untreated straw, with a tendency for total gas to increase (P = 0.06) with enzymes. Ammonia fiber expansion increased (P < 0.01) total volatile fatty acid (VFA) production and the molar proportion of propionate, while it decreased (P < 0.01) acetate and the acetate-to-propionate ratio. The AFEX-treated straw had lower relative quantities of fungi, methanogens, and Fibrobacter succinogenes (P < 0.01) and fewer protozoa (P < 0.01) compared to untreated straw. The pH of fermenters fed AFEX-treated straw was lower (P < 0.01) than those fed untreated straw. Both AFEX (P < 0.01) and enzymes (P = 0.02) decreased xylanase activity. There was an enzyme × straw interaction (P = 0.02) for endoglucanase activity. Enzymes increased endoglucanase activity of AFEX-treated wheat straw, but had no effect on untreated straw. The addition of enzymes lowered the relative abundance of Ruminococcus flavefaciens , but increased F. succinogenes. These results indicate that AFEX increased the ruminal disappearance of wheat straw and improved fermentation and microbial protein synthesis in the RUSITEC. [ABSTRACT FROM AUTHOR]

This study investigated the effect of treatment of wheat straw using ammonia fiber expansion (AFEX) and exogenous fibrolytic enzymes (Viscozyme) on fiber digestibility, rumen fermentation, microbial protein synthesis, and microbial populations in an artificial rumen system [Rumen Simulation Technique (RUSITEC)]. Four treatments were assigned to 16 vessels (4 per treatment) in 2 RUSITEC apparatuses in a randomized block design. Treatments were arranged as a 2 × 2 factorial using untreated or AFEX-treated wheat straw with or without exogenous fibrolytic enzymes [0 or 500 μg of protein/g straw dry matter (DM)]. Fibrolytic enzymes were applied to straw, prior to sealing in nylon bags. The concentrate mixture was provided in a separate bag within each fermentation vessel. The RUSITECs were adapted for 8 d and disappearance of DM, neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) was measured after 48 h of incubation. Ammonia fiber expansion increased (P < 0.01) the disappearance of wheat straw DM (69.6 vs. 38.3%), NDF (65.6 vs. 36.8%), ADF (61.4 vs. 36.0%), and CP (68.3 vs. 24.0%). Total dietary DM, organic matter (OM), and NDF disappearance was also increased (P ≤ 0.05) by enzymes. Total microbial protein production was greater (P < 0.01) for AFEX-treated (72.9 mg/d) than untreated straw (63.1 mg/d). Total gas and methane (CH4) production (P < 0.01) were also greater for AFEX-treated wheat straw than untreated straw, with a tendency for total gas to increase (P = 0.06) with enzymes. Ammonia fiber expansion increased (P < 0.01) total volatile fatty acid (VFA) production and the molar proportion of propionate, while it decreased (P < 0.01) acetate and the acetate-to-propionate ratio. The AFEX-treated straw had lower relative quantities of fungi, methanogens, and Fibrobacter succinogenes (P < 0.01) and fewer protozoa (P < 0.01) compared to untreated straw. The pH of fermenters fed AFEX-treated straw was lower (P < 0.01) than those fed untreated straw. Both AFEX (P < 0.01) and enzymes (P = 0.02) decreased xylanase activity. There was an enzyme × straw interaction (P = 0.02) for endoglucanase activity. Enzymes increased endoglucanase activity of AFEX-treated wheat straw, but had no effect on untreated straw. The addition of enzymes lowered the relative abundance of Ruminococcus flavefaciens , but increased F. succinogenes. These results indicate that AFEX increased the ruminal disappearance of wheat straw and improved fermentation and microbial protein synthesis in the RUSITEC. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate the impact of essential oils (EO) from Lippia turbinata (LT) and Tagetes minuta (TM) as well as the rotation of both EO on fermentation parameters in vitro. Daily addition of LT, TM, or a 3-day rotation between them (TM/LT), as well as a control (without EO), was evaluated using the rumen simulation technique (Rusitec). The experiment lasted 19 days, with a 7-day adaptation period, followed by 12 days of treatment (Days 0–12). The EO were dissolved in ethanol (70% vol/vol) to be added daily to fermenters (300 μL/L) from Day 0. Daily measurements included methane concentration, total gas production, apparent DM disappearance and pH, which started 2 days before the addition of treatments. On Days 0, 4, 8 and 12 apparent crude protein disappearance and neutral detergent fibre disappearance, ammonia and volatile fatty acid concentration and composition were determined. Methane production was significantly inhibited shortly after addition of both EO added individually, and persisted over time with no apparent adaptation to EO addition. The TM/LT treatment showed a similar effect on methane production, suggesting that rotating the EO did not bring further improvements in reduction or persistency compared with the inclusion of the EO individually. Gas production, total volatile fatty acid concentration and composition and apparent crude protein disappearance were not affected by EO addition. Compared with the control, a 5% reduction of apparent DM disappearance and a 15% reduction of neutral detergent fibre disappearance were observed with the addition of EO. Only TM and TM/LT reduced ammonia concentration. Given the significant and persistent antimethanogenic activity of both EO, and the potential of T. minuta to modify nitrogen metabolism, EO from these plant species are of interest for developing new feed additives with potential application in ruminant nutrition that are also likely to be acceptable to consumers. Ruminants developed the ability to produce meat and milk by utilising feeds that humans cannot use. However, during ruminal fermentation of feed, methane is produced, which is a potent greenhouse gas and waste of energy. Strategies to decrease methane emissions from ruminants must be implemented and the use of natural products is likely to be more acceptable to the public than the use of in-feed antibiotics. The present work has demonstrated that essential oils from novel sources can decrease methane production by ruminal microbes in vitro. [ABSTRACT FROM AUTHOR]