Severity of Rolling Reconstituted High Moisture Barley on Ensiling Characteristics and In Vitro Ruminal Fermentation.

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 barley was rolled through a roller gap width of 1.40 (RHBF), 1.86 (RHBM), or 2.31 mm (RHBC) and 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 artificial rumen 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 dry rolled barley. 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, organic matter, 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 PI using a smaller roller gap width, yielded similar DM and OM disappearance suggesting a lesser risk for low ruminal pH without compromising digestibility.

Changes in the solid-associated bacterial and fungal communities following ruminal in vitro fermentation of winery by-products: aspects of the bioactive compounds and feed safety.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Sigla storax (Liquidambar orientalis) mitigates in vitro methane production without disturbances in rumen microbiota and nutrient fermentation in comparison to monensin.

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.

Characterization of microbial intolerances and ruminal dysbiosis towards different dietary carbohydrate sources using an in vitro model.

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.

Proper motility enhances rumen fermentation and microbial protein synthesis with decreased saturation of dissolved gases in rumen simulation technique.

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.

Ensiling sorghum with unsalable pumpkin improves feed digestibility with minimal influence on the rumen microbial population using the rumen simulation technique.

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.

Effects of supplemental plant oils on rumen bacterial community profile and digesta fatty acid composition in a continuous culture system (RUSITEC).

Lipid supplementation of ruminant diets may trigger changes in the ruminal microbiota and in anaerobic digestion. Changes in the bacterial community composition and in the fatty acid hydrogenation caused by the addition of different supplemental plant oils to a high concentrate diet were investigated in vitro using RUSITEC (rumen simulation technique) fermenters. The control (CTR) diet was a high-concentrate total mixed ration for dairy sheep, with no supplementary oil. The other experimental diets were supplemented with olive (OLV), sunflower (SFL) or linseed (LNS) oils at 6% (dry matter basis). Four RUSITEC fermenters were used for each experimental diet, all inoculated with rumen digesta of sheep. Extent of dry matter and fat degradation, composition of the bacterial community and long-chain fatty acids in digesta were determined. The addition of plant oils increased (P < 0.001) apparent degradation of fat in the fermenters, whereas fermentation kinetics (gas production and average fermentation rate) were lower (P < 0.05) with the LNS than with the CTR diet. Hydrogenation of C18 unsaturated fatty acids (P < 0.05), in particular that of oleic acid (P < 0.001), and stearic acid proportion (P < 0.001) were reduced, and oleic acid proportion was increased (P < 0.001) with all oil supplements. Addition of OLV decreased linoleic and LNS increased α-linolenic (P < 0.001), whereas conjugated linoleic was increased with SFL oil (P = 0.025) and vaccenic increased with both SFL and LNS oils (P = 0.008). Addition of 6% OLV and LNS reduced (P < 0.05) microbial community diversity and quantity of total bacteria relative to the control. Some specific microbial groups were affected (P < 0.001) by oil addition, with less relative abundance of Clostridiales and Actinobacteria and increased Bacteroidales, Aeromonadales and Lactobacillales species. In conclusion, the supplementation of high-concentrate ruminant diets with plant oils, in particular from sunflower or linseed, causes shifts in the rumen microbiota and fatty acid hydrogenation in the rumen increasing the formation of vaccenic and conjugated linoleic acids.

Effects of commercial aldehydes from green leaf volatiles (green odour) on rumen microbial population and fermentation profile in an artificial rumen (Rusitec).

The effects of plant metabolites on rumen metabolism vary greatly depending on their antimicrobial spectrum and applied doses. In this study, the minimum inhibitory concentrations (MICs) of commercial aldehydes, trans-2-hexenal (T2H), cis-3-hexenal (C3H), trans-2-nonenal (T2N), and trans-2-decenal (T2D) from green leaf volatiles, were tested on rumen bacteria. These compounds were found more effective on Gram-positive rumen bacteria than the Gram-negatives, and C3H was the most effective compound. Then, for 14 days, the in vitro effects of C3H compared with monensin (5 mg/day) on the rumen microbial population and ruminal fermentation at 187.5, 375 and 750 mg/day doses were tested based on the MIC value (500 µg/mL) by using the rumen simulation technique (Rusitec). Supplementation with C3H at 375 mg/day increased the cell numbers of Butyrivibrio fibrisolvens significantly. The addition of C3H at 375 and 750 mg/day doses also increased Streptecoccus bovis cell counts. The use of monensin did not affect the cell numbers of these bacteria. On the other hand, C3H did not change the counts of total bacteria, methanogens, or hyper-ammonia-producing (HAP) bacteria like monensin. The numbers of Ruminococcus albus and Ruminococcus flavefaciens were also stable in the presence of C3H but decreased significantly with the addition of monensin (P < 0.05). Fibrobacter succinogenes, Megasphaera elsdenii, and Selenomonas ruminantium cell counts were not affected by either application. In addition, C3H increased the acetate and methane production along with the acetate-to-propionate ratio at all tested concentrations, unlike monensin. Supplementation with C3H decreased propionate production significantly, except at the 187.5 mg/day dose. Butyrate production increased (P < 0.05) only in the presence of 187.5 and 375 mg/day doses of C3H. Production of total volatile fatty acids (VFA) and dry matter digestibility (DMD) did not change in treatment groups. Also, the total protozoa numbers and ammonia-N concentrations significantly decreased (P < 0.05) in C3H-treated samples, similar to monensin. Although C3H did not have favorable effects on energy efficiency, it suppressed rumen protozoa and mitigated rumen ammonia without adversely effecting ruminal fermentation in all applied doses. Based on the result, C3H has the potential to improve protein utilization in the rumen.

Effects of supplementing corn silage with different nitrogen sources on ruminal fermentation and microbial populations in vitro.

Compared to grass silage (GS)-, corn silage (CS)-based diets appear to increase the efficiency of microbial protein synthesis (EMPS) in the rumen. Opposite results for the EMPS obtained in vitro have raised the question of whether an inadequate supply of amino N for microbes might explain the low EMPS. We examined the effects of supplementation with different N sources in CS on the EMPS and microbial populations in vitro. GS and CS were used as substrates for in vitro incubation. CS was non-supplemented or supplemented with urea, mixed amino acids (AA), peptone, or protein to adjust the N content to that of GS. Degradation of organic matter (OM) and crude protein (CP) revealed a positive effect of all N supplements, except protein. Additionally, N supplementation increased fiber degradation of CS. Peptone primarily stimulated hemicellulolytic activity and urea stimulated cellulolytic activity. The EMPS of CS was improved by all N supplements, with peptone and urea exhibiting the highest increase (57% and 54%, respectively), followed by AA mix (40%) and protein (11%) compared to that of CS alone (111 g microbial CP kg-1 fermented OM). However, the level of EMPS detected with GS (200 g microbial CP kg-1 fermented OM) was not achieved. Protozoal 18S rRNA gene copy numbers were negatively correlated with the EMPS, whereas no correlation was found between total bacteria and the EMPS. A stimulating effect of urea, AA mix, and peptone was detected for Ruminococcus albus and Prevotella bryantii, whereas Fibrobacter succinogenes was inhibited by N supplementation. This indicated that neither the amount of available N nor the N source was the only limiting factor in the low EMPS values of CS in vitro. Information is also provided on the stimulating effects of different N sources on several microbial species in mixed rumen culture.

Graded replacement of maize grain with molassed sugar beet pulp modulated ruminal microbial community and fermentation profile in vitro.

BACKGROUND: Molassed sugar beet pulp (Bp) is a viable alternative to grains in cattle nutrition for reducing human edible energy input. Yet little is known about the effects of high inclusion rates of Bp on rumen microbiota. This study used an in vitro approach and the quantitative polymerase chain reaction technique to establish the effects of a graded replacement of maize grain (MG) by Bp on the ruminal microbial community, fermentation profile and nutrient degradation. RESULTS: Six different amounts of Bp (0-400 g kg-1 ), which replaced MG in the diet, were tested using the in vitro semi-continuous rumen simulation technique. The increased inclusion of Bp resulted in greater dietary content and degradation of neutral detergent fibre (P < 0.01). Further, Bp feeding enhanced (P < 0.01) the abundance of genus Prevotella and shifted (P < 0.01) the short-chain fatty acid patterns in favour of acetate and propionate and at the expense of butyrate. A total replacement of MG with Bp resulted in an increased daily methane production (P < 0.01). CONCLUSION: Results suggest positive effects of the replacement of MG by Bp especially in terms of stimulating ruminal acetate and propionate fermentation. However, high replacement rates of Bp resulted in lowered utilization of ammonia and higher ruminal methane production. © 2017 Society of Chemical Industry.

Rumen microbial abundance and fermentation profile during severe subacute ruminal acidosis and its modulation by plant derived alkaloids in vitro.

Rumen microbiota have important metabolic functions for the host animal. This study aimed at characterizing changes in rumen microbial abundances and fermentation profiles using a severe subacute ruminal acidosis (SARA) in vitro model, and to evaluate a potential modulatory role of plant derived alkaloids (PDA), containing quaternary benzophenanthridine and protopine alkaloids, of which sanguinarine and chelerythrine were the major bioactive compounds. Induction of severe SARA strongly affected the rumen microbial composition and fermentation variables without suppressing the abundance of total bacteria. Protozoa and fungi were more sensitive to the low ruminal pH condition than bacteria. Induction of severe SARA clearly depressed degradation of fiber (P < 0.001), which came along with a decreased relative abundance of fibrolytic Ruminococcus albus and Fibrobacter succinogenes (P < 0.001). Under severe SARA conditions, the genus Prevotella, Lactobacillus group, Megasphaera elsdenii, and Entodinium spp. (P < 0.001) were more abundant, whereas Ruminobacter amylophilus was less abundant. SARA largely suppressed methane formation (-70%, P < 0.001), although total methanogenic 16S rRNA gene abundance was not affected. According to principal component analysis, Methanobrevibacter spp. correlated to methane concentration. Addition of PDA modulated ruminal fermentation under normal conditions such as enhanced (P < 0.05) concentration of total SCFA, propionate and valerate, and increased (P < 0.05) degradation of crude protein compared with the unsupplemented control diet. Our results indicate strong shifts in the microbial community during severe SARA compared to normal conditions. Supplementation of PDA positively modulates ruminal fermentation under normal ruminal pH conditions.

Substitution of common concentrates with by-products modulated ruminal fermentation, nutrient degradation, and microbial community composition in vitro.

A rumen simulation technique was used to evaluate the effects of the complete substitution of a common concentrate mixture (CON) with a mixture consisting solely of by-products from the food industry (BP) at 2 different forage-to-concentrate ratios on ruminal fermentation profile, nutrient degradation, and abundance of rumen microbiota. The experiment was a 2×2 factorial arrangement with 2 concentrate types (CON and BP) and 2 concentrate levels (25 and 50% of diet dry matter). The experiment consisted of 2 experimental runs with 12 fermentation vessels each (n=6 per treatment). Each run lasted for 10d, with data collection on the last 5d. The BP diets had lower starch, but higher neutral detergent fiber (NDF) and fat contents compared with CON. Degradation of crude protein was decreased, but NDF and nonfiber carbohydrate degradation were higher for the BP diets. At the 50% concentrate level, organic matter degradation tended to be lower for BP and CH4 formation per unit of NDF degraded was also lower for BP. The BP mixture led to a higher concentration of propionate and a lower acetate-to-propionate ratio, whereas concentrations of butyrate and caproate decreased. Concentrate type did not affect microbial community composition, except that the abundance of bacteria of the genus Prevotella was higher for BP. Increasing the concentrate level resulted in higher degradation of organic matter and crude protein. At the higher concentrate level, total short-chain fatty acid formation increased and concentrations of isobutyrate and valerate decreased. In addition, at the 50% concentrate level, numbers of protozoa increased, whereas numbers of methanogens, anaerobic fungi, and fibrolytic bacteria decreased. No interaction was noted between the 2 dietary factors on most variables, except that at the higher concentrate level the effects of BP on CH4 and CO2 formation per unit of NDF degraded, crude protein degradation, and the abundance of Prevotella were more prominent. In conclusion, the results of this study suggest that BP in the diet can adequately substitute CON with regard to ruminal fermentation profile and microbiota, showing even favorable fermentation patterns when fed at 50% inclusion rate.

Treatment of grain with organic acids at 2 different dietary phosphorus levels modulates ruminal microbial community structure and fermentation patterns in vitro.

Recent data indicate positive effects of treating grain with citric (CAc) or lactic acid (LAc) on the hydrolysis of phytate phosphorus (P) and fermentation products of the grain. This study used a semicontinuous rumen simulation technique to evaluate the effects of processing of barley with 50.25 g/L (wt/vol) CAc or 76.25 g/L LAc on microbial composition, metabolic fermentation profile, and nutrient degradation at low or high dietary P supply. The low P diet [3.1g of P per kg of dry matter (DM) of dietary P sources only] was not supplemented with inorganic P, whereas the high P diet was supplemented with 0.5 g of inorganic P per kg of DM through mineral premix and 870 mg of inorganic P/d per incubation fermenter via artificial saliva. Target microbes were determined using quantitative PCR. Data showed depression of total bacteria but not of total protozoa or short-chain fatty acid (SCFA) concentration with the low P diet. In addition, the low P diet lowered the relative abundance of Ruminococcus albus and decreased neutral detergent fiber (NDF) degradation and acetate proportion, but increased the abundance of several predominantly noncellulolytic bacterial species and anaerobic fungi. Treatment of grain with LAc increased the abundance of total bacteria in the low P diet only, and this effect was associated with a greater concentration of SCFA in the ruminal fluid. Interestingly, in the low P diet, CAc treatment of barley increased the most prevalent bacterial group, the genus Prevotella, in ruminal fluid and increased NDF degradation to the same extent as did inorganic P supplementation in the high P diet. Treatment with either CAc or LAc lowered the abundance of Megasphaera elsdenii but only in the low P diet. On the other hand, CAc treatment increased the proportion of acetate in the low P diet, whereas LAc treatment decreased this variable at both dietary P levels. The propionate proportion was significantly increased by LAc at both P levels, whereas butyrate increased only with the low P diet. Treatments with CAc or LAc reduced the degradation of CP and ammonia concentration compared with the control diet at both P levels. In conclusion, the beneficial effects of CAc and LAc treatment on specific ruminal microbes, fermentation profile, and fiber degradation in the low P diet suggest the potential for the treatment to compensate for the lack of inorganic P supplementation in vitro. Further research is warranted to determine the extent to which the treatment can alleviate the shortage of inorganic P supplementation under in vivo conditions.

Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec.

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.

Metagenomic insights into the mechanistic differences of plant polyphenols and nitrocompounds in reducing methane emissions using the rumen simulation technique.

Methane (CH4) emissions from ruminants contribute significantly to greenhouse gas levels and also result in considerable feed energy losses. Plant polyphenols and nitrocompounds are two typical types of methane inhibitors. The study investigates the mechanistic differences between 2-nitroethanol (NE) and proanthocyanidins (PAC) in reducing methane emissions from ruminant livestock using the rumen simulation technique (RUSITEC) combined with metagenomic analyses. The experiment was performed as a complete randomized block design with 3 runs. Run was used as a blocking factor. The treatments included a control (CON) with no additive, NE at 0.5 g/kg dry matter (DM), and PAC at 20 g/kg DM, all incubated in vitro for 24 h (h) with eight replicates per treatment. The results showed that NE significantly reduced CH4 production by 94.9 % (P < 0.01) and total volatile fatty acid (TVFA) concentration by 11.1 % (P < 0.05) compared to the control. NE also decreased the acetate-to-propionate ratio (A/P) from 1.93 to 1.60 (P < 0.01), indicating a shift towards more efficient fermentation. In contrast, PAC reduced methane production by 11.7 % (P < 0.05) and decreased the A/P (P < 0.05) while maintaining microbial diversity and fermentation stability, with no significant impact on TVFA concentration (P > 0.05). Metagenomic analysis revealed that NE markedly suppressed the abundance of key genera involved in carbohydrate metabolism, including Prevotella and Bacteroides, leading to reduced acetate and butyrate pathways. NE also selectively inhibited methanogenic archaea, particularly Methanobrevibacter spp., which are integral to the hydrogenotrophic pathway (P < 0.01). On the other hand, PAC showed selective inhibition of Methanosphaera spp., targeting the methylotrophic pathway (P < 0.01). These findings provide valuable insights into the distinct microbial and metabolic pathways modulated by NE and PAC, offering potential strategies for developing effective dietary interventions to mitigate methane emissions in ruminant livestock.

In Vitro Evaluation of Chito-Oligosaccharides on Disappearance Rate of Nutrients, Rumen Fermentation Parameters, and Micro-Flora of Beef Cattle.

The study aimed to investigate the effect of dietary chitosan oligosaccharides (COS) meal levels on the nutrient disappearance rate, rumen fermentation, and microflora of beef cattle in vitro. A total of 24 fermentation tanks were randomly divided into four treatments containing 0% COS (CON), 0.02% COS, 0.04% COS, and 0.08% COS for an 8-day experiment period, with each treatment comprising six replicates. The disappear rates of DM, CP, EE, and total gas production were quadratically increased with increasing COS levels. The disappear rates of DM, CP, EE, and ADF were greatest, whereas the total gas production was lowest in the 0.08% COS group. The pH, NH3-N, MCP, the content of propionate, isobutyrate, butyrate, valerate, and the A/P were quadratically increased with increasing COS levels, while the A/P were linearly decreased. The pH, MCP, and the content of propionate, and butyrate were highest, whereas the NH3-N and the content of acetate, isobutyrate, valerate, and the A/P were lowest in the 0.08% COS group. Microbiomics analysis showed that the rumen microbial diversity was not altered between the CON and the 0.08% COS group. However, the relative abundance of Methanosphaera, Ruminococcus, Endomicrobium, and Eubacterium groups was increased, and the relative abundance of pathogenic bacteria Dorea and Escherichia-Shigella showed a decrease in the 0.08% COS group. Overall, the 0.08% COS was the most effective among the three addition levels, resulting in an increase in the disappearance rate of in vitro fermented nutrients and improvements in rumen fermentation indexes and microbial communities. This, in turn, led to the maintenance of rumen health.

Novel oxidising feed additives reduce in vitro methane emissions using the rumen simulation technique.

Enteric methane (CH4) produced by ruminant livestock is a potent greenhouse gas and represents significant energy loss for the animal. The novel application of oxidising compounds as antimethanogenic agents with future potential to be included in ruminant feeds, was assessed across two separate experiments in this study. Low concentrations of oxidising agents, namely urea hydrogen peroxide (UHP) with and without potassium iodide (KI), and magnesium peroxide (MgO2), were investigated for their effects on CH4 production, total gas production (TGP), volatile fatty acid (VFA) profiles, and nutrient disappearance in vitro using the rumen simulation technique. In both experiments, the in vitro diet consisted of 50:50 grass silage:concentrate on a dry matter basis. Treatment concentrations were based on the amount of oxygen delivered and expressed in terms of fold concentration. In Experiment 1, four treatments were tested (Control, 1× UHP + KI, 1× UHP, and 0.5× UHP + KI), and six treatments were assessed in Experiment 2 (Control, 0.5× UHP + KI, 0.5× UHP, 0.25× UHP + KI, 0.25× UHP, and 0.12× MgO2). All treatments in this study had a reducing effect on CH4 parameters. A dose-dependent reduction of TGP and CH4 parameters was observed, where treatments delivering higher levels of oxygen resulted in greater CH4 suppression. 1× UHP + KI reduced TGP by 28 % (p = 0.611), CH4% by 64 % (p = 0.075) and CH4 mmol/g digestible organic matter by 71 % (p = 0.037). 0.12× MgO2 reduced CH4 volume by 25 % (p > 0.05) without affecting any other parameters. Acetate-to-propionate ratios were reduced by treatments in both experiments (p < 0.01). Molar proportions of acetate and butyrate were reduced, while propionate and valerate were increased in UHP treatments. High concentrations of UHP affected the degradation of neutral detergent fibre in the forage substrate. Future in vitro work should investigate alternative slow-release oxygen sources aimed at prolonging CH4 suppression.

Effects of Garlic Oil and Cinnamaldehyde on Sheep Rumen Fermentation and Microbial Populations in Rusitec Fermenters in Two Different Sampling Periods.

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.

The effect of Asparagopsis taxiformis, Ascophyllum nodosum, and Fucus vesiculosus on ruminal methanogenesis and metagenomic functional profiles in vitro.

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.

Effects of hop varieties on ruminal fermentation and bacterial community in an artificial rumen (rusitec).

BACKGROUND: There is a growing interest in the use of hops (Humulus lupulus) as an alternative to antibiotics to manipulate ruminal fermentation. However, the effects of different hop varieties on ruminal fermentation and bacterial populations have not been studied. Here the effects of three hop varieties, Cascade (CAS), Millennium (MIL) and Teamaker (TM), at a level of 800 µg mL(-1) inoculum on ruminal fermentation and microbial populations in an artificial rumen system (rusitec) fed a barley silage-based total mixed ration were investigated. Bacterial populations were assessed using real-time polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies. RESULTS: All hops reduced (P < 0.001) total gas, methane and the acetate:propionate ratio. Liquid-associated Fibrobacter succinogenes, Ruminococcus albus and Streptococcus bovis were reduced (P < 0.05) by MIL and TM. Feed particle-associated S. bovis was reduced (P < 0.01) by MIL and TM, but TM and CAS increased (P < 0.01) Ruminobacter amylophilus and Prevotella bryantii respectively. Methanogens were decreased (P < 0.05) by MIL in both liquid and solid fractions and by CAS in the solid fraction. The total amount of α- and ß-acids in hops affected the ruminal fermentation. CONCLUSION: Hop-induced changes in fermentation and microbial populations may improve energy efficiency use in the rumen. Further research is needed to determine the effects of hops on in vivo ruminal fermentation, microbial populations and animal performance.