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Background Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H 2 ) + carbon dioxide and from H 2 + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H 2 that accumulates less than expected under inhibited methanogenesis. Results The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H 2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H 2 -producing bacteria to regulate the amount of H 2 production. No previously reported alternative H 2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. Conclusion By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H 2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. -iX6YPbp4qKjzQQ25nvbCQ Video Abstract

Livestock emits copious quantities of methane, a major constituent of the greenhouse gases currently driving climate change. Methanogens within the bovine rumen produce methane during the breakdown of feed. While the red seaweed Asparagopsis taxiformis (AT) can significantly reduce methane emissions when fed to cows, its effects appear short-lived. This study revealed that the effective reduction of methane emissions by AT was accompanied by the near-total elimination of methane-generating Methanosphaera . However, Methanosphaera populations subsequently rebounded due to their ability to inactivate bromoform, a major inhibitor of methane formation found in AT. This study presents novel findings on the contribution of Methanosphaera to ruminal methanogenesis, the mode of action of AT, and the possibility for complementing different strategies to effectively curb methane emissions.

The objective of this study was to evaluate the effects of supplementing myristic acid in dairy cow rations on ruminal methanogenesis and the fatty acid profile in milk. Twelve multiparous Holstein dairy cows (710±17.3kg of live weight; 290±41.9 d in milk) housed in a tie-stall facility were used in the study. The cows were paired by parity and days in milk and allocated to 1 of 2 treatments: 1) the regular milking cow total mixed ration (control diet), and 2) the regular milking cow total mixed ration supplemented with 5% myristic acid on a dry matter basis (MA diet). The cows were fed and milked twice daily (feeding, 0830 and 1300h; milking, 0500 and 1500h). The experiment was conducted as a completely randomized design and consisted of a 7-d pretrial period when cows were fed the control diet to obtain baseline measurements, a 10-d dietary adaptation period, and a 1-d, 8-h measurement period. The MA diet reduced methane (CH4) production by 36% (608.2 vs. 390.6±56.46 L/d, control vs. MA diet, respectively) and milk fat percentage by 2.4% (4.2 vs. 4.1±0.006%, control vs. MA diet, respectively). The MA diet increased 14:0 in milk by 139% and cis-9 14:1 by 195%. There was a correlation (r=−0.58) between the 14:0 content in milk and CH4 production and cis-9 14:1 and CH4 production (r=−0.47). Myristic acid had no effect on the contents of CLA or trans-10 18:1 and trans-11 18:1 isomers in milk. These results suggest that MA could be used to inhibit the activities of methanogens in ruminant animals without altering the conjugated linoleic acid and trans-18:1 fatty acid profile in milk.

Background: Thiamine supplementation in high-concentrate diets (HC) was confirmed to attenuate ruminal subacute acidosis through promoting carbohydrate metabolism, however, whether thiamine supplementation in HC impacts methane metabolism is still unclear. Therefore, in the present study, thiamine was supplemented in the high-concentrate diets to investigate its effects on ruminal methanogens and methanogenesis process. Methods: an in vitro fermentation experiment which included three treatments: control diet (CON, concentrate/forage = 4:6; DM basis), high-concentrate diet (HC, concentrate/forage = 6:4; DM basis) and high-concentrate diet supplemented with thiamine (HCT, concentrate/forage = 6:4, DM basis; thiamine supplementation content = 180 mg/kg DM) was conducted. Each treatment concluded with four repeats, with three bottles in each repeat. The in vitro fermentation was sustained for 48h each time and repeated three times. At the end of fermentation, fermentable parameters, ruminal bacteria and methanogens community were measured. Results: HC significantly decreased ruminal pH, thiamine and acetate content, while significantly increasing propionate content compared with CON (p < 0.05). Conversely, thiamine supplementation significantly increased ruminal pH, acetate while significantly decreasing propionate content compared with HC treatment (p < 0.05). No significant difference of ruminal methanogens abundances among three treatments was observed. Thiamine supplementation significantly decreased methane production compared with CON, while no significant change was found in HCT compared with HC. Conclusion: thiamine supplementation in the high-concentrate diet (HC) could efficiently reduce CH4 emissions compared with high-forage diets while without causing ruminal metabolic disorders compared with HC treatment. This study demonstrated that supplementation of proper thiamine in concentrate diets could be an effective nutritional strategy to decrease CH4 production in dairy cows.

Six plant sources of hydrolyzable tannins (HT) or HT and condensed tannins (CT; designated as HT1, HT2, HT3, HT + CT1, HT + CT2, and HT + CT3) were evaluated to determine their effects in vitro on CH4 production and on ruminal archaeal and protozoa populations, and to assess potential differences in biological activities between sources containing HT only or HT and CT. Samples HT1, HT2, and HT3 contained only HT, whereas samples HT + CT1, HT + CT2, and HT + CT3 contained HT and CT. In experiment 1, in vitro incubations with samples containing HT or HT + CT resulted in a decrease in CH4 production of 0.6 and 5.5%, respectively, compared with that produced by incubations containing the added tannin binder polyethylene glycol-6000. Tannin also suppressed the population of methanogenic archaea in all incubations except those with HT2, with an average decrease of 11.6% in HT incubations (15.8, 7.09, and 12.0 in HT1, HT2, and HT3) and 28.6% in incubations containing HT + CT (35.0, 40.1, and 10.8 in HT + CT1, HT + CT2, and HT + CT3) when compared with incubations containing added polyethylene glycol-6000. The mean decrease in protozoal counts was 12.3% in HT and 36.2% in HT + CT incubations. Tannins increased in vitro pH, reduced total VFA concentrations, increased propionate concentrations, and decreased concentrations of iso-acids. In experiment 2, when a basal diet was incubated with graded levels of HT + CT1, HT + CT2, and HT + CT3, the total gas and CH4 production and archaeal and protozoal populations decreased as the concentration of tannins increased. Our results confirm that tannins suppress methanogenesis by reducing methanogenic populations in the rumen either directly or by reducing the protozoal population, thereby reducing methanogens symbiotically associated with the protozoal population. In addition, tannin sources containing both HT and CT were more potent in suppressing methanogenesis than those containing only HT.

Secondary plant metabolites (SPMs) can influence the reduction of enteric methane ([CH.sub.4]) emissions in ruminants. This study aimed to evaluate the effects of supplementing diet with cyanogenic glucoside linamarin (LIN), essential oil eugenol (EU), and their combination (LIN+EU) on methanogenic microorganisms, [CH.sub.4] production, and rumen fermentation parameters in vitro. The basal diet (BD) (alfalfa [Medicago sativa L.] hay and oat [Avena sativa L.] grain in a 3:1 ratio) was supplemented with LIN, EU, and LIN+EU mixtures and placed in amber bottles. The experimental treatments were: T1, BD (control); T2, BD-LIN (20 mg [L.sup.-1]); T3, BD-LIN (40 mg [L.sup.-1]); T4, BD-EU (400 mg [L.sup.-1]); T5, BD-LIN+EU (20 mg [L.sup.-1] + 400 mg [L.sup.-1]); and T6, BD-LIN+EU (40 mg [L.sup.-1] + 400 mg [L.sup.-1]). The treatments were inoculated with rumen fluid and incubated for 6, 12, and 24 h to assess abundance of total bacteria (TB), methanogenic archaea, and ruminal fermentation parameters. The [CH.sub.4] production was significantly reduced (p < 0.001) with inclusion of LIN, EU, and LIN+EU, particularly at 12 and 24 h incubation. However, at these same time points, in vitro DM disappearance (IVDMD) was also reduced (p < 0.001), except when LIN was included alone, highlighting its advantage in this variable. Rumen pH remained relatively stable at 6 and 12 h but decreased (p < 0.001) at 24 h with LIN. The pH values ranged between 6.65 (minimum) and 6.85 (maximum), which are optimal for microbial activity. Total bacterial abundance (TB) was not affected (p > 0.05) by treatments, but methanogenic archaea abundance was significantly reduced (p < 0.001) at 24 h with the LIN+EU mixtures (T5 and T6), coinciding with the highest [CH.sub.4] reductions of 36.9% and 56.7%, respectively. At 24 h, IVDMD with LIN alone was 51.83%, whereas EU alone resulted in 46.48%. In conclusion, LIN+EU mixtures T5 and T6 had a synergistic effect, effectively reducing [CH.sub.4] emissions, demonstrating the combined impact of different SPMs mechanisms of action.

The objective of this study was to evaluate the effects of different concentrations of hydrogen-rich water (HRW) on in vitro rumen fermentation characteristics and the dynamics of bacterial communities. The experiment included four treatment groups: a control (CON) and hydrogen-rich water (HRW) at 200, 400, and 800 ppb. Each group was analyzed at 12-hour (h) and 48-hour (h) time points with five replicates, totaling 40 samples. The experimental results highlighted the HRW 800ppb group as the top production in terms of gas production and CH 4 content. In contrast, the HRW 200ppb group exhibited significantly lower methane levels at both 12 h and 48 h ( P  < 0.05). Regarding rumen fermentation, the HRW 400ppb group significantly increased the levels of ammonia nitrogen (NH 3 -N) and microbial crude protein (MCP) at 12 h fermentation, but reduced the dry matter degradation rate ( P  < 0.05). After 48 h, the HRW 400ppb group had highest MCP content ( P  < 0.05), but no significant differences in NH 3 -N and dry matter degradation rate compared with the CON group ( P  > 0.05). Although HRW did not significantly benefit the synthesis of total volatile fatty acids (TVFA) and individual VFA, the HRW 800ppb group significantly increased the ratio of acetate to propionate ( P  < 0.05). Based on CH 4 emissions and MCP synthesis, we selected the HRW 400ppb group for subsequent bacterial community analysis. Bacterial community analysis showed that at 12 h, compared with the CON group, the Bacterial community analysis revealed that the HRW 400ppb group had significant increases in the Simpson index, Firmicutes, Streptococcus, Schwartzia, Prevotellaceae_YAB2003_group , and Oribacterium , and decreases in Prevotella , Ruminobacter , Succinivibrio , unclassified_Succinivibrionaceae , and Prevotellaceae_UCG-003 ( P  < 0.05). At 48 h, the Prevotellaceae_YAB2003_group and Oribacterium abundances continued to rise significantly, while Rikenellaceae_RC9_gut_group and Succiniclasticum abundances fell in the HRW 400ppb group ( P  < 0.05). Correlation analysis indicated a negative link between CH 4 and Streptococcus , and a positive correlation between the abundance of Rikenellaceae_RC9_gut_group and CH 4 . Collectively, these results indicate that HRW can modulate rumen fermentation and microbial community structure to reduce methane emissions without significantly affecting VFA synthesis, highlighting its potential as drinking water for enhancing ruminant nutrition and mitigating the environmental impact of livestock farming.

Ruminal microbiota (RM) were co-inoculated with anaerobic sludge (AS) at different ratios to study the digestion of rice straw in batch experiments. The CH 4 yield reached 273.64 mL/g volatile solid (VS) at a co-inoculum ratio of 1:1. The xylanase and cellulase activities were 198.88–212.88 and 24.51–29.08 U/mL in co-inoculated samples, respectively, and were significantly different compared to the results for single inoculum ( p  < 0.05). Higher ratios of AS enhanced acetoclastic methanogenesis, and propionate accumulation could be the main reason for the longer lag phase observed in samples with a higher RM ratio. The microbial compositions were clearly altered after digestion. Fibrobacter , Ruminococcus and Butyrivibrio from the rumen did not settle in the co-inoculated system, whereas Clostridiales members became the main polysaccharide degraders. Microbial interactions involving hydrolytic bacteria and acetoclastic methanogens in the residue were considered to be significant for hydrolysis activities and methane production. Syntrophy involving propionate oxidizers with associated methanogens occurred in the liquid phase. Our findings provide a better understanding of the anaerobic digestion of rice straw that is driven by specific microbial populations.

Holocellulose (HC) fraction extracted from date-pits was evaluated as a novel feed additive for ruminant feeding. This study was performed to investigate the effectiveness of the HC additive on rumen fermentation, methane (CH4) production, and diet degradability over 24 h of in vitro incubation. Three independent incubation trials were conducted over three consecutive weeks, employing the same in vitro methodology to assess four treatment doses in a completely randomized design. The experimental diet incorporated four increasing doses of HC, containing HC at 0 (HC0), 10 (HC10), 20 (HC20), and 30 (HC30) g/kg dry matter (DM). In vitro gas production (GP) and CH4 production, volatile fatty acids (VFAs) concentration, protozoa accounts, degraded organic matter (DOM), metabolizable and net energy (ME and NE), and hydrogen (H2) estimates were measured. No significant differences in ruminal pH were observed as the HC doses gradually increased. All incremental doses of HC additive over 24 h resulted in a linear increase in GP (P < 0.001), DOM (P < 0.001), total VFAs (P = 0.011), and propionate (P < 0.001) concentrations, as well as estimated energy (ME and NE) (P < 0.05) and microbial protein (P = 0.017) values. However, the inclusion of increasing doses of HC in the diet displayed linear reductions in the net CH4 production (ml/kg DOM; P = 0.002), protozoa abundance (P = 0.027); acetate (P = 0.029), and butyrate (P < 0.001) concentrations, the acetate-to-propionate ratio (P < 0.001), and the estimated net H2 production concentration (P = 0.049). Thus, the use of date-pits HC additive generated positive ruminal fermentability, including increased total VFAs and a reduction in the acetate-to-propionate ratio, leading to decreased CH4 output over 24 h of in vitro incubation. Hence, HC could be considered a potent feed additive (at up to 30 g/kg DM), demonstrating promising CH4-mitigating competency and thereby enhancing energy-use efficiency in ruminants.

The effects of a composite polyphenolic-rich extract (CPRE) on ruminal fermentation, nutrient utilisation, growth performance, excretion of nitrogen and phosphorus and methane emission were studied in growing buffaloes. Four herbal dry extracts prepared from Acacia arabica (babul; bark), Acacia catechu (cutch; bark), Punica granatum (pomegranate; peel) and Eugenia jambolana (Indian blackberry; seeds) were mixed in an equal proportion (1:1:1:1) to prepare the CPRE that contained mainly phenolic compounds (146 g/kg), flavonoids (41.7 g/kg) and saponins (40.5 g/kg). First, in vitro tests were performed for ruminal fermentation and feed degradability using ruminal fluid as inocula and CPRE at 0 to 40 g/kg substrate to decide an optimal dose of CPRE for an in vivo study on buffaloes. In the animal study, 20 buffaloes were randomly assigned to two groups ( n  = 10)—a control diet and a CPRE diet (control diet added with extra 20 g/kg of CPRE). The in vitro tests suggested that addition of CPRE at 20 g/kg substrate increased degradability of substrate, short-chain fatty acid concentration and propionate proportion, and reduced methane production, acetate proportion, acetate:propionate ratio and ammonia concentration in fermentation media, which were also noted in the rumen of buffaloes. Feeding CRPE to buffaloes did not affect feed intake, but increased daily body weight gain, dry matter and crude protein digestibility and nitrogen and phosphorus retention in the body. Total bacteria, methanogens and protozoal numbers were similar between two groups, but Fibrobacter succinogenes increased in the rumen of buffaloes fed CPRE. Concentrations of total, essential, non-essential and glucogenic amino acids were greater in the plasma of CPRE-fed buffaloes. Cell-mediated immune response improved in the CPRE-fed buffaloes compared with the control group. Estimated methane production and excretion of nitrogen and phosphorus per unit of body weight gain decreased in the CPRE group. The comprehensive results of this study clearly suggested that the composite polyphenol-rich feed additive at 20 g/kg diet improved growth performance, ruminal fermentation, immunity and plasma amino acids profile, whereas it reduced indicators of environmental impacts of buffalo production.