Dietary selection of metabolically distinct microorganisms drives hydrogen metabolism in ruminants.

Ruminants are important for global food security but emit the greenhouse gas methane. Rumen microorganisms break down complex carbohydrates to produce volatile fatty acids and molecular hydrogen. This hydrogen is mainly converted into methane by archaea, but can also be used by hydrogenotrophic acetogenic and respiratory bacteria to produce useful metabolites. A better mechanistic understanding is needed on how dietary carbohydrates influence hydrogen metabolism and methanogenesis. We profiled the composition, metabolic pathways, and activities of rumen microbiota in 24 beef cattle adapted to either fiber-rich or starch-rich diets. The fiber-rich diet selected for fibrolytic bacteria and methanogens resulting in increased fiber utilization, while the starch-rich diet selected for amylolytic bacteria and lactate utilizers, allowing the maintenance of a healthy rumen and decreasing methane production (p < 0.05). Furthermore, the fiber-rich diet enriched for hydrogenotrophic methanogens and acetogens leading to increased electron-bifurcating [FeFe]-hydrogenases, methanogenic [NiFe]- and [Fe]-hydrogenases and acetyl-CoA synthase, with lower dissolved hydrogen (42%, p < 0.001). In contrast, the starch-rich diet enriched for respiratory hydrogenotrophs with greater hydrogen-producing group B [FeFe]-hydrogenases and respiratory group 1d [NiFe]-hydrogenases. Parallel in vitro experiments showed that the fiber-rich selected microbiome enhanced acetate and butyrate production while decreasing methane production (p < 0.05), suggesting that the enriched hydrogenotrophic acetogens converted some hydrogen that would otherwise be used by methanogenesis. These insights into hydrogen metabolism and methanogenesis improve understanding of energy harvesting strategies, healthy rumen maintenance, and methane mitigation in ruminants.

Application of 3-nitrooxypropanol and canola oil to mitigate enteric methane emissions of beef cattle results in distinctly different effects on the rumen microbial community.

BACKGROUND: The major greenhouse gas from ruminants is enteric methane (CH4) which in 2010, was estimated at 2.1 Gt of CO2 equivalent, accounting for 4.3% of global anthropogenic greenhouse gas emissions. There are extensive efforts being made around the world to develop CH4 mitigating inhibitors that specifically target rumen methanogens with the ultimate goal of reducing the environmental footprint of ruminant livestock production. This study examined the individual and combined effects of supplementing a high-forage diet (90% barley silage) fed to beef cattle with the investigational CH4 inhibitor 3-nitrooxypropanol (3-NOP) and canola oil (OIL) on the rumen microbial community in relation to enteric CH4 emissions and ruminal fermentation. RESULTS: 3-NOP and OIL individually reduced enteric CH4 yield (g/kg dry matter intake) by 28.2% and 24.0%, respectively, and the effects were additive when used in combination (51.3% reduction). 3-NOP increased H2 emissions 37-fold, while co-administering 3-NOP and OIL increased H2 in the rumen 20-fold relative to the control diet. The inclusion of 3-NOP or OIL significantly reduced the diversity of the rumen microbiome. 3-NOP resulted in targeted changes in the microbiome decreasing the relative abundance of Methanobrevibacter and increasing the relative abundance of Bacteroidetes. The inclusion of OIL resulted in substantial changes to the microbial community that were associated with changes in ruminal volatile fatty acid concentration and gas production. OIL significantly reduced the abundance of protozoa and fiber-degrading microbes in the rumen but it did not selectively alter the abundance of rumen methanogens. CONCLUSIONS: Our data provide a mechanistic understanding of CH4 inhibition by 3-NOP and OIL when offered alone and in combination to cattle fed a high forage diet. 3-NOP specifically targeted rumen methanogens and partly inhibited the hydrogenotrophic methanogenesis pathway, which increased H2 emissions and propionate molar proportion in rumen fluid. In contrast, OIL caused substantial changes in the rumen microbial community by indiscriminately altering the abundance of a range of rumen microbes, reducing the abundance of fibrolytic bacteria and protozoa, resulting in altered rumen fermentation. Importantly, our data suggest that co-administering CH4 inhibitors with distinct mechanisms of action can both enhance CH4 inhibition and provide alternative sinks to prevent excessive accumulation of ruminal H2.

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.

Comparison of in situ ruminal straw fiber degradation and bacterial community between buffalo and Holstein fed with high-roughage diet.

Buffalo exhibits great efficiency in utilizing low-quality roughage, which can be due to the combined effect of host physiological feature and roughage diet fed. The present study was designed to compare the ruminal fiber degradation and the bacterial community attached to straws in buffalo and Holstein when fed with the same high-roughage diet using in situ ruminal incubation technique. Rice and wheat straws were selected as the incubation substrates and sampled at 0, 4, 12, 24, 48, 72, 120, and 216 h of incubation time to measure the kinetics of dry matter (DM) and neutral detergent fiber (NDF) disappearance. Additional two bags were incubated and sampled at 4 and 48 h of incubation time to evaluate the bacterial community attached to straws. The results showed that buffalo exhibited a greater (p ≤ 0.05) fraction of rapidly soluble and washout nutrients and effective ruminal disappearance for both DM and NDF of straw than Holstein, together with a greater (p ≤ 0.05) disappearance rate of potentially degradable nutrient fraction for NDF. Principal coordinate analysis indicated that both host and incubation time altered the bacterial communities attached to straws. Buffalo exhibited greater (p ≤ 0.05) 16S rRNA gene copies of bacteria and greater (p ≤ 0.05) relative abundance of Ruminococcus attached to straw than Holstein. Prolonging incubation time increased (p ≤ 0.05) the 16S rRNA gene copies of bacteria, and the relative abundance of phyla Proteobacteria and Fibrobacters by comparing 4 vs. 48 h of incubation time. In summary, buffalo exhibits greater ruminal fiber degradation than Holstein through increasing bacterial population and enriching Ruminococcus, while prolonging incubation time facilitates fiber degradation through enriching phyla Proteobacteria and Fibrobacteres.

Combined effects of 3-nitrooxypropanol and canola oil supplementation on methane emissions, rumen fermentation and biohydrogenation, and total tract digestibility in beef cattle.

The individual and combined effects of 3-nitrooxypropanol (3-NOP) and canola oil (OIL) supplementation on enteric methane (CH4) and hydrogen (H2) emissions, rumen fermentation and biohydrogenation, and total tract nutrient digestibility were investigated in beef cattle. Eight beef heifers (mean body weight ± SD, 732 ± 43 kg) with ruminal fistulas were used in a replicated 4 × 4 Latin square with a 2 (with and without 3-NOP) × 2 (with and without OIL) arrangement of treatments and 28-d periods (13 d adaption and 15 d measurements). The four treatments were: control (no 3-NOP, no OIL), 3-NOP (200 mg/kg dry matter [DM]), OIL (50 g/kg DM), and 3-NOP (200 mg/kg DM) plus OIL (50 g/kg DM). Animals were fed restrictively (7.6 kg DM/d) a basal diet of 900 g/kg DM barley silage and 100 g/kg DM supplement. 3-NOP and OIL decreased (P < 0.01) CH4 yield (g/kg DM intake) by 31.6% and 27.4%, respectively, with no 3-NOP × OIL interaction (P = 0.85). Feeding 3-NOP plus OIL decreased CH4 yield by 51% compared with control. There was a 3-NOP × OIL interaction (P = 0.02) for H2 yield (g/kg DM intake); the increase in H2 yield (P < 0.01) due to 3-NOP was less when it was combined with OIL. There were 3-NOP × OIL interactions for molar percentages of acetate and propionate (P < 0.01); individually, 3-NOP and OIL decreased acetate and increased propionate percentages with no further effect when supplemented together. 3-NOP slightly increased crude protein (P = 0.02) and starch (P = 0.01) digestibilities, while OIL decreased the digestibilities of DM (P < 0.01) and neutral detergent fiber (P < 0.01) with no interactions (P = 0.15 and 0.10, respectively). 3-NOP and OIL increased (P = 0.04 and P < 0.01, respectively) saturated fatty acid concentration in rumen fluid, with no interaction effect. Interactions for ruminal trans-monounsaturated fatty acids (t-MUFA) concentration and percentage were observed (P = 0.02 and P < 0.01); 3-NOP had no effect on t-MUFA concentration and percentage, while OIL increased the concentration (P < 0.01) and percentage (P < 0.01) of t-MUFA but to a lesser extent when combined with 3-NOP. In conclusion, the CH4-mitigating effects of 3-NOP and OIL were independent and incremental. Supplementing ruminant diets with a combination of 3-NOP and OIL may help mitigate CH4 emissions, but the decrease in total tract digestibility due to OIL may decrease animal performance and needs further investigation.

In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio.

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

Association of fibre degradation with ruminal dissolved hydrogen in growing beef bulls fed with two types of forages.

The present study investigated the association between fibre degradation and the concentration of dissolved molecular hydrogen (H2) in the rumen. Napier grass (NG) silage and corn stover (CS) silage were compared as forages with contrasting structures and degradation patterns. In the first experiment, CS silage had greater 48-h DM, neutral-detergent fibre (NDF) and acid-detergent fibre degradation, and total gas and methane (CH4) volumes, and lower 48-h H2 volume than NG silage in 48-h in vitro incubations. In the second experiment, twenty-four growing beef bulls were fed diets including 55 % (DM basis) NG or CS silages. Bulls fed the CS diet had greater DM intake (DMI), average daily gain, total-tract digestibility of OM and NDF, ruminal dissolved methane (dCH4) concentration and gene copies of protozoa, methanogens, Ruminococcus albus and R. flavefaciens, and had lower ruminal dH2 concentration, and molar proportions of valerate and isovalerate, in comparison with those fed the NG diet. There was a negative correlation between dH2 concentration and NDF digestibility in bulls fed the CS diet, and a lack of relationship between dH2 concentration and NDF digestibility with the NG diet. In summary, the fibre of CS silage was more easily degraded by rumen microorganisms than that of NG silage. Increased dCH4 concentration with the CS diet presumably led to the decreased ruminal dH2 concentration, which may be helpful for fibre degradation and growth of fibrolytic micro-organisms in the rumen.

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa.

QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, United States) and RBB + C (Yu and Morrison, 2004) methodologies are widely employed to extract microbial DNA from rumen samples and can exhibit different efficiencies of obtaining DNA yield, quality, and downstream amplicon sequence analysis. No study has conducted to investigate the contributions of chemical and mechanical lysis on DNA extraction, which included chemical lysis from QIAamp Fast DNA Stool Mini Kit (QIA) and RBB + C (YM), bead (BB), and sand beating (SB). Effects of chemical lysis and bead beating (BB) were investigated by conducting a 2 × 2 factorial-designed experiment with four methodologies, including QIA without (QIA-) and with BB (QIA + BB), and YM without (YM-) and with BB (YM + BB). Comparisons between bead and sand were conducted by comparing methodologies of YM + BB and YM + SB. Comparing with QIA, YM had lower (P ≤ 0.10) OD260 / 280 and diversity of ZOTUs and length polymorphism of protozoal amplicons but harvested greater (P ≤ 0.086) DNA from fibrolytic bacteria such as Ruminococcaceae lineages. Including BB increased (P = 0.001) total DNA yield without affecting (P ≥ 0.55) OD260 / 280 and richness of bacterial ZOTUs but decreased (P ≤ 0.08) richness of both ZOTUs and length polymorphism of protozoal amplicon. Bead beating and SB showed no difference (P ≥ 0.19) in DNA yield and quality and bacterial and protozoal community. In summary, chemical lysis provided by RBB + C and QIAamp Fast DNA Stool Mini Kit should be better to extract DNA for analyzing bacterial and protozoal community, respectively. Sand can be an alternative beater for DNA extraction, and mechanical lysis is not recommended for protozoal community analysis.

3-Nitrooxypropanol supplementation had little effect on fiber degradation and microbial colonization of forage particles when evaluated using the in situ ruminal incubation technique.

3-Nitrooxypropanol (3-NOP) is an investigational compound that acts as an enzyme inhibitor to decrease ruminal methanogenesis. We hypothesized that when feeding 3-NOP to cattle fed a high-forage diet, H2 would accumulate in the rumen, which could suppress microbial colonization of feed particles and fiber degradation. Therefore, the study investigated the effects of supplementing a high-forage diet with 3-NOP on ruminal fiber degradability and microbial colonization of feed particles using the in situ technique. Eight ruminally cannulated beef cattle were allocated to 2 groups (4 cattle/group) in a crossover design with 2 periods and 2 dietary treatments. The treatments were control (basal diet) and 3-NOP (basal diet supplemented with 3-NOP, 150 mg/kg of dry matter). The basal diet consisted of 45% barley silage, 45% chopped grass hay, and 10% concentrate (dry matter basis). Samples of dried, ground barley silage and grass hay were incubated in the rumen of each animal for 0, 4, 12, 24, 36, 48, 96, 120, 216, and 288 h to determine neutral detergent fiber (NDF) degradation kinetics. An additional 2 bags were incubated for 4 and 48 h to evaluate the bacterial community attached to the incubated forages. Dietary supplementation of 3-NOP decreased (-53%) the dissolved methane concentration and increased (+780%) the dissolved H2 concentration in ruminal fluid, but did not substantially alter in situ NDF degradation. The addition of 3-NOP resulted in a decrease in the α-diversity of the microbial community with colonizing communities showing reduced numbers of amplicon sequence variants and phylogenetic diversity compared with control diets. Principal coordinate analysis plots indicated that forages incubated in animals fed 3-NOP resulted in highly specific changes to targeted microbes compared with control diets based on unweighted analysis (considering only absence and presence of taxa), but did not alter the overall composition of the colonizing community based on weighted UniFrac distances; unchanged relative abundances of major taxa included phyla Bacteroidetes, Firmicutes, and Fibrobacteres. The effect of 3-NOP on colonizing methanogenic microbes differed depending upon the forage incubated, as abundance of genus Methanobrevibacter was decreased for barley silage but not for grass hay. In conclusion, 3-NOP supplementation of a high-forage diet decreased ruminal methanogenesis and increased dissolved H2 concentration, but had no negative effects on ruminal fiber degradation and only minor effects on relative abundances of the major taxa of bacteria adhered to forage substrates incubated in the rumen.

Liquid hot water treatment of rice straw enhances anaerobic degradation and inhibits methane production during in vitro ruminal fermentation.

Liquid hot water (LHW) treatment can be used to disrupt the fiber structure of rice straw. This in vitro ruminal batch culture study investigated the effect of LHW treatment on feed degradation, methane (CH4) production, and microbial populations. Rice straw was treated by LHW, and in vitro ruminal fermentation was performed using an automatic system with 72 h of incubation. Scanning electron microscopy showed that LHW treatment disrupted the physical structure of rice straw. Liquid hot water treatment decreased neutral detergent fiber and hemicellulose contents of the rice straw and increased neutral detergent solubles, water-soluble carbohydrates, and arabinose contents. Liquid hot water treatment increased dry matter degradation and volatile fatty acid concentration and decreased the acetate:propionate ratio, CH4 production, hydrogen accumulation, neutral detergent fiber degradation, and populations of protozoa, fungi, and cellulolytic bacteria. In summary, LHW treatment disrupted the cellulose-hemicellulose-lignin structure matrix of rice straw, leading to increased substrate degradability and decreased CH4 production. Therefore, the LHW treatment is a potential strategy to improve the nutritive value of forage such as rice straw and decrease the CH4 emissions in ruminants.

Corn oil supplementation enhances hydrogen use for biohydrogenation, inhibits methanogenesis, and alters fermentation pathways and the microbial community in the rumen of goats.

Enteric methane (CH4) emissions are not only an important source of greenhouse gases but also a loss of dietary energy in livestock. Corn oil (CO) is rich in unsaturated fatty acid with >50% PUFA, which may enhance ruminal biohydrogenation of unsaturated fatty acids, leading to changes in ruminal H2 metabolism and methanogenesis. The objective of this study was to investigate the effect of CO supplementation of a diet on CH4 emissions, nutrient digestibility, ruminal dissolved gases, fermentation, and microbiota in goats. Six female goats were used in a crossover design with two dietary treatments, which included control and CO supplementation (30 g/kg DM basis). CO supplementation did not alter total-tract organic matter digestibility or populations of predominant ruminal fibrolytic microorganisms (protozoa, fungi, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes), but reduced enteric CH4 emissions (g/kg DMI, -15.1%, P = 0.003). CO supplementation decreased ruminal dissolved hydrogen (dH2, P < 0.001) and dissolved CH4 (P < 0.001) concentrations, proportions of total unsaturated fatty acids (P < 0.001) and propionate (P = 0.015), and increased proportions of total SFAs (P < 0.001) and acetate (P < 0.001), and acetate to propionate ratio (P = 0.038) in rumen fluid. CO supplementation decreased relative abundance of family Bacteroidales_BS11_gut_group (P = 0.032), increased relative abundance of family Rikenellaceae (P = 0.021) and Lachnospiraceae (P = 0.025), and tended to increase relative abundance of genus Butyrivibrio_2 (P = 0.06). Relative abundance (P = 0.09) and 16S rRNA gene copies (P = 0.043) of order Methanomicrobiales, and relative abundance of genus Methanomicrobium (P = 0.09) also decreased with CO supplementation, but relative abundance (P = 0.012) and 16S rRNA gene copies (P = 0.08) of genus Methanobrevibacter increased. In summary, CO supplementation increased rumen biohydrogenatation by facilitating growth of biohydrogenating bacteria of family Lachnospiraceae and genus Butyrivibrio_2 and may have enhanced reductive acetogenesis by facilitating growth of family Lachnospiraceae. In conclusion, dietary supplementation of CO led to a shift of fermentation pathways that enhanced acetate production and decreased rumen dH2 concentration and CH4 emissions.

Effect of traditional Chinese medicine compounds on rumen fermentation, methanogenesis and microbial flora in vitro.

This study was conducted to investigate the effects of traditional Chinese medicine compounds (TCMC) on rumen fermentation, methane emission and populations of ruminal microbes using an in vitro gas production technique. Cablin patchouli herb (CPH), Atractylodes rhizome (AR), Amur Cork-tree (AC) and Cypsum were mixed with the weight ratios of 1:1:1:0.5 and 1:1:1:1 to make up TCMC1 and TCMC2, respectively. Both TCMC were added at level of 25 g/kg of substrate dry matter. In vitro gas production was recorded and methane concentration was determined at 12 and 24 h of incubation. After 24 h, the incubation was terminated and the inoculants were measured for pH, ammonia nitrogen, volatile fatty acids (VFA). Total deoxyribonucleic acid of ruminal microbes was extracted from the inocula, and populations were determined by a real-time quantitative polymerase chain reaction. Populations of total rumen methanogens, protozoa, total fungi, Ruminococcus albus, Fibrobacter succinogenes and Ruminococcus flavefaciens were expressed as a proportion of total rumen bacterial 16S ribosomal deoxyribonucleic acid. Compared with the control, the 2 TCMC decreased (P ≤ 0.05) total VFA concentration, acetate molar proportion, acetate to propionate ratio, gas and methane productions at 12 and 24 h, hydrogen (H) produced and consumed, and methanogens and total fungi populations, while the 2 TCMC increased (P ≤ 0.05) propionate molar proportion. Traditional Chinese medicine compound 1 also decreased (P ≤ 0.05) R. flavefaciens population. From the present study, it is inferred that there is an effect of the TCMC in suppressing methanogenesis, probably mediated via indirect mode by channeling H2 utilized for methanogenesis to synthesis of propionate and direct action against the rumen microbes involved in methane formation. In addition, the relative methane reduction potential (RMRP) of TCMC2 was superior to that of TCMC1.

Technical note: Evaluation of interval between measurements and calculation method for the quantification of enteric methane emissions measured by respiration chamber.

Respiration chambers share one analyzer working in parallel, and methane (CH4) concentrations have to be measured at certain intervals. The maximum and minimum values in the kinetics of CH4 emissions can be missed during the interval between measurements, which may influence the quantification of CH4 emissions. Chambers must be opened for morning feeding and cleaning, which causes a loss of CH4 data. Calculation methods are needed to estimate the lost CH4 emission data, which may influence the estimated amount of daily CH4 emissions. In this study, we measured the CH4 emissions of 10 growing Chinese Holstein dairy heifers in respiration chambers. Methane concentrations were measured every 0.5 min to obtain the 23-h kinetics of CH4 emissions, which were further selected at different intervals between measurements (i.e., 5, 30, 60, 120, 180, and 240 min) to evaluate the effects of interval on quantification of CH4 emissions. The missing 1-h kinetics of CH4 emissions before feeding were not measured, and 2 calculation methods were used to estimate the missing 1-h kinetics of CH4 emissions: mean value of measuring period (the mean method) and the nearest value of measurement just before chamber opening (the nearest method). The results showed that the rates of CH4 emission from 10 heifers varied from 4.56 to 11.42 g/h. The increment of intervals decreased maximum rate of CH4 emission and increased minimum rate of CH4 emission. Interval caused less than 5% of the difference in measuring CH4 emissions. Although the mean method had greater estimated daily CH4 emission than the nearest method, the difference was within 3%. The interval between measurements (≤3 h) and calculation method had little influence on enteric CH4 emission measurements.

Molecular hydrogen produced by elemental magnesium inhibits rumen fermentation and enhances methanogenesis in dairy cows.

Hydrogen is a key metabolite that connects microbial fermentation and methanogenesis in the rumen. This study was to investigate the effects of elevated H2 produced by elemental Mg on rumen fermentation and methanogenesis in dairy cows. Four nonlactating Chinese Holstein dairy cows were employed for this experiment in a replicated crossover design. The 2 dietary treatments included a basal diet supplemented with Mg(OH)2 (14.5 g/kg of feed dry matter) or elemental Mg (6.00 g/kg of feed dry matter). When compared with Mg(OH)2 treatment, cows fed diet with elemental Mg had similar rumen Mg2+ concentration, but higher rumen dissolved H2 and methane concentrations at 2.5 h after morning feeding. Also, elemental Mg supplementation decreased feed digestibility, rumen volatile fatty acid concentration, and relative abundance of group Ruminococcaceae_UCG-014, genus Bifidobacterium, and group Mollicutes_RF9, increased acetate to propionate ratio, succinate concentration, and abundance of family Christensenellaceae. Elemental Mg supplementation increased enteric CH4 emission, altered methanogen community with increased abundance of order Methanomassiliicoccales, 16S ribosomal RNA gene copies of methanogens, and order Methanobacteriales. In summary, the pulse of elevated dissolved H2 after feeding produced by elemental Mg inhibited rumen fermentation and feed digestibility by decreasing the abundance of carbohydrate-degrading bacteria, promoted H2 incorporation into succinate by increasing family Christensenellaceae and genus Bacteroidales_BS11, and increased H2 utilization for methanogenesis by favoring growth of methanogens.

Effects of rumen cannulation on dissolved gases and methanogen community in dairy cows.

Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows. Eight Holstein cows were used in the experiment. Four cows were surgically fitted with rumen cannulas, whereas the other 4 intact cows were used as control. Rumen cannulation decreased gaseous hydrogen and methane concentrations, dissolved carbon dioxide concentration, and relative abundances of Methanosphaera, and increased the saturation factor of dissolved hydrogen and dissolved methane, dissolved methane concentration, volatile fatty acid concentration, 16S ribosomal RNA gene copies of methanogens, and Simpson index of methanogen community. In summary, rumen cannulation causes a reduction in headspace gaseous hydrogen and gaseous methane, which may not decrease dissolved gas concentrations due to an increase in saturation factors. Furthermore, rumen cannulation alters methanogen community with increased methanogen population and decreased relative abundances of Methanosphaera.

Nitrate improves ammonia incorporation into rumen microbial protein in lactating dairy cows fed a low-protein diet.

Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control. Eight lactating cows were blocked in 4 pairs according to days in milk, parity, and milk yield and allocated to urea (7.0 g urea/kg of dry matter of basal diet) or nitrate (14.6 g of NO3-/kg of dry matter of basal diet, supplemented as sodium nitrate) treatments, which were formulated on 75% of metabolizable protein requirements. Nitrate supplementation decreased ammonia concentration in the rumen liquids (-33.1%) and plasma (-30.6%) as well as methane emissions (-15.0%) and increased dissolved hydrogen concentration (102%), microbial N (22.8%), propionate molar percentage, milk yield, and 16S rRNA gene copies of Selenomonas ruminantium. Ruminal dissolved hydrogen was positively correlated with the molar proportion of propionate (r = 0.57), and negatively correlated with acetate-to-propionate ratio (r = -0.57) and estimated net metabolic hydrogen production relative to total VFA produced (r = -0.58). Nitrate reduction to ammonia redirected metabolic hydrogen away from methanogenesis, enhanced ammonia incorporation into rumen microbial protein, and shifted fermentation from acetate to propionate, along with increasing S. ruminantium 16S rRNA gene copies, likely leading to the increased milk yield.

Inferring the Skeletal Muscle Developmental Changes of Grazing and Barn-Fed Goats from Gene Expression Data.

Thirty-six Xiangdong black goats were used to investigate age-related mRNA and protein expression levels of some genes related to skeletal muscle structural proteins, MRFs and MEF2 family, and skeletal muscle fiber type and composition during skeletal muscle growth under grazing (G) and barn-fed (BF) feeding systems. Goats were slaughtered at six time points selected to reflect developmental changes of skeletal muscle during nonrumination (days 0, 7, and 14), transition (day 42), and rumination phases (days 56 and 70). It was observed that the number of type IIx in the longissimus dorsi was increased quickly while numbers of type IIa and IIb decreased slightly, indicating that these genes were coordinated during the rapid growth and development stages of skeletal muscle. No gene expression was affected (P > 0.05) by feeding system except Myf5 and Myf6. Protein expressions of MYOZ3 and MEF2C were affected (P < 0.05) by age, whereas PGC-1α was linearly decreased in the G group, and only MYOZ3 protein was affected (P < 0.001) by feeding system. Moreover, it was found that PGC-1α and MEF2C proteins may interact with each other in promoting muscle growth. The current results indicate that (1) skeletal muscle growth during days 0-70 after birth is mainly myofiber hypertrophy and differentiation, (2) weaning affects the expression of relevant genes of skeletal muscle structural proteins, skeletal muscle growth, and skeletal muscle fiber type and composition, and (3) nutrition or feeding regimen mainly influences the expression of skeletal muscle growth genes.

Shifts in Rumen Fermentation and Microbiota Are Associated with Dissolved Ruminal Hydrogen Concentrations in Lactating Dairy Cows Fed Different Types of Carbohydrates.

BACKGROUND: Different carbohydrates ingested greatly influence rumen fermentation and microbiota and gaseous methane emissions. Dissolved hydrogen concentration is related to rumen fermentation and methane production. OBJECTIVES: We tested the hypothesis that carbohydrates ingested greatly alter the rumen environment in dairy cows, and that dissolved hydrogen concentration is associated with these changes in rumen fermentation and microbiota. METHODS: Twenty-eight lactating Chinese Holstein dairy cows [aged 4-5 y, body weight 480 ± 37 kg (mean ± SD)] were used in a randomized complete block design to investigate effects of 4 diets differing in forage content (45% compared with 35%) and source (rice straw compared with a mixture of rice straw and corn silage) on feed intake, rumen fermentation, and microbial populations. RESULTS: Feed intake (10.7-12.6 kg/d) and fiber degradation (0.584-0.692) greatly differed (P ≤ 0.05) between cows fed the 4 diets, leading to large differences (P ≤ 0.05) in gaseous methane yield (27.2-37.3 g/kg organic matter digested), dissolved hydrogen (0.258-1.64 µmol/L), rumen fermentation products, and microbiota. Ruminal dissolved hydrogen was negatively correlated (r < -0.40; P < 0.05) with molar proportion of acetate, numbers of fungi, abundance of Fibrobacter succinogenes, and methane yield, but positively correlated (r > 0.40; P < 0.05) with molar proportions of propionate and n-butyrate, numbers of methanogens, and abundance of Selenomonas ruminantium and Prevotella spp. Ruminal dissolved hydrogen was positively correlated (r = 0.93; P < 0.001) with Gibbs free energy changes of reactions producing greater acetate and hydrogen, but not correlated with those reactions producing more propionate without hydrogen. CONCLUSIONS: Changes in fermentation pathways from acetate toward propionate production and in microbiota from fibrolytic toward amylolytic species were closely associated with ruminal dissolved hydrogen in lactating dairy cows. An unresolved paradox was that greater dissolved hydrogen was associated with greater numbers of methanogens but with lower gaseous methane emissions.

Supersaturation of Dissolved Hydrogen and Methane in Rumen of Tibetan Sheep.

Hydrogen (H2) is an essential substrate for methanogens to produce methane (CH4), and also influences pathways of volatile fatty acids (VFA) production in the rumen. Dissolved H2 (H2 (aq)) is the form of H2 available to microbes, and dissolved CH4 (CH4 (aq)) is important for indicating methanogens activity. Rumen H2 (aq) concentration has been estimated by assuming equilibrium with headspace gaseous H2 (H2 (g)) concentration using Henry's law, and has also been directly measured in the liquid phase in some in vitro and in vivo experiments. In this in vivo study, H2 (aq) and CH4 (aq) concentration measured directly in rumen fluid and their corresponding concentrations estimated from their gaseous phase concentrations, were compared to investigate the existence of equilibrium between the gas and liquid phases. Twenty-four Tibetan sheep were randomly assigned to two mixed diets containing the same concentrate mixed with oat grass (OG diet) or barley straw (BS diet). Rumen gaseous phase and contents were sampled using rumenocentesis and oral stomach tubing, respectively. Rumen H2 (aq) and CH4 (aq) concentration and VFA profile differed between sheep fed OG and BS diets. Measured H2 (aq) and CH4 (aq) concentration were greater than H2 (aq) and CH4 (aq) concentrations estimated using gas concentrations, indicating lack of equilibrium between gas and liquid phase and supersaturation of H2 and CH4 in rumen fluid. As a consequence, Gibbs energy changes (ΔG) estimated for various metabolic pathways were different when calculated using dissolved gases concentrations directly measured and when using dissolved gases concentrations assuming equilibrium with the gaseous phase. Dissolved CH4, but not CH4 (g), was positively correlated with H2 (aq). Both H2 (aq) and H2 (g) concentrations were positively correlated with the molar percentage of butyrate and negatively correlated with the molar percentage of acetate. In summary, rumen fluid was supersaturated with both H2 and CH4, and H2 (aq) was closely associated with the VFA profile and CH4 (aq) concentration. The assumption of equilibrium between dissolved gases and gaseous phase affected ΔG estimation.

Effects of three methane mitigation agents on parameters of kinetics of total and hydrogen gas production, ruminal fermentation and hydrogen balance using in vitro technique.

Methane (CH4 ) can be mitigated through directly inhibiting methanogen activity and starving methanogens by hydrogen (H2 ) sink. Three types of mechanism (i.e. bromoethanesulphonate (BES), nitrate and emodin) and doses of CH4 mitigation agents were employed to investigate their pathways of CH4 inhibition. Results indicated that both BES and emodin inhibited CH4 production and altered H2 balance, which could be accompanied by decreased dry matter disappearance (DMD), fractional rate of gH2 formation, volatile fatty acid (VFA) production, ability to produce and use reducing equivalences and molecular H2 , and increased final asymptotic gH2 production, time to the peak of gH2 , discrete lag time of gH2 production and fermentation efficiency. However, emodin decreased gas volume produced by rapidly fermentable components of substrate and the rate of fermentation at early stage of incubation, while BES supplementation inhibited gas volume produced by both rapidly and slowly fermentable components of substrate and the rate of fermentation at middle or late stage of incubation. The nitrate supplementation inhibited CH4 production without affecting VFA profile, because of its dual role as H2 sink and being toxic to methanogens. Nitrate supplementation had more complicated pattern of fermentation, VFA production and profile and H2 balance in comparison to BES and emodin supplementation.