Stephane Duval

An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production

Significance Methane from enteric fermentation in the ruminant digestive system is a major contributor to anthropogenic greenhouse gas emissions in the United States and worldwide. Methane is also a net loss of feed energy to the animal. This study was undertaken to investigate the effect of a methane inhibitor on enteric methane emissions from lactating dairy cows. The experiment demonstrated that, under industry-relevant conditions, the inhibitor persistently decreased by 30% enteric methane emissions, without negatively affecting animal productivity. The spared methane energy was partially used for tissue synthesis, which led to a greater body weight gain by the inhibitor-treated cows. If adopted, this mitigation practice could lead to a substantial reduction of greenhouse gas emissions from the ruminant livestock sector. A quarter of all anthropogenic methane emissions in the United States are from enteric fermentation, primarily from ruminant livestock. This study was undertaken to test the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP), on enteric methane emission in lactating Holstein cows. An experiment was conducted using 48 cows in a randomized block design with a 2-wk covariate period and a 12-wk data collection period. Feed intake, milk production, and fiber digestibility were not affected by the inhibitor. Milk protein and lactose yields were increased by 3NOP. Rumen methane emission was linearly decreased by 3NOP, averaging about 30% lower than the control. Methane emission per unit of feed dry matter intake or per unit of energy-corrected milk were also about 30% less for the 3NOP-treated cows. On average, the body weight gain of 3NOP-treated cows was 80% greater than control cows during the 12-wk experiment. The experiment demonstrated that the methane inhibitor 3NOP, applied at 40 to 80 mg/kg feed dry matter, decreased methane emissions from high-producing dairy cows by 30% and increased body weight gain without negatively affecting feed intake or milk production and composition. The inhibitory effect persisted over 12 wk of treatment, thus offering an effective methane mitigation practice for the livestock industries.

Effects of 3-nitrooxypropanol on methane emission, digestion, and energy and nitrogen balance of lactating dairy cows

The objective was to measure effects of 3-nitrooxypropanol (3 NP) on methane production of lactating dairy cows and any associated changes in digestion and energy and N metabolism. Six Holstein-Friesian dairy cows in mid-lactation were fed twice daily a total mixed ration with maize silage as the primary forage source. Cows received 1 of 3 treatments using an experimental design based on two 3 × 3 Latin squares with 5-wk periods. Treatments were a control placebo or 500 or 2,500 mg/d of 3 NP delivered directly into the rumen, via the rumen fistula, in equal doses before each feeding. Measurements of methane production and energy and N balance were obtained during wk 5 of each period using respiration calorimeters and digestion trials. Measurements of rumen pH (48 h) and postprandial volatile fatty acid and ammonia concentrations were made at the end of wk 4. Daily methane production was reduced by 3 NP, but the effects were not dose dependent (reductions of 6.6 and 9.8% for 500 and 2,500 mg/d, respectively). Dosing 3 NP had a transitory inhibitory effect on methane production, which may have been due to the product leaving the rumen in liquid outflow or through absorption or metabolism. Changes in rumen concentrations of volatile fatty acids indicated that the pattern of rumen fermentation was affected by both doses of the product, with a decrease in acetate:propionate ratio observed, but that acetate production was inhibited by the higher dose. Dry matter, organic matter, acid detergent fiber, N, and energy digestibility were reduced at the higher dose of the product. The decrease in digestible energy supply was not completely countered by the decrease in methane excretion such that metabolizable energy supply, metabolizable energy concentration of the diet, and net energy balance (milk plus tissue energy) were reduced by the highest dose of 3 NP. Similarly, the decrease in N digestibility at the higher dose of the product was associated with a decrease in body N balance that was not observed for the lower dose. Milk yield and milk fat concentration and fatty acid composition were not affected but milk protein concentration was greater for the higher dose of 3 NP. Twice-daily rumen dosing of 3 NP reduced methane production by lactating dairy cows, but the dose of 2,500 mg/d reduced rumen acetate concentration, diet digestibility, and energy supply. Further research is warranted to determine the optimal dose and delivery method of the product.

Ruminal methane inhibition potential of various pure compounds in comparison with garlic oil as determined with a rumen simulation technique (Rusitec).

Ruminants represent an important source of methane (CH(4)) emissions; therefore, CH(4) mitigation by diet supplementation is a major goal in the current ruminant research. The objective of the present study was to use a rumen simulation technique to evaluate the CH(4)-mitigating potential of pure compounds in comparison with that achieved with garlic oil, a known anti-methanogenic supplement. A basal diet (15 g DM/d) consisting of ryegrass hay, barley and soyabean meal (1:0·7:0·3) was incubated with the following additives: none (negative control); garlic oil (300 mg/l incubation liquid; positive control); allyl isothiocyanate (75 mg/l); lovastatin (150 mg/l); chenodeoxycholic acid (150 mg/l); 3-azido-propionic acid ethyl ester (APEE, 150 mg/l); levulinic acid (300 mg/l); 4-[(pyridin-2-ylmethyl)-amino]-benzoic acid (PABA, 300 mg/l). Fermentation profiles (SCFA, microbial counts and N turnover) and H(2) and CH(4) formation were determined. Garlic oil, allyl isothiocyanate, lovastatin and the synthetic compound APEE decreased the absolute daily CH(4) formation by 91, 59, 42 and 98 %, respectively. The corresponding declines in CH(4) emitted per mmol of SCFA were 87, 32, 40 and 99 %, respectively, compared with the negative control; the total SCFA concentration was unaffected. Garlic oil decreased protozoal numbers and increased bacterial counts, while chenodeoxycholic acid completely defaunated the incubation liquid. In vitro, neutral-detergent fibre disappearance was lower following chenodeoxycholic acid and PABA treatments (- 26 and - 18 %, respectively). In conclusion, garlic oil and APEE were extremely efficient at mitigating CH(4) without noticeably impairing microbial nutrient fermentation. Other promising substances were allyl isothiocyanate and lovastatin.

Effects of ethyl-3-nitrooxy propionate and 3-nitrooxypropanol on ruminal fermentation, microbial abundance, and methane emissions in sheep

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3 NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3 NP (40 and 80 μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500 mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3 NP at one dose (100mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48 h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3 NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals.

The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation.

The objective of the current study was to determine the effects of adding 3-nitrooxypropanol to the diet of lactating Holstein cows on methane emissions, rumen fermentation, ruminal microbial profile, and milk production. Twelve ruminally cannulated Holstein cows in midlactation were used in a crossover design study with 28-d periods. Cows were fed a diet containing 38% forage on a dry matter basis with either 2,500 mg/d of 3-nitrooxypropanol (fed as 25 g of 10% 3-nitrooxypropanol on silicon dioxide) or 25 g/d of silicon dioxide (control). After a 21-d diet adaptation period, dry matter intake (DMI) and milk yield were recorded daily. Rumen fluid and digesta were collected on d 22 and 28 for volatile fatty acid analysis and microbial profiling. Enteric methane emissions were measured on d 23 to 27 using the sulfur hexafluoride tracer gas technique. Feeding 3-nitrooxypropanol did not affect DMI; however, methane production was reduced from 17.8 to 7.18 g/kg of DMI. No change in milk or milk component yields was observed, but cows fed 3-nitrooxypropanol gained more body weight than control cows (1.06 vs. 0.39 kg/d). Concentrations of total volatile fatty acids in ruminal fluid were not affected by treatment, but a reduction in acetate proportion and a tendency for an increase in propionate proportion was noted. As such, a reduction in the acetate-to-propionate ratio was observed (2.02 vs. 2.36). Protozoa counts were not affected by treatment; however, a reduction in methanogen copy count number was observed when 3-nitrooxypropanol was fed (0.95 vs. 2.69 × 10(8)/g of rumen digesta). The data showed that feeding 3-nitrooxypropanol to lactating dairy cows at 2,500 mg/d can reduce methane emissions without compromising DMI or milk production.

The potential of 3-nitrooxypropanol to lower enteric methane emissions from beef cattle.

This study evaluated if 3-nitrooxypropanol reduces enteric methane (CH4) emissions when added to the diet of beef cattle. The effects of 3-nitrooxypropanol on related variables including diet digestibility, ruminal fermentation, and ruminal microorganisms were also investigated. Eight ruminally cannulated Angus heifers (549 ± 64.3 kg [mean BW ± SD]) were fed a high forage diet (backgrounding diet) supplemented with 4 levels of 3-nitrooxypropanol (0, 0.75, 2.25 and 4.50 mg/kg BW). The experiment was designed as a duplicated 4 × 4 Latin square with 2 groups of heifers and four 28-d periods. Methane emissions were measured during 3 consecutive days using metabolic chambers. Up to a 5.8% reduction in ad libitum DMI was observed when 2.5 mg/kg BW of 3-nitrooxypropanol was fed (P = 0.03). Increasing level of 3-nitrooxypropanol linearly (P < 0.001) reduced CH4, with 33% less CH4 (corrected for DMI) at the highest level of supplementation compared with the control. Feed energy lost as CH4 was also reduced when 3-nitrooxypropanol was supplemented (P < 0.001). Molar proportion of acetate was reduced (P < 0.001) and that for propionate increased (P < 0.001) with increasing dose of 3-nitrooxypropanol, which in turn led to a reduction in the acetate to propionate ratio (P < 0.001). Total copy numbers of 16S ribosomal RNA (rRNA) genes for bacteria, methanogens, and 18S rRNA genes for protozoa in ruminal contents were not affected by 3-nitrooxypropanol supplementation (P ≥ 0.31). There was no effect of 3-nitrooxypropanol on DM (P = 0.1) digestibility in the total tract. The use of 4.5 mg/kg BW of 3-nitrooxypropanol in beef cattle consuming a backgrounding diet was effective in reducing enteric CH4 emissions without negatively affecting diet digestibility.

Sustained reduction in methane production from long-term addition of 3-nitrooxypropanol to a beef cattle diet.

The objective was to evaluate whether long-term addition of 3-nitrooxypropanol (NOP) to a beef cattle diet results in a sustained reduction in enteric CH4 emissions in beef cattle. Eight ruminally cannulated heifers (637 ± 16.2 kg BW) were used in a completely randomized design with 2 treatments: Control (0 g/d of NOP) and NOP (2 g/d of NOP). Treatments were mixed by hand into the total mixed ration (60% forage, DM basis) at feeding time. Feed offered was restricted to 65% of ad libitum DMI (slightly over maintenance energy intake) and provided once per day. The duration of the experiment was 146 d, including an initial 18-d covariate period without NOP use; a 112-d treatment period with NOP addition to the diet, divided into four 28-d time intervals (d 1 to 28, 29 to 56, 57 to 84, and 85 to 112); and a final 16-d recovery period without NOP use. During the covariate period and at the end of each interval and the end of the recovery period, CH4 was measured for 3 d using whole animal metabolic chambers. The concentration of VFA was measured in rumen fluid samples collected 0, 3, and 6 h after feeding, and the microbial population was evaluated using rumen samples collected 3 h after feeding on d 12 of the covariate period, d 22 of each interval within the treatment period, and d 8 of the recovery period. Average DMI for the experiment was 7.04 ± 0.27 kg. Methane emissions were reduced by 59.2% when NOP was used (9.16 vs. 22.46 g/kg DMI; P < 0.01). Total VFA concentrations were not affected (P = 0.12); however, molar proportion of acetate was reduced and that for propionate increased when NOP was added (P < 0.01), which reduced the acetate to propionate ratio (3.0 vs. 4.0; P < 0.01). The total copy number of the 16S rRNA gene of total bacteria was not affected (P = 0.50) by NOP, but the copy number of the 16S rRNA gene of methanogens was reduced (P < 0.01) and the copy number of the 18S rRNA gene of protozoa was increased (P = 0.03). The residual effect of NOP for most of the variables studied was not observed or was minimal during the recovery period. These results demonstrated that the addition of NOP to a diet for beef cattle caused a sustained decrease of methanogenesis, with no sign of adaptation, and that these effects were reversed once NOP addition was discontinued

Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

Significance Methane emission from the ruminant livestock sector—a by-product from enteric fermentation of plant biomass in the ruminant digestive system—is produced by methanogenic archaea and represents not only a significant amount of anthropogenic greenhouse gases contributing to climate change but also an energy loss and a reduction in feed efficacy. The present study elucidates the development and the unique mode of action of the highly specific inhibitor 3-nitrooxypropanol (3-NOP), which is targeting the nickel enzyme methyl-coenzyme M reductase in rumen archaea that catalyzes the methane-forming reaction. At the very low effective concentrations recently applied in vivo (dairy and beef cattle), 3-NOP appears to inhibit only methanogens and thus to be attractive for development as a feed supplement. Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.

Effect of 3-nitrooxypropanol on methane and hydrogen emissions, methane isotopic signature, and ruminal fermentation in dairy cows

The objective of this crossover experiment was to investigate the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP), on enteric methane emission, methane isotopic composition, and rumen fermentation and microbial profile in lactating dairy cows. The experiment involved 6 ruminally cannulated late-lactation Holstein cows assigned to 2 treatments: control and 3NOP (60 mg/kg of feed dry matter). Compared with the control, 3NOP decreased methane emission by 31% and increased hydrogen emission from undetectable to 1.33 g/d. Methane emissions per kilogram of dry matter intake and milk yield were also decreased 34% by 3NOP. Milk production and composition were not affected by 3NOP, except milk fat concentration was increased compared with the control. Concentrations of total VFA and propionate in ruminal fluid were not affected by treatment, but acetate concentration tended to be lower and acetate-to-propionate ratio was lower for 3NOP compared with the control. The 3NOP decreased the molar proportion of acetate and increase those of propionate, butyrate, valerate, and isovalerate. Deuterium-to-hydrogen ratios of methane and the abundance of (13)CH3D were similar between treatments. Compared with the control, minor (4‰) depletion in the (13)C/(12)C ratio was observed for 3NOP. Genus composition of methanogenic archaea (Methanobrevibacter, Methanosphaera, and Methanomicrobium) was not affected by 3NOP, but the proportion of methanogens in the total cell counts tended to be decreased by 3NOP. Prevotella spp., the predominant bacterial genus in ruminal contents in this experiment, was also not affected by 3NOP. Compared with the control, Ruminococcus and Clostridium spp. were decreased and Butyrivibrio spp. was increased by 3NOP. This experiment demonstrated that a substantial inhibition of enteric methane emission by 3NOP in dairy cows was accompanied with increased hydrogen emission and decreased acetate-to-propionate ratio; however, neither an effect on rumen archaeal community composition nor a significant change in the isotope composition of methane was observed.

Effects of sustained reduction of enteric methane emissions with dietary supplementation of 3-nitrooxypropanol on growth performance of growing and finishing beef cattle

The study objective was to evaluate the effects of sustained reduction of enteric methane (CH) emissions with dietary supplementation of the inhibitor 3-nitrooxypropanol (NOP) on growth rate and feed conversion efficiency of growing and finishing beef cattle. Eighty-four crossbred steers were used in a 238-d feeding study and fed a backgrounding diet for the first 105 d (backgrounding phase) and transition diets for 28 d followed by a finishing diet for 105 d (finishing phase) with 3 doses of NOP (0, 100, and 200 mg/kg DM). The experiment was a completely randomized design using 21 pens (4 cattle/pen) with 7 pens per treatment. When cattle were fed the backgrounding diet, pen DMI was reduced ( < 0.01) whereas G:F tended to improve ( = 0.06) with increasing dose of NOP supplementation. During the finishing phase, DMI ( = 0.06) and ADG ( = 0.07) tended to decrease with increasing dose of NOP supplementation. Although both levels of NOP were effective in reducing CH emissions from the backgrounding diet ( < 0.01), only NOP supplemented at the highest dose was effective in reducing total CH emissions from the finishing diet ( < 0.01). Methane yield (g/kg DMI) was reduced whereas hydrogen emissions were increased at the highest dose of NOP supplementation with both backgrounding and finishing diets ( < 0.01). Overall, these results demonstrate efficacy of NOP in reducing enteric CH emissions from cattle fed backgrounding and finishing diets, and these effects were negated once NOP supplementation was discontinued.