3-Nitrooxypropanol Decreased Enteric Methane Production From Growing Beef Cattle in a Commercial Feedlot: Implications for Sustainable Beef Cattle Production

Effects of the investigational methane (CH4) inhibitor 3-nitrooxypropanol (3-NOP) on animal performance, health and enteric CH4 production of beef cattle were evaluated in a commercial feedlot. Two concurrent studies were conducted: a large pen study (4,048 cattle, eight pen replicates per experimental group) to measure animal performance and health and a small pen study (a subset of 50 cattle from the large pen study, n = 25 per experimental group) to measure enteric CH4 emissions. Within the study, animals (body weight ± SD, 282 ± 8 kg) were assigned in a completely randomized design to one of two groups: control, fed a backgrounding diet (70% corn or barley silage, 30% steam-flaked barley grain concentrate; dry matter (DM) basis) and 3-NOP, fed the backgrounding diet containing 3-NOP. The treatment group in the large pen study was adapted to 3-NOP (12 ± 3 d) before receiving the final target level of 200 mg/kg of DM, which was fed for 108 ± 8 d. Animals in the small pen CH4 emissions study received a basal diet or a basal diet with 3-NOP, with the dose increased every 28 d: low (150 mg/kg DM; 1.27 g/d), medium (175 mg/kg DM; 2.25 g/d), and high (200 mg/kg DM; 2.75 g/d). Intake in the small pens was monitored by electronic feeding bunks and CH4 was measured using the GreenFeed system. In the large pen study, total weight gained, average daily gain, and animal health variables were not affected by 3-NOP, but DM intake (DMI) tended to decrease (P = 0.06) by 2.6% relative to control (8.07 kg/d), while gain:feed ratio tended to be improved (P = 0.06) by 2.5% relative to control (0.161 kg weight gain/kg DMI). In the small pen study, average daily consumption of 3-NOP increased with inclusion rate whereas average DMI was decreased by 5.4% (P = 0.02) compared with control (10.4 kg/d). On average, addition of 3-NOP decreased (P = 0.001) CH4 emissions (g/d) by 25.7% and yield (g CH4/kg DMI) by 21.7%. In conclusion, supplementing a backgrounding diet with 3-NOP decreased CH4 yield and tended to improve feed efficiency of beef cattle fed in a commercial feedlot with no negative impacts on animal health.

Effects of the investigational methane (CH 4 ) inhibitor 3-nitrooxypropanol (3-NOP) on animal performance, health and enteric CH 4 production of beef cattle were evaluated in a commercial feedlot. Two concurrent studies were conducted: a large pen study (4,048 cattle, eight pen replicates per experimental group) to measure animal performance and health and a small pen study (a subset of 50 cattle from the large pen study, n = 25 per experimental group) to measure enteric CH 4 emissions. Within the study, animals (body weight ± SD, 282 ± 8 kg) were assigned in a completely randomized design to one of two groups: control, fed a backgrounding diet (70% corn or barley silage, 30% steam-flaked barley grain concentrate; dry matter (DM) basis) and 3-NOP, fed the backgrounding diet containing 3-NOP. The treatment group in the large pen study was adapted to 3-NOP (12 ± 3 d) before receiving the final target level of 200 mg/kg of DM, which was fed for 108 ± 8 d. Animals in the small pen CH 4 emissions study received a basal diet or a basal diet with 3-NOP, with the dose increased every 28 d: low (150 mg/kg DM; 1.27 g/d), medium (175 mg/kg DM; 2.25 g/d), and high (200 mg/kg DM; 2.75 g/d). Intake in the small pens was monitored by electronic feeding bunks and CH 4 was measured using the GreenFeed system. In the large pen study, total weight gained, average daily gain, and animal health variables were not affected by 3-NOP, but DM intake (DMI) tended to decrease (P = 0.06) by 2.6% relative to control (8.07 kg/d), while gain:feed ratio tended to be improved (P = 0.06) by 2.5% relative to control (0.161 kg weight gain/kg DMI). In the small pen study, average daily consumption of 3-NOP increased with inclusion rate whereas average DMI was decreased by 5.4% (P = 0.02) compared with control (10.4 kg/d). On average, addition of 3-NOP decreased (P = 0.001) CH 4 emissions (g/d) by 25.7% and yield (g CH 4 /kg DMI) by 21.7%. In conclusion, supplementing a backgrounding diet with 3-NOP decreased CH 4 yield and tended to improve feed efficiency of beef cattle fed in a commercial feedlot with no negative impacts on animal health.
Keywords: beef cattle, enteric methane, environmental sustainability, greenhouse gas emissions, methane inhibitor INTRODUCTION As countries move toward greenhouse gas (GHG) emission neutrality by 2050 there is increasing pressure on ruminant livestock production to reduce enteric methane (CH 4 ) emissions. The Special Report of the International Panel on Climate Change [Intergovernmental Panel on Climate Change (IPCC), 2018] calls for a 24-27% reduction in CH 4 emissions from agriculture in order to limit a potential temperature increase to 1.5 • C. Methane has a much shorter lifetime (half-life; 8.6 years) than carbon dioxide (CO 2 ) in the atmosphere (>100 years, Muller and Muller, 2017), which makes it attractive for short-term gains in global warming abatement. Enteric CH 4 from ruminants comprises ∼4-6% of global anthropogenic GHG emissions (40% of all livestock emissions; Gerber et al., 2013). As a result, there is an urgent need to develop technologies and mitigation strategies that can be cost-effectively adopted by cattle producers to lessen the contribution of ruminant livestock to GHG emissions (Beauchemin et al., 2020).
Ruminant livestock produce enteric CH 4 as an end product of feed digestion. In the rumen, polysaccharides (mainly cellulose, hemicellulose, and starch) are hydrolyzed to glucose and other hexoses and pentoses, with the monosaccharides further metabolized to volatile fatty acids, CO 2 and dihydrogen (H 2 ). The volatile fatty acids are used by the animal as a main source of energy, while CO 2 and H 2 are used to form CH 4 , which is eructated to the atmosphere via the breath. This process allows ruminants to derive nutrients from forages and other cellulosic materials, thereby avoiding direct competition with humans. However, CH 4 is a potent GHG with a global warming potential of 28-times that of CO 2 [over a 100-year period, Intergovernmental Panel on Climate Change (IPCC), 2013].
A broad range of potential mitigation strategies has been proposed to decrease CH 4 emissions as outlined by comprehensive reviews (Hristov et al., 2013;Beauchemin et al., 2020). Among the strategies proposed, the investigational CH 4 inhibitor 3-nitrooxypropanol (3-NOP; DSM Nutritional Products Ltd., Kaiseraugst, Switzerland) shows tremendous promise with 20-80% decreases in CH 4 production depending upon the type of animal, diet composition, dose and method of supplementation (Hristov et al., 2015;Vyas et al., 2016aVyas et al., , 2018Dijkstra et al., 2018;McGinn et al., 2019). This inhibitor reduces methanogenesis in the rumen by inactivating the enzyme methyl-coenzyme M reductase used by archaea (Duin et al., 2016). The decrease in CH 4 production was shown to persist over several months when 3-NOP was included in the diets of lactating dairy cows (25-32% decrease, 12-week study ;Hristov et al., 2015) and growing beef cattle (37-42% decrease over 238 d; Vyas et al., 2018).
Incorporating 3-NOP into beef cattle diets to decrease CH 4 production could allow producers to participate in the carbon market, such as the carbon pricing system in Canada (Government of Canada, 2019), by trading CO 2 equivalents. Additionally, any improvement in animal performance [body weight (BW) gain, feed conversion efficiency] resulting from feeding 3-NOP could help offset the additional costs of using the feed additive. Therefore, it is important to evaluate, at a commercial scale, the results for CH 4 mitigation and animal performance reported in controlled research studies that evaluated 3-NOP. Previous beef cattle studies were conducted using individually housed animals (Romero-Perez et al., 2014 or small pens (≤10 cattle/pen) of animals (Vyas et al., 2016a(Vyas et al., ,b, 2018) that minimize social interactions among animals and thus do not reflect the conditions of commercial feedlots where animals are housed in large groups (>100 cattle/pen). Thus, the objectives of this study were to evaluate the effects of feeding 3-NOP to backgrounded beef cattle on feed consumption, animal performance, animal health and enteric CH 4 production in a commercial feedlot setting.

Research Overview, Site Description, and Diet
Two concurrent studies were conducted using procedures in accordance with the guidelines of the Canadian Council on Animal Care (2009) and with approval of the Veterinary Drug Directorate of Health Canada (DSTS No. 207171). The animal performance study evaluated the effects of 3-NOP supplementation on dry matter intake (DMI), average daily gain (ADG), feed conversion efficiency (gain:feed ratio, G:F), and health using cattle housed in 16 commercial feedlot pens. The effects of 3-NOP on enteric CH 4 production were evaluated in a second study using a subset of cattle maintained in two small pens. The animal performance study was conducted between November 2017 and September 2018 for a total of 108 ± 8 d (12 ± 3 d of adaptation, 89 ± 8 d at the final concentration for measurements), while the CH 4 production study was conducted between May and September, 2018 for a total of 112 d (28 d of adaptation, 84 d of measurement).
The research was conducted at a commercial beef cattle feedlot located near Nanton, Alberta, Canada. The basic design of the feedlot is representative of standard designs used in western Canada where animals are housed in open-air, dirtfloor pens arranged side-by-side with central feed alleys and 20% porosity wood-fence windbreaks. The 16 large pens used in the performance study had dimensions of ∼61 m × 70 m with a capacity of ∼250 animals per pen and were equipped with a concrete feed bunk along one side of the pen. The two small pens used in the CH 4 production study had dimensions of ∼15.5 m × 29.9 m with a capacity of 25 animals per pen and were equipped with four electronic feed bunks (GrowSafe, Calgary, Alberta, Canada) per pen. One of the pens was equipped with a GreenFeed emission monitoring (GEM) system (C-Lock Inc., Rapid City, SD, USA). There were three animal handling facilities located at the site. Each facility contained a hydraulic chute equipped with an individual animal scale, a chute-side computer with individual animal data collection and management software (iFHMS © ; Feedlot Health Management Systems, Okotoks, Alberta, Canada), and separation alleys to facilitate the return of animals to designated pens. Open-air containment pens are located adjacent to each facility.
A total of 4,048 mixed breed steers were assigned to the two concurrent studies. In the large pen study, the cattle were fed a backgrounding diet containing 70% whole crop barley (Hordeum vulgare L.) silage and 30% barley-based concentrate [dry matter (DM) basis; Table 1]. In the small pen study, the initial diet formulation contained barley silage but due to an unexpected shortage, corn silage (Zea mays) was used midway (starting August 19, 2018) until the end of the experiment. Micro-ingredients (ionophore, antibiotic, minerals, vitamins, and 3-NOP) were added to the ration via a water-based microingredient machine. The ionophore monensin (Rumensin, Elanco Canada Limited, Guelph, Ontario, Canada) was included at 25 mg/kg DM and chlortetracycline (Chlor 100, Bio-Agri Mix, Mitchell, Ontario, Canada) was included at 35 mg/kg DM for liver abscess control. The 3-NOP was a dry granular product with an active ingredient concentration of 100 g 3-NOP/kg. The rations were mixed thoroughly before delivery into the feed bunks, and prior to starting the experiment, the mixer underwent testing to validate the weigh scale and mixing consistency.

Animal Performance and Health Study
The animal performance and health study was conducted in large pens (average 253 animals/pen; range: 244-263 animals/pen), with eight replicates. Each replicate was assigned to two pens; control and 3-NOP, and thus the experimental unit was the pen (n = 8/treatment group). Animals (mixed breed beef steer calves) for the study were sourced from various auction markets across Canada and randomly allocated to treatments upon arrival to the feedlot or at re-handling (39 d after arrival). The average (±SD) initial individual animal live weight of study pens was 283 ± 9 and 282 ± 8 kg for the control and 3-NOP groups, respectively. Animals in the control group (2,025 animals) were fed the TABLE 1 | Ingredient inclusion rate and diet composition (mean ± SD) for the basal diet used in the animal performance and methane production studies.

Item
Basal diet a backgrounding basal diet without 3-NOP whereas animals in the 3-NOP group (2,023 animals) were fed the same basal diet containing 3-NOP at a concentration of 200 mg/kg DM basis. Animals in the 3-NOP group were incrementally adapted to reach the final concentration of 3-NOP: 100 mg 3-NOP/kg DM for 7-10 d, 150 mg 3-NOP/kg DM for 7 d, and the final concentration of 200 mg/kg DM until the end of the study. The dose of 3-NOP was based on previous studies including Vyas et al. (2016aVyas et al. ( ,b, 2018 and Romero-Perez et al. (2014. Feed was delivered twice daily (and topped-up throughout the day when needed) by mixer trucks equipped with load cells. Feed bunks were monitored every day before morning feeding and feed adjustment was made based on slick-bunk management (Schwartzkopf-Genswein et al., 2003;Schutz et al., 2011). Daily DMI was calculated as the total feed delivered to the pen daily, adjusted for DM content, divided by the number of animals in the pen. As minimal feed remained in the feed bunk prior to the next day's feed allocation, it was not necessary to account for feed refusals.
Animal health was monitored by experienced herdsmen and veterinarians. The herdsmen conducted health monitoring on a daily basis and used subjective criteria based on modified DART system (Depression, Appetite, Respiratory signs and Temperature; Step et al., 2008) for identification and further evaluation and treatment of sick animals. Animals were weighed before feeding (non-fasted) once at the start and once at the end of the study to determine body weight (BW) gain. The weight gain was divided by number of days on the study to calculate ADG (shrunk), and feed conversion efficiency was measured as G:F (kg:kg) ratio by dividing ADG by daily DMI.

Methane Production Study
A subset of animals allocated to one of the replicates (Replicate 8) in the animal performance and health study (80 animals, 40 from each experimental group) were removed from the large pens at the start of the study, weighed over two consecutive days (nonfasted), and ranked by BW. Twenty-five candidate animals from each group were then selected based on BW to provide two balanced groups for the CH 4 production study. The remaining 15 animals from each experimental group were returned to the appropriate large pen. The selected animals (initial BW = 328 ± 29 kg) were maintained on their respective experimental group assignments throughout the study: control and 3-NOP. Prior to starting the study, the animals in the 3-NOP group received the backgrounding diet containing 100 mg 3-NOP/kg DM for 7 d. Once assigned to the CH 4 production study, they were provided a 21-d adaptation (adaptation phase) during which they continued to receive the diet containing 150 mg 3-NOP/kg DM. The adaptation phase allowed the animals to become familiar with the CH 4 emission monitoring system described below. Following the adaptation phase, the study was conducted in three 28-d phases, with the concentration of 3-NOP in the basal diet increased in each phase: low (150 mg/kg DM) in Phase 1, medium (175 mg/kg DM) in Phase 2, and high (200 mg/kg DM, final dose) in Phase 3. An additional 2 weeks of adaptation was added between Phases 1 and 2, during which barley silage was replaced with corn silage due to a shortage of barley silage at the feedlot. Animals were fed twice a day (0900 and 1,500 h) for ad libitum intake (5% orts). Orts were removed and weighed weekly. Because individual animal data were obtained for gas measurements and DMI, the experimental/observational unit was the animal (Bello and Renter, 2017).

Emission Measurements
Methane and H 2 were measured using the GEM system. Each week the two pens of animals were rotated, such that each pen of cattle had access to the GEM system for 1 week, every second week. This approach controlled any possible pen effects and has been implemented previously (Alemu et al., 2019(Alemu et al., , 2020. Within a phase, each pen had access to the system for two 7-d periods. The GEM system allows free movement of animals (in and out of the system) and gasses are measured only when the animal's head is in the "head chamber" unit as determined by the proximity sensor. The system is equipped with a radio-frequency reader that identifies the electronic ear tag of each animal. During a visit to the GEM system, animals are provided with pellets from an overhead hopper (as bait). The interval between pellet drops was set to 35 s to keep the animals in the hood for sufficient time (3-7 min) to obtain a full measurement. Maximum daily pellet drops per animal (36 drops, 6 drops/visit) was set to restrict the amount of pellet consumption. The total amount of pellet DM consumed per animal per day was added to the animal's intake of basal diet DM from the GrowSafe bunks, to calculate total DMI. Animals could visit the system any time but they were eligible for pellet drops only during six visits that were spaced a minimum of 4 h apart during each 24-h cycle. Thus, animals were required to wait 4 h before getting their next pellet drop. The pellet was composed of ground barley, canola meal and oil, dried molasses and salt with a composition of (% of DM): 14.4 ± 1.10 crude protein (CP), 22.0 ± 1.62 neutral detergent fiber (NDF), 8.23 ± 0.48 acid detergent fiber (ADF), and 20.5 ± 0.29 MJ/kg gross energy.
Once the animal's head is in the hood of the GEM system, air is drawn past the nose and mouth of the animal at about 26-40 L/s into the collection pipe. The system measures CH 4 , CO 2 , and H 2 continuously together with air flow, temperature, atmospheric pressure, and relative humidity. Each gas is analyzed by a separate non-dispersive infrared analyzer, which was calibrated weekly using a zero (semipure nitrogen) and span CH 4 and CO 2 gases, with nitrogen as the balance gas. The purpose of the calibration was to define sensor responses to known concentration of gasses. Five times during the experiment, the air flux sensor was calibrated by releasing a gravimetrically determined quantity of CO 2 into the system using a 90-g prefilled CO 2 cylinder for 3 min (at least three times). The amount released was compared to the calculated capture (96.7% CO 2 recovery, SD = 4.6, n = 5). To maintain a consistent airflow rate between 27 and 40 L/s in the collection tube (Velazco et al., 2016), the air filter was cleaned and changed regularly (every 3-5 days).
To calculate mass flux of CH 4 , the measured increase in concentration in the animal's breath relative to that in the ambient (background) air was multiplied by the measured air flow rate, and then the ideal gas law was applied.
Where, F C is the dimensional factor; 1/C R is the capture rate of emissions into collection pipes determined using a tracer (%); t is time period over which emissions are measured (1 s); CH 4−average is average concentration of CH 4 (%); CH 4−background is background concentration of CH 4 (%); and Q air is air flow rate (flow per unit time). Once the volume flow rates of CH 4 are determined, the mass flux is determined by applying the ideal gas law. Daily CH 4 emission for individual animals was calculated over the 7-d by aggregating and averaging the visit fluxes by time of day (for each 4-h block). Within each phase, only cattle with ≥10 useful/good visits with visits in at least five of the six 4-h time blocks were used in the analysis to ensure that the full diurnal cycle of emission was represented, as CH 4 emissions fluctuate over the 24-h cycle (Gunter and Bradford, 2015;Manafiazar et al., 2016). Hydrogen was calculated using an "arithmetic averaging method, " which is a straight averaging of the visit fluxes (Manafiazar et al., 2016).

Sample Collection and Analysis
Feed ingredients and the basal diet offered were sampled every day during the CH 4 emission study and composited by week for further chemical analyses. A sample of pellets from the GEM system was collected every 2 weeks for chemical analysis. The chemical composition of ingredients and basal diet are presented in

Statistical Analysis
For the large pen study, the animal performance data (DMI, ADG and G:F) for each pen were analyzed using the GLIMMIX procedure of SAS (SAS Inst. Inc., Cary, NC) where experimental group (control, 3-NOP) was considered a fixed effect and replicate (1-8) a random effect. Morbidity and mortality data  were analyzed using the GENMOD procedure in SAS where experimental group was considered a fixed effect and pen within replicate as a clustering effect. For the small pen CH 4 study, DMI and gas data for each animal were analyzed by phase and overall using the MIXED procedure of SAS. Within phase, the model included experimental group (control, 3-NOP), week (1-4 for DMI, 1 and 2 for gases), and their interaction as fixed effects, with week considered as a repeated effect in the model. Kenward-Roger's option was used in the model statement to estimate denominator degrees of freedom. Best time-series covariance structure was selected on the basis of the lowest Akaike and Bayesian information criteria and compound symmetry was used. Residual plots were used to check the validity of the underlying statistical assumptions of homogeneity of variances and normality. Statistical significance was declared at P < 0.05 and trends are discussed at P ≤ 0.10.

Animal Performance Study
Inclusion of 3-NOP tended to decrease DMI (2.6%, P = 0.06), without affecting total weight gain (P = 0.87) or ADG (P = 0.87) ( Table 2). As a result, G:F tended to improve (2.5%; P = 0.06) by feeding 3-NOP compared with the control (0.16 kg liveweight gain/kg DMI). The pattern of DMI over the study showed that the cattle fed 3-NOP generally had lower intake during the first 6 weeks of the feeding period (Figure 1). There were no differences in morbidity or mortality detected between the experimental groups (Table 3). However, there was a tendency for animals fed 3-NOP to have a slightly greater percentage (P = 0.07) of treatment of animals for relapse without a fever.

Methane Production Study
The measured concentrations of 3-NOP in the basal diet offered in the GrowSafe bunks and calculated concentrations in the total feed consumed (basal diet plus pellet offered in the GEM system) during the study are presented in Table 4. Recovery of 3-NOP in the basal diet averaged 105.7%, ranging from 89.7 to 120.7%, which is within an acceptable range for most feed additives. Calculated concentration of 3-NOP in the total DMI, which accounts for intake of the basal diet and the pellets offered in the GEM system, was 124.6, 192.8, and 226.8 mg/kg DM for the low, a Basal diet was provided in the GrowSafe feed bunks and total diet refers to basal diet + pellets provided in the GreenFeed system. 3-NOP concentration in the control diet was zero. b Animals adapted to 3-NOP and the GreenFeed emission monitoring system for 28 d. c Calculated as: (measured 3-NOP concentration/target concentration) × 100. d Daily 3-NOP intake was calculated from the measured concentration in the basal diet and the measured total DMI of each animal. medium and high doses respectively. Average daily consumption of 3-NOP increased with inclusion rate as expected, 1.27 g/d for the low, 2.25 g/d for medium, and 2.75 g/d for the high dose. Dry matter intake for the low (phase 1), medium (phase 2), and high (phase 3) doses of 3-NOP is reported in Table 5. On average, total DMI was lower (P = 0.02) by 5.3% for 3-NOP as compared to the control treatment (10.43 kg/d). Intake was not affected by the low dose but was 5.8% less for the medium dose compared with control (11.55 kg/d) and 5.9% lower for the high dose compared with control (12.06 kg/d). For animals visiting the GEM system, average pellet consumption tended to be greater (P = 0.07) for the 3-NOP group (0.36 kg/d) relative to the control group (0.27 kg/d).
Of the 25 animals assigned to each treatment, on average, 76% of animals (ranging between 60 and 84%) for control and 88% of animals (ranging between 80 and 92%) for 3-NOP visited the GEM system ( Table 6). Over the study period, the average total visits were 1,156 and 1,556 for control and 3-NOP groups, respectively. The number of weekly visits to the GEM system and the number of 4-h blocks in which visits occurred were not affected by treatment group in any phase (P ≥ 0.20). The visits to the GEM system were relatively consistent throughout the 24-h period, with the exception of between 0300 and 0400 h (Figure 2). On average, each visit to the GEM system lasted slightly more than 4 min and did not differ (P ≥ 0.48) between treatment groups ( Table 6). a Visits were compiled into six 4-h blocks corresponding to time of day. Only "useful/good" visits of at least 3-min were used to calculate weekly visits per animal (Arthur et al., 2017;Beck et al., 2018). Only animals with ≥10 "useful/good" weekly visits, with visits in at least 5 of the six 4-h time blocks were selected for final analysis. b Low dose = 150 mg 3-NOP/kg DM, medium dose = 175 mg 3-NOP/kg DM, high dose = 200 mg 3-NOP/kg DM. 3-Nitrooxypropanol was added only to the basal diet.  DMI, dry matter intake. a Low dose = 150 mg 3-NOP/kg DM, medium dose = 175 mg 3-NOP/kg DM, high dose = 200 mg 3-NOP/kg DM. 3-Nitrooxypropanol was added only to the basal diet. b Only animals with ≥ 10 "useful/good" weekly visits, with visits in at least 5 of the six 4-h time blocks were selected for CH 4 analysis. Methane and H 2 production (g/d) and yield (g/kg DMI) for the low (phase 1), medium (phase 2), and high (phase 3) doses of 3-NOP are reported in Table 7. Methane production (g/d) decreased (P ≤ 0.01) by 17.4, 28.8, and 28.1% for the low, medium and high doses of 3-NOP, respectively, compared with control, averaging 255.2 g/d. Thus, over the entire study, 3-NOP reduced CH 4 production by an average of 25.7% (P = 0.001). Methane yield (g/kg DMI) followed a similar pattern with decreases of 17.2, 25.7, and 21.3% for low, medium and high doses of 3-NOP, respectively, with a 21.7% decrease overall (P = 0.001). When CH 4 energy was expressed as percentage of gross energy intake, feeding 3-NOP decreased emissions by 19.1, 23.9, and 18.4% for the three doses respectively, and by 20.8% overall (P ≤ 0.01). The reduction in CH 4 yield by phase is compared with previous literature for beef cattle fed high-forage diets in Figure 3.The average diurnal pattern of CH 4 emissions during the study by treatment is presented in Figure 4A.
With the decrease in CH 4 production due to 3-NOP, there was a concomitant increase in H 2 production, which increased from 0.39 g/d for control to 1.50 g/d (P < 0.001) for 3-NOP overall. The increases in H 2 emissions were consistent within each phase. The average diurnal pattern of H 2 production during the study by treatment is presented in Figure 4B.

Animal Performance and Health
Two previous controlled research studies conducted in small pens examined the effects of including 3-NOP in high-forage backgrounding diets on animal performance (Vyas et al., 2016a(Vyas et al., , 2018. In a 105-day study, Vyas et al. (2016a) reported that FIGURE 4 | Mean hourly enteric methane emissions (A) and hydrogen production (B) from feedlot steers in the methane production study fed a backgrounding diet without (control) and with 3-nitrooxypropanol (3-NOP) supplemented at increasing concentrations in the basal diet: low (150 mg 3-NOP/kg DM, Phase 1), medium (175 mg 3-NOP/kg DM, Phase 2), and high (200 mg 3-NOP/kg DM, Phase 3). Animals were fed twice a day at 0900 h (indicated as 0) and 1500 h (indicated by the arrow), and error bars indicated standard error.
inclusion of 3-NOP (200 mg/kg DM) reduced DMI by 8.0% and increased G:F by 7.7%. Using the same concentration of 3-NOP, Vyas et al. (2018) reported 9.2% reduction in DMI and 6.5% improvement in G:F for beef steers relative to control. Several factors including improved energy status of the animal as a result of decreased energy loss in the form of CH 4 and a shift in metabolic precursors may account for the observed reduction in DMI and improved efficiency Alemu et al., 2020). The present study is the first to examine the effects of 3-NOP in a commercial feedlot setting in which animals were fed a backgrounding diet and group-housed in large pens that are representative of the scale that exists in commercial production scenarios, which may create considerable competition among animals at the feed bunk. For beef cattle fed forage-based diets, Custodio et al. (2017) reported that feeding behavior of cattle can be influenced by housing system (individual vs. collective pen). Before beef producers use 3-NOP to mitigate CH 4 emissions they need information on the health and performance of animals fed 3-NOP in commercial conditions, in addition to efficacy of CH 4 reduction.
Using a 3-NOP concentration of 200 mg/kg DM in the performance study reduced DMI by 2.6%, which was considerably less than that observed in previous small-scale research studies (Vyas et al., 2016a(Vyas et al., , 2018. The relatively small decrease in DMI of animals in the performance study (2.6%) also contrasts with the observed 5.9% reduction in DMI of animals in the CH 4 study, when fed the high dose (200 mg 3-NOP/kg DM). The greater decline in DMI of cattle fed 3-NOP in smallpen studies does not appear to be due to lack of adaptation of animals to 3-NOP, because in the present studies the animals were gradually transitioned to 3-NOP. Furthermore, during CH 4 measurements, for which the reduction in DMI was greatest, the transition was gradual with use of an adaption period followed by a phased step-up. As DMI relative to BW was similar for the control animals in both the performance and CH 4 studies, the reasons for the greater decline in DMI of animals fed 3-NOP in the small pens are not clear. It is possible that the differing response is related to differences in competition at the feed bunk caused by the type of feeder (long trough in the large pens vs. feed bins in the small pens), feed bunk management (slick-bunk vs. ad lib, Schwartzkopf-Genswein et al., 2003;Schutz et al., 2011) and the frequency of feed allocation (top-up throughout the day in large pens vs. 2-times daily in small pens). The smaller decline in DMI observed in the animal production study may account for the relatively smaller (2.5%) improvement in feed conversion efficiency compared with the 6.5-7.7% improvement observed in previously reported small-pen studies (Vyas et al., 2016a(Vyas et al., , 2018.
Animal health in response to 3-NOP supplementation has not been previously documented. Although the finding of no increased risk of mortality and morbidity in cattle fed 3-NOP has important implications for future use of the product by commercial feedlots, the observed tendency for the treatment of animals with relapse without a fever requires further investigation.

Gaseous Emissions
A meta-analysis of data from 11 experiments (Dijkstra et al., 2018) indicated that with a mean inclusion rate of 123 mg 3-NOP/kg DM, enteric CH 4 production (g/d) and yield (g/kg DM) were reduced by 22.2 and 17.1%, respectively, in beef cattle fed a range of diets. Those authors also indicated that the effect of 3-NOP on enteric CH 4 production was positively associated with dose and inversely associated with diet NDF concentration. Thus, the response to 3-NOP at a particular level of 3-NOP would be expected to be less for backgrounded cattle fed higher fiber diets compared with finishing cattle fed grainbased diets. The reduction in CH 4 yield observed in the present study (decrease of 20.1, 25.5, and 21.1% for low, medium and high doses of 3-NOP; 21.7% decrease overall) is consistent with the previous literature (Figure 3), although a linear response to 3-NOP concentration was not observed in the present study. When examined over nine beef studies (including the present study) with 17 treatment means of cattle fed high-forage diets (≥60% forage DM), the linear response to targeted dietary concentration of 3-NOP is: Reduction in CH 4 yield = 0.1389 × mg 3-NOP/kg DM (R 2 = 0.73). The reason for the lack of linear response to 3-NOP dose in the present study is not clear. One factor may be the study design wherein each dose of 3-NOP was evaluated sequentially, rather than simultaneously, and hence the cattle were exposed to 3-NOP for a differing number of days at each dose, making it difficult to compare the animal responses across dose.
Methane is the largest sink of H 2 in the rumen. When methanogenesis is inhibited gaseous H 2 can accumulate (Hristov et al., 2015;Vyas et al., 2018), as was observed in the present study where H 2 was on average 1.50 g/d for the 3-NOP group compared with 0.39 g/d for control cattle. Although the release of gaseous H 2 represents an inefficiency of energy utilization, the loss of energy as H 2 was only 4% of the energy potentially available from the decrease in CH 4 production (65.7 g CH 4 , 55 kJ/g vs. 1.11 g H 2 , 142 kJ/g). In terms of reducing equivalents, 65.7 g/d of spared CH 4 is equivalent to releasing 16 g/d H 2 . It appears that <2% of the spared H 2 was released as gas, indicating that >98% was diverted toward dissolved H 2 and alternate H 2 sinks in the rumen (e.g., such as formate, propionate, valerate, caproate, heptanoate, unsaturated fatty acids, nitrate and sulfate reduction, and microbial protein synthesis; Guyader et al., 2017). This shift in H 2 flow within the rumen toward nutritionally beneficial sinks may partially account for the observed improvement in G:F.

Implications for Sustainable Beef Production
Approximately 80% of Nationally Determined Contributions to meet the commitments of the Paris Agreement specifically mention agriculture [(Food and Agriculture Organization of the United Nations (FAO), 2019a)], highlighting its important role in mitigating GHG emissions. Additionally, 54 countries have set goals of decreasing emissions from livestock (Richards et al., 2015). Consequently, beef production, which has the greatest GHG emissions per gram of protein produced (Poore and Nemecek, 2018), is under increasing pressure to decrease emissions. The GHG intensity (kg CO 2 equivalent/kg carcass) of beef production continues to decrease over time in many countries (e.g., 14.5% decrease between 1981 and 2011 in Canada; Legesse et al., 2016), due to improvements in management, health, nutrition, and genetics of animals, as well as manure management, grazing management, crop production and decreased land conversion (Mayberry et al., 2019). However, a decrease in GHG intensity due to improved efficiency of production will not be sufficient to meet targets for absolute GHG reductions if animal production continues to expand to meet the demand for food security. As enteric CH 4 represents more than 50% of farm-based GHG emissions of beef production (Beauchemin and McGeough, 2013), reducing enteric CH 4 emissions has been identified as a key means of reducing emissions from the red meat sector (Mayberry et al., 2019). Thus, providing beef producers with effective mitigation options is critical. The present study conducted under commercial feedlot conditions confirms previous small scale research studies that show 3-NOP has tremendous potential for CH 4 mitigation for beef production (Vyas et al., 2016a(Vyas et al., , 2018. Achieving carbon neutral beef production will undoubtedly increase the cost of production as well as the retail price of meat (Mayberry et al., 2019). Some of the additional costs to farmers of using CH 4 inhibiting feed additives may be at least partially offset by revenues from participating in voluntary carbon offset markets. For example, beef feedlots in Alberta, Canada, can participate in the Alberta Emission Offset System (www.alberta.ca/alberta-emission-offset-system.aspx) by quantifying reductions in CH 4 using scientifically valid methodologies. Furthermore, improvements in animal performance would lead to greater revenues per animal sold. The observed tendency in feed conversion efficiency improvement (2.5%) in the present study would be economically significant to the cattle industry, as feed costs represent the largest source of total input costs (>60%). Feed conversion efficiency has a substantial impact on revenue per animal sold (Retallick et al., 2013). For example, using current feed costs, a 1% improvement in feed conversion efficiency is estimated to save the Canadian feedlot sector $11.1 million annually (Buchanan-Smith and Wood, 2019). The Canadian beef industry is the 11th largest beef-producing country and the 5th largest exporter of beef globally [(Food and Agriculture Organization of the United Nations (FAO), 2019b)] with over 2.5 million cattle finished annually (Statistics Canada, 2019). In the U.S., where there are over 11 million beef cattle on feed at any one time (Cowley et al., 2019), the impact of feeding 3-NOP on reducing CH 4 emissions and improving feed conversion efficiency could be substantial. Furthermore, improvements in feed conversion efficiency decrease the demand for feed inputs resulting in fewer GHG emissions from feed production, less land required for feed production, and decreased manure output . Thus, the observed 2.5% improvement in G:F and 21.7% decrease in CH 4 yield in the present study could have both significant environmental and economic implications for beef production systems in North America and elsewhere, if 3-NOP is approved by licensing authorities and made commercially available. However, further studies are needed to validate the efficacy of 3-NOP for CH 4 mitigation and determine its effects on animal performance when used in commercial beef production systems with varying animal types, diets (including high-grain finishing diets), and management conditions.

CONCLUSIONS
This research is the first to show the effects of feeding 3-NOP on feed consumption, animal performance, animal health and enteric CH 4 production of beef cattle fed a backgrounding diet in a commercial feedlot. Feeding 3-NOP tended to reduce DMI but improved G:F by 2.5%. No negative impacts on animal health (mortality and morbidity) were observed. Feeding 3-NOP resulted in a sustained reduction in enteric CH 4 yield of 22%, on average (ranging from 20 to 26% depending upon dose). Assuming it becomes commercially available, 3-NOP has great potential to reduce GHG emissions from the beef industry, particularly the feedlot sector where use of feed additives and nutritional supplements is commonplace.