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Greenhouse gas and ammonia emissions from stored manure from beef cattle supplemented 3-nitrooxypropanol and monensin to reduce enteric methane emissions


The investigative material 3-nitrooxypropanol (3-NOP) can reduce enteric methane emissions from beef cattle. North American beef cattle are often supplemented the drug monensin to improve feed digestibility. Residual and confounding effects of these additives on manure greenhouse gas (GHG) emissions are unknown. This research tested whether manure carbon and nitrogen, and GHG and ammonia emissions, differed from cattle fed a typical finishing diet and 3-NOP [125–200 mg kg−1 dry matter (DM) feed], or both 3-NOP (125–200 mg kg−1 DM) and monensin (33 mg kg−1 DM) together, compared to a control (no supplements) when manure was stockpiled or composted for 202 days. Consistent with other studies, cumulative GHGs (except nitrous oxide) and ammonia emissions were higher from composted compared to stockpiled manure (all P < 0.01). Dry matter, total carbon and total nitrogen mass balance estimates, and cumulative GHG and ammonia emissions, from stored manure were not affected by 3-NOP or monensin. During the current experiment, supplementing beef cattle with 3-NOP did not significantly affect manure GHG or NH3 emissions during storage under the tested management conditions, suggesting supplementing cattle with 3-NOP does not have residual effects on manure decomposition as estimated using total carbon and nitrogen losses and GHG emissions.


Agricultural activities represent roughly one third of global anthropogenic greenhouse gas (GHG) emissions1. Cattle are responsible for up to 17% of global methane (CH4) production, most of which is from ruminant enteric fermentation2. Methane from enteric fermentation and manure in beef cattle production represents between 40 and 70% of the total GHGs from North American beef operations3,4, and animal housing and manure storage accounts for between 5 and 15% of total nitrous oxide (N2O) emissions5.

In North America, finishing beef cattle are fed mixed diets in commercial feedlots where they regularly receive additives. The ionophore monensin (Rumensin, Elanco Animal Health, Guelph, Ontario, Canada) is one of the most common additives administered to cattle as it improves feed efficiency6 and reduces the risk of bloat and rumen acidosis7,8. Ionophores like monensin may also reduce enteric CH4 production by decreasing the availability of primary substrates (hydrogen and formate) for ruminant methanogens8,9. In some cases, dietary supplementation of monensin has been found to alter stored manure chemical composition10 and may lower CH4 emissions from manure11, but the response is influenced by cattle diet, age and breed12.

3-Nitrooxypropanol (3-NOP, DSM Nutritional Products AG; Kaiseraugst, Switzerland) is an investigative material that has been developed to reduce enteric CH4 production. 3-Nitrooxypropanol works by specifically inhibiting the methyl-coenzyme M reductase, an enzyme in methanogenic archaea that catalyses the CH4-forming reaction in the rumen13. The inhibitory effects of 3-NOP in the rumen are transient13,14,15 as 3-NOP is degraded in the rumen by its own mode of action and therefore it is highly unlikely to reach the lower digestive tract to be excreted in manure13. Past beef cattle studies have reported that 3-NOP does not significantly affect the digestibility of organic matter, crude protein, neutral detergent fibre, acid detergent fibre or starch16. However, it is unknown whether 3-NOP supplementation alters manure chemical properties through its effects on rumen fermentation in such a way that it will affect manure decomposition during storage.

Storage affects manure decomposition and therefore manure GHG and ammonia (NH3) emissions. In a typical western Canadian beef feedlot, cattle are held in pens, and the dry solid manure is removed from the pens and spread onto nearby fields as a source of nutrients and organic matter17. Manure is often stockpiled or composted prior to application to fields17,18. Stockpiles heap manure into stacks with no active management of the pile19, while manure piles that are composted are turned or otherwise aerated20. Composting introduces oxygen (O2) into the manure piles, so it is generally expected that composted manure emits more aerobically-produced gases like carbon dioxide (CO2) and NH3,19,21 while stockpiled manure is expected to emit more anaerobically-produced gases like CH420. Both high18,22,23 and low24 N2O emissions from composted manure have been reported. Emissions of these gases are related to manure carbon (C) and nitrogen (N) components, the O2 concentrations and other environmental conditions during decomposition.

No studies have assessed GHG and NH3 emissions from stored manure from cattle supplemented with 3-NOP. It is not known whether supplementing cattle with 3-NOP and monensin affects manure decomposition during storage. Mass balance estimates of manure C and N adjust concentrations to compensate for losses in dry matter during storage17. Total C and N mass balances could help evaluate whether processes during decomposition differ for manure from cattle supplemented with 3-NOP compared to manure from cattle that did not receive the supplement. If there are differences in manure C and N mass balances during storage because of the addition of 3-NOP and monensin to the diet, the resulting manure GHG and NH3 emissions may also be affected. Whether the GHG reductions achieved from lower enteric CH4 production with 3-NOP supplementation are negated by alterations to GHG and NH3 emissions during manure storage has not been assessed.

The objective of this research was to test if total carbon (TC) and total nitrogen (TN) mass balances, and GHG (CH4, CO2 and N2O) and NH3 emissions, from stockpiled and composted manure differed when manure piles were constructed with manure from cattle that received dietary 3-NOP or 3-NOP and monensin (3-NOP + Mon) compared to manure from cattle that were not fed these additives.


Climate and pile characteristics

During the 202-day trial (September 6, 2016, to March 28, 2017), there were 40 days of measurable precipitation (total = 116.8 mm, rain = 22.6 mm, snow = 116.8 cm, Fig. 1a). Daily average air temperature followed typical seasonal trends for the Canadian prairies from September to March, ranging from − 30 to 20 °C (Fig. 1b).

Figure 1

(a) Precipitation as rain and snow and (b) air temperature and mean pile temperatures by treatment (control, no supplements; 3-NOP, 3-nitrooxypropanol; 3-NOP + Mon, 3-nitrooxypropanol and monensin) for the stockpile and compost from September 2016 to March 2017.

At the start of the trial, the manure was dry (0.34 kg kg−1). The composted manure surface area ranged from 36 to 38 m2 (average initial length = 6.72 m, width = 4.94 m and height = 1.37 m) and the stockpiled manure surface area averaged 26 m2 (average initial length = 5.31 m, width = 4.93 m and height = 1.32 m). The initial wet mass of the composted manure piles averaged 14,278 kg (± 292 kg s.d.) and the initial wet mass of the stockpiled manure piles averaged 9,665 kg (± 201 kg).

All manure piles reached internal temperatures of ~ 60 °C (Fig. 1b). Mean internal pile temperature between days 1 and 104 was 8 °C higher in the composted manure than in the stockpiled manure. Between days 1 and 104, the average pile temperature of the composted manure was 47 °C higher and stockpiled manure was 39 °C higher than between days 105 and 202 as decomposition rates, and therefore internal pile temperature, decreased after day 105 (Fig. 1b).

Gas emissions

Carbon dioxide

Between days 1 and 104, CO2 fluxes from the composted manure averaged 66.9 g C m−2 d−1 and from the stockpiled manure averaged 40.3 g C m−2 d−1 (Fig. 2a, b). Carbon dioxide fluxes were lower between days 105 and 202, averaging 1.2 g C m−2 d−1 from the composted manure and 0.7 g C m−2 d−1 from the stockpiled manure.

Figure 2

Daily surface-to-atmosphere carbon dioxide fluxes from the (a) composted and (b) stockpiled manure and methane fluxes from the (c) composted and (d) stockpiled manure from days 0 to 202. Treatment daily means (control is no supplements, 3-NOP is 3-nitrooxypropanol and 3-NOP + Mon is 3-nitrooxypropanol and monensin) are presented ± the standard error of the mean.

Between days 1 and 104, CO2 flux trends differed by handling method over time. Stockpiled manure CO2 fluxes were high at the beginning of the trial and generally declined with time (Fig. 2a). Composted manure CO2 fluxes were low at the start of the trial and increased after compost turning (Fig. 2b). The control, 3-NOP and 3-NOP + Mon treatments showed similar patterns of variability over time within the same handling method with some daily between-treatment variation noted within the first 20 days.

Cumulative CO2 emissions did not significantly differ by additive treatment, but they were affected by handling method when averaged across all additive treatments (Table 1). Compared to stockpiled manure, cumulative CO2 emissions from composted manure were on average 102% higher when expressed as kg C m−2 (P < 0.001), 179% higher when expressed as kg C pile−1 (pile surface area, P < 0.001) and 162% higher when expressed as CO2 kg C tonne−1 of initial C (P < 0.001).

Table 1 Cumulative gas emissions over the entire trial by additive treatment and pile handling method.


Between days 1 and 104, CH4 fluxes averaged 2.6 g C m−2 d−1 from the composted manure and 0.7 g C m−2 d−1 from the stockpiled manure (Fig. 2c, d). Methane fluxes were negligible between days 105 and 202, averaging 7.5 × 10−4 g m−2 d−1 from the composted manure and 8.5 × 10−5 g C m−2 d−1 from the stockpiled manure.

Daily CH4 fluxes varied throughout the trial from the composted manure, with maximum fluxes occurring within one to two weeks after pile construction for all additives (Fig. 2c). While the timing of the maximum peaks in CH4 fluxes varied among additives, the temporal trends in CH4 fluxes from the composted manure were similar from all additives after the first compost turning on day 19. Generally, stockpiled manure CH4 emissions were low, with small spikes occurring at days 1, 8 and 49 of the trial (Fig. 2d). On those days, the largest CH4 spikes occurred from the 3-NOP treatment.

When averaged over all additives, cumulative CH4 emissions were 5.8 fold higher from the composted manure compared to the stockpiled manure when expressed as kg C m−2 (Table 1, P < 0.001). When expressed as CH4 kg C pile−1, cumulative CH4 emissions were 7.1 fold higher from the composted manure than from the stockpiled manure (P = 0.003). The CH4 kg C tonne−1 of initial C from the composted manure were 6.4 fold higher than from the stockpiled manure (P < 0.001).

Nitrous oxide

Between days 1 and 104, N2O fluxes averaged 0.5 g N m−2 d−1 from the composted manure and 0.4 g N m−2 d−1 from the stockpiled manure (Fig. 3a, b). From day 105 to 202, N2O fluxes averaged 0.03 g m−2 d−1 from the composted manure and 0.02 g m−2 d−1 from the stockpiled manure.

Figure 3

Daily surface-to-atmosphere nitrous oxide fluxes from the (a) composted and (b) stockpiled manure and ammonia fluxes from the (c) composted and (d) stockpiled manure from days 0 to 202. Treatment daily means (control in no supplements, 3-NOP is 3-nitrooxypropanol and 3-NOP + Mon is 3-nitrooxypropanol and monensin) are presented ± the standard error of the mean.

Daily N2O fluxes from composted manure were low throughout the trial. The exceptions were the measurements taken after compost turning where N2O fluxes from all additive treatments were between 6.2- to 24.8-fold higher when compared to measurements made the day prior to turning (Fig. 3a). From the stockpiled manure, daily N2O fluxes increased in all additives after day 28, but there were some differences in flux trends over time between additives: N2O fluxes from stockpiled 3-NOP manure were generally lower than the other additive treatments on day 40, and higher than other additive treatments on day 76 (Fig. 3b).

Despite some temporal discrepancies in daily N2O fluxes (Fig. 3a, b), there were no differences in cumulative N2O emitted from different handling methods, additive treatments, or any handling method by additive treatment interactions detected when emissions were presented as kg N m−2, kg N pile−1 or the kg N tonne−1 initial N (Table 1).


Between days 1 and 104, NH3 fluxes averaged 2.9 g N m−2 d−1 and 2.2 g N m−2 d−1 from the composted and stockpiled manure, respectively (Fig. 3c, d). Between days 105 and 202, NH3 fluxes averaged 0.1 g N m−2 d−1 from both composted and stockpiled manure.

Daily NH3 fluxes from composted and stockpiled manure were high early in the trial, peaking within the first six days after pile construction in all additive treatments (Fig. 3c, d). Within the respective handling methods, daily NH3 fluxes from all three additives were similar in magnitude throughout most of the trial and followed similar undulating trends.

There were no significant differences in cumulative NH3 emissions between the dietary additive treatments (Table 1). Cumulative NH3 emissions averaged over all additives on a kg m−2 basis were 83% higher from the composted compared to the stockpiled manure (P < 0.001). Cumulative NH3 emissions extrapolated over the initial surface area of the piles (kg pile −1) averaged over all additives were 158% higher from the composted compared to the stockpiled manure (P < 0.001). Compared to the stockpiled manure, the NH3 kg N tonne−1 initial N was 142% higher (P < 0.001) from the composted manure.

Manure chemistry

When averaged over all storage methods and additive treatments, manure TC concentrations, TN concentrations, C:N ratio, inorganic N concentrations, pH and electrical conductivity (EC) differed between days 1 and 202 (Table 2). All measured variables were higher on day 1 than on day 202 except pH and EC, which were higher on day 202. Manure storage affected TC concentrations; stockpiled manure TC concentrations at the end of the trial were lower (P < 0.10) than in initial samples, and composted manure TC concentrations at the end of the trial were lower (P < 0.10) than both the initial composted manure concentrations and the final stockpiled manure concentrations.

Table 2 Effects of manure handling method and additive treatments on manure chemical properties presented as mean ± standard error of the mean in parentheses.

Manure TC concentrations were significantly affected by the additive treatment (Table 2). When averaged over the entire trial, TC in the control (mean = 157.9 g C kg−1) and 3-NOP (mean = 158.1 g C kg−1) were lower (P ≤ 0.05) than in 3-NOP + Mon (mean = 170.5 g C kg−1).

Concentrations of TN were affected by day, decreasing between days 1 and 202 (P ≤ 0.10) in both the composted and stockpiled manure (Table 2), but TN was not affected by additive treatment or manure handling (Table 2).

Manure TC, TN and C:N ratios from the additive treatments were different between the beginning and end of the trial (Table 2). The TN, TC and C:N ratio for all additive treatments were lower on day 202 than on day 1 (P ≤ 0.10). The TN and C:N ratio for 3-NOP + Mon were higher than 3-NOP (P ≤ 0.10) on day 1, but by day 202, there were no differences between the additive treatments.

Stored manure carbon and nitrogen mass balance estimates

The mass balance estimates considered the changes in concentrations to compensate for losses in mass from the manure piles during the trial. Mass losses of DM as well as TC and TN tended to be higher in composted compared to stockpiled manure, however, these differences were not significant (Table 3).

Table 3 Mean ± standard error of the mean in parentheses showing the percent losses of dry matter, total carbon and total nitrogen from each pile handling method (P) and additive treatment (T) between the start and end of the trial (n = 18) determined by the mass balance analysis.

From the composted manure, the mass balance showed an equivalent of 90.9 kg C tonne−1 was lost from the control and 94.3 kg C tonne−1 was lost from 3-NOP + Mon, while only 55.9 kg C tonne−1 was lost from 3-NOP. Likewise, from the composted manure, an equivalent of 5.9 kg N tonne−1 was lost from the control and 5.3 kg N tonne−1 was lost from 3-NOP + Mon, while 3.2 kg N tonne−1 was lost from 3-NOP. While 3-NOP showed slightly lower mass losses of TC and TN compared to the control and 3-NOP + Mon in the composted manure, due to the high variability, these differences were not statistically significant (Table 3). The combination of slightly greater TC and TN losses from 3-NOP resulted in greater (P < 0.10) DM losses in 3-NOP compared to 3-NOP+Mon (Table 3).

There were discrepancies between the losses calculated using the mass balance approach (Table 3) and the GHGs lost as a fraction of the initial TC and TN in the compost (Table 1). It appears that more TC was lost as CO2 and CH4 than were lost as a fraction of the initial TC when calculated using the mass balance approach. The mass losses of TN were higher than the cumulative losses of N as NH3 and N2O (Table 1).

Across all additive treatments in the stockpiled manure, the mass balance calculations estimated that total losses of C averaged 58.3 kg C tonne−1 and total losses of N averaged 3.1 kg N tonne−1 (Table 3). The total losses of C estimated using the mass balance approach from the stockpiled manure were similar to the cumulative losses of CO2 and CH4 expressed as kg C tonne−1 initial C from the stockpiled manure (equating to 65.2 kg C tonne−1 C). The TN losses calculated using the mass balance approach (Table 3) were higher than those determined by cumulative losses of N2O or NH3 as kg N tonne−1 initial N from the stockpiled manure (Table 1).


This was the first study to assess whether GHG and NH3 emissions from composted and stockpiled beef cattle manure are affected by dietary supplementation with 3-NOP or a combination of 3-NOP and monensin. While 3-NOP and monensin supplementation can reduce enteric CH4 production from ruminant animals10,13,14, we found that they did not significantly affect cumulative GHGs or NH3 emissions from stored manure. Stored manure TC concentrations were affected by these dietary additives. However, the additives did not significantly alter mass balance estimates of TC and TN in composted or stockpiled manure.

In our study, a combination of 3-NOP and monensin resulted in significantly higher manure TC concentrations compared to the control and 3-NOP. This suggests there could be differences in the relative amounts of recalcitrant versus labile forms of C in manure from cattle supplemented with 3-NOP and monensin. A previous study noted 3-NOP-induced increases in excreted neutral (hemicellulose, cellulose and lignin) and acid detergent fibre (cellulose and lignin) in fresh manure from lactating dairy cows14, and monensin has been shown to alter digestibility of NDF and ADF in cattle10. Future studies should consider using more sophisticated methods to gather insights into these differences. When mass losses were considered, we did not find significant differences in TC and TN between the additive treatments suggesting that microbial activity during manure decomposition was not significantly affected by the additive treatments.

This was the first study to evaluate the potential impacts of 3-NOP on GHG and NH3 emissions from stored manure. Therefore, there are no directly comparable data sets. Additive treatments did not significantly affect cumulative GHGs or NH3 emissions. This is consistent with the lack of mass balance differences in TC and TN for stored manure between additive treatments. For GHG or NH3 emissions to be affected by additives, differences in substrates or microbial communities and/or microbial activity within the manure pile would be required. Since changes in stored manure TC and TN are largely the result of microbial activity, there is no evidence that 3-NOP had a significant effect on these factors in our study.

It was expected that there would be differences between the composted and stockpiled manure. There was slightly greater thermophilic decomposition in the composted manure compared to the stockpiled manure as indicated by a longer duration of relatively higher internal pile temperatures25,26. However, we did not observe differences in mass losses of DM, TC or TN as a result of the different storage methods (Table 3). This is in contrast to the results of other studies which have noted greater losses of TC and TN from composted manure compared to stockpiled manure17. Our results may be a consequence of low manure moisture content initially and throughout which were less-than-optimal for manure composting27. They may also be attributed to the low compost turning frequency (three times) compared to the six to eight times in other studies as greater turning frequency leads to greater decomposition and therefore greater DM, TC and TN losses28.

Trends in daily fluxes observed during our trial were similar to those observed during other studies. Similar to our results, others have also reported CH4 emissions from composted and stockpiled cattle manure spiked shortly after pile creation18,29 and that CO2 emissions from composted and stockpiled beef cattle manure were initially high and decreased over time18,24. Composting manure has been shown to result in high variability of N2O emissions18,24, and others have similarly reported N2O fluxes increased after NH3 fluxes decreased24,30.

Composting manure resulted in higher cumulative CO2 and NH3 emissions compared to stockpiled manure which is consistent with other studies22,28. Compost turning increases aeration and aerobic decomposition of organic material and hydrolysis of organic N resulting in the production of CO2 and NH324,26. Because turning introduces O2 into the compost, it was a little surprising that we observed higher cumulative CH4 emissions from the composted manure compared to the stockpiled manure.

Methane production requires the absence of O2 as a precondition for production31 and stockpiled manure can have high CH4 emissions20. However, anaerobic conditions do not always form in stockpiled manure18 and high CH4 production within manure piles does not always equate to greater surface-to-atmosphere CH4 emissions23. In the current study, low CH4 emissions from our stockpiled manure can be partially attributed to the initial pile characteristics as the manure was mixed with straw bedding and the initial moisture content was ~ 0.34 kg kg−1 which is lower than the > 0.60 kg kg−1 initial manure moisture values reported in other studies that measured high CH4 emissions from stockpiled manure22,32,33. These characteristics can contribute to low density and high porosity in the manure piles, which allows diffusion of O2 into the pile from the atmosphere. This may have allowed aerobic conditions to predominate inside the stockpiled manure resulting in low CH4 emissions34. Likewise in these conditions, CH4 produced within the pile may be oxidized before being emitted to the atmosphere23. The lower cumulative CH4 emissions from the stockpiled compared to the composted manure may also be attributed to the relatively smaller pile size of the stockpiles as CH4 emissions have been found to be lower when manure piles are smaller30.

We found no differences in cumulative N2O emissions between the composted manure and stockpiled manure. There was some evidence that reducing conditions were developing within the stockpiled manure after day 30 as shown by the increase in N2O fluxes, while in the composted manure most of the N2O was emitted after turning. During manure storage, nitrification on the pile surface leads to accumulation of NO2- and NO3- and these substrates are reintroduced into the compost pile during turning contributing to spikes in N2O fluxes from denitrification23,26. The data suggests the environmental conditions and processes occurring in the composted and stockpiled manure differed, however, they emitted similar amounts of N2O.

The mass balance estimates suggest more N was lost from both the composted and stockpiled manure than was accounted for as NH3 or N2O. Some of the discrepancies could be related to gases that we have not accounted for (i.e. nitric oxide and nitrogen gas)35. The losses of TC from the stockpiled manure estimated by the mass balance (All = 58.3 kg tonne−1) were similar to the total losses of CO2 + CH4 kg C tonne−1 TC (All = 65.1 kg C tonne−1). However, mass balance estimates of TC losses (All = 80.4 kg tonne−1) and the losses of CO2 + CH4 kg C tonne−1 TC (All = 174.3 kg C tonne−1) were not comparable in the composted manure. Previous studies have found similarities between these values36. The mass balance estimates use the initial and final manure samples and mass balances are derived from an empirical model37. This method has been shown to sometimes cause erroneous values when manure samples are not representative of the entire pile, or when soil is introduced into the compost piles during turning38. These factors may have impacted our results. Despite these discrepancies, both the gas and mass balance data consistently showed no significant effects on C or N cycling in stored manure piles resulting from supplementing beef cattle with 3-NOP or 3-NOP and monensin. Future studies should consider placing manure piles on concrete pads to prevent mixing soil with the manure, and collecting multiple samples at various areas of the manure piles to ensure samples are representative.

Measurement of GHGs using static chambers can contribute to errors. There are inaccuracies associated with extrapolating 40 min static chamber measurements made one to three times a week over the entire 202 day trial. Temporal extrapolation of GHG fluxes from static chambers are associated with some error in cumulative GHG emissions when used on soils39. Likewise, the area covered by the chambers was small so we only measured a fraction of the total surface area of the manure piles.

Our study did not evaluate the potential effects of differences in 3-NOP and monensin dosage, and we also did not consider differences in cattle diets. The effects of 3-NOP on the digestibility of organic matter in manure have been found to vary slightly by dose16 and enteric CH4 production has been found to be related to diet40. Thus our results might not be replicated under different 3-NOP doses and different diets.

The presence of residual monensin in the manure was not considered during our study. Monensin in its active form can be excreted with manure, and supplementation with monensin could alter GHG emissions from manure11,41. However, both composting and stockpiling manure have been shown to reduce monensin concentrations in stored manure42. Future studies should consider incorporating extractions to test for monensin over time during manure storage to properly evaluate whether GHG emissions from stored manure are directly affected by monensin.

Management activities such as adding water to the manure piles, swapping straw for other materials29,32, changing cattle diet43 and changing compost turning frequency28 affect the degradation of residual dietary additives in manure, as well as manure components and GHG and NH3 emissions. These factors have not been considered in the current study.

In summary, our study found some differences in manure chemistry as well as higher CO2, CH4 and NH3 emissions from composted compared to stockpiled manure, which can be attributed to differences in conditions resulting from differences in handling strategies and pile characteristics. We found that supplementing cattle with 3-NOP and monensin affected the initial manure TC concentrations, but they did not significantly influence estimates of TC or TN mass balances in stored manure. As a result, there were no significant differences between additive treatments for cumulative GHGs and NH3 emissions from composted and stockpiled manure. The lack of differences between additive treatments in our trial suggests that manure from cattle supplemented 3-NOP does not require manure storage that differs from current strategies. Our trial results provide assurance that enteric CH4 reductions achieved through dietary supplementation with 3-NOP are not negated by increases in GHG emissions during manure storage. This study can serve as a reference for policy makers that will want to know about any confounding effects that supplements have on GHG emissions and nutrient transformations in manure from beef cattle feedlots.


This experiment was conducted in semiarid Lethbridge, Alberta, Canada (49°42′03.3"N, 112°45′51.0"W). Precipitation data was acquired from an Agriculture and Agri-Food Canada weather station located < 1 km from the trial.

Manure collection

Manure mixed with cereal straw (manure:straw ratio of 4:1) was collected from a congruent feeding trial that was conducted from November 2015 to August 201644. All cattle were fed the same high-forage diet. Steers were fed a backgrounding diet for 105 days consisting of 65% barley (Hordem vulgare) silage, 5% supplement (vitamins, minerals and crude protein), and 30% dry rolled barley and then were transitioned to a finishing diet over 28 days. The finishing diet consisted of 8% barley silage, 5% supplements, and 87% dry rolled barley grain for 105 days. A full profile of feed ingredients and chemical composition of dietary ingredients are presented in Vyas et al.44.

In separate pens, three treatments were established based on the addition or exclusion of additives. The control group did not receive additional supplements, the “3-NOP” group received 3-NOP, and the “3-NOP + Mon” group were supplemented with 3-NOP and monensin. During backgrounding, 3-NOP was supplemented at 200 mg kg−1 dry matter (DM) and monensin was supplemented at 33 mg kg−1 DM. During finishing, 3-NOP was supplemented at 125 mg kg−1 DM and monensin was supplemented at 33 mg kg−1 DM.

Experimental setup

Manure was removed from the feedlot on September 6, 2016, at the end of the associated feeding trial44. Manure from each treatment was separately transported from the feedlot to the experimental site. Each pile was separated by a buffer of 3.65 m in the east–west direction and 7.62 m in the north–south direction. The piles were divided into two blocks by handling method: composting and stockpiling. Three replicates of each handling method (composting and stockpiling) and treatment (control, 3-NOP and 3-NOP + Mon) were randomly distributed throughout their respective blocks for a total of 18 manure piles.

The initial masses of each compost pile and stockpile were determined during the pile creation from the manure spreader’s on-board scale. Dimensions of each pile were determined by manually measuring the length, width, circumference and height of piles with a tape measure. This data was used to determine pile surface area.

For the stockpiles, manure was heaped into piles roughly conical in shape. For the composted manure, windrows were created. All manure piles were unconfined and situated on soil.

The piles were uncovered for the duration of the trial (202 days). The trial duration was dictated by the temperature, which decreases when manure decomposition slows. The manure remained stored until the spring—even after decomposition had slowed—when it could be applied to the soil. The compost piles were turned on September 26, 2016 (day 19), October 17, 2016 (day 40) and November 15, 2016 (day 69) using an Earthsaver windrow turner (Fuel Harvesters Equipment, Midland, TX, USA). The stockpiles were left undisturbed for the duration of the trial.

Manure properties and analyses

Air and internal manure pile temperatures at the top, middle and bottom of the piles were measured hourly during the trial using T-wire thermocouples recorded by a datalogger (CR800 with an AM16/32B channel relay multiplexer, Campbell Scientific, Logan, UT, USA). The thermocouples were briefly removed from the compost piles during turning. Pile temperatures were averaged into one temperature representative of each treatment and handling method.

At the start and end of the trial, each manure pile was cut perpendicular to its length to expose a vertical face. Manure was sampled to determine manure properties from the top, middle and bottom of each pile. The values were averaged for each treatment and handling method.

Gravimetric moisture content was determined by drying 100 g of manure at 60 °C for one week. The moisture content was expressed on a wet basis while manure C and N concentrations were expressed on a dry basis.

Manure pH and EC (ds cm−1) were obtained by mixing 15 g of fresh sample and 150 mL ultrapure water for 30 min, then centrifuging (10,000 rpm for 10 min). The pH and EC of the supernatant were measured on a Orion Star A215 pH & EC meter (Thermo Fisher Scientific, Waltham, MA, USA). The sample was then filtered (0.45 µm) and the supernatant solution was analysed for soluble inorganic N (ammonium, NH4+; nitrite, NO2-; nitrate, NO3-) using ion chromatography (ICS-1000 Ion Chromatography System, Dionex, Sunnyvale, CA, USA).

Manure TC and TN (kg tonne−1 DM manure) were determined from freeze-dried ground (≤ 0.15 mm) manure and measured using a CN analyser (Carlo Erba Instruments, Rodano, Italy) and expressed on a dry weight basis.

Mass balance estimates

Mass losses (%) of DM, TC and TN were estimated from measurements of the initial and final samples from all additive treatments and both manure storage practices using equations outlined in Larney et al.17. Briefly, assuming an initial mass of 1000 kg of fresh manure, and using initial and final moisture measurements, DM losses were derived by assuming all mass losses were organic material and estimated according to an empirically derived relationship37. The TC and TN losses were derived by calculating the percent difference in their initial and the final concentrations relative to the initial and final masses of the manure piles.

Greenhouse gas and ammonia sampling

Ammonia and GHG fluxes were measured one to three times a week from September 7, 2016 (day 1) until November 29, 2016 (the first 83 days, the thermophilic phase when internal manure pile temperatures were > 45 °C) then three to six more times thereafter until March 28, 2017 (day 202). For each handling method and additive treatment, this resulted in a total of 14 NH3 measurements. For stockpiled manure, we measured GHGs 15 times for each additive treatment, and from composted manure, we measured GHGs 18 times for each additive treatment. The three additional measurements made from the composted manure occurred immediately after the compost was turned.

Ammonia fluxes were measured using cylindrical PVC vented chambers (chamber area = 0.019 m2) with bases inserted 3–5 cm into the manure.

The chambers were equipped with two 2.5 cm thick polyurethane foam disks cut fit-to-seal to the inner diameter of the PVC chambers and infused with 50 mL of phosphoric-glycerol solution to trap gaseous NH3. The “measurement disk” was situated above the pile surface to capture NH3 emitted off the manure, and a “scrubbing disk” was inserted between the measurement disk and the atmosphere to prevent contamination of the measurement disk. Each chamber was covered with a canopy to prevent precipitation from washing the acid solution from the disks.

For measurements, the foam disks were brought to the laboratory in sealed bags, weighed, and the amount of solution absorbed into each disk was determined using before and after deployment weights. Disk NH3 concentrations were extracted by saturating each disk with 100 mL of 0.5 M KCl solution for 30 min. Extractions were frozen at − 20 °C prior to analysis. The extracts were analysed using the indophenol blue method with a MultiSkan Go Microplate Spectrophotometer at 650 nm (Thermo Scientific, Waltham, MA, USA). Ammonia fluxes were calculated by dividing the extraction concentrations by the chamber area and deployment time.

Greenhouse gas samples were collected from vented static chambers (area = 0.07 m2, volume = 7 L). Chamber bases (inner diameter = 30 cm) were inserted 5 cm into the manure pile. The bases were briefly removed and reinstalled before and after compost turning.

For GHG measurements, chamber covers were clipped to the bases and samples taken at 0, 10, 20 and 40 min after covering. From each chamber, 11 mL of gas was extracted from the headspace using a polypropylene syringe and immediately injected into a pre-evacuated (− 1 atm) 5.8 mL Exetainer (Labco Ltd., Lampeter, United Kingdom). Gas concentrations were determined using a gas chromatograph equipped with electron capture, thermal conductivity and flame ionization detectors (Varian 3800, Varian Instruments, Palo Alto, CA, USA). The injector and column temperatures were kept at 55 °C. The carrier was P10 gas (10% methane, balance argon) for N2O and helium for CO2 and CH4. The channel was maintained at a static pressure of 150 kPa.

Greenhouse gas fluxes from the surface of manure piles to the atmosphere were calculated using air temperature, the ideal gas law, chamber area and volume, and the change in gas concentration over time for each chamber. The change in gas concentration over time was assessed using both quadratic (QR) and linear regression (LR). Unless the second derivative of the model was ≤ 0 according to the LINEST function in Microsoft Excel (version 2019), fluxes were calculated with QR45,46. The minimum detectable fluxes were determined for CO2, CH4 and N2O47. The minimum detectable fluxes for quadratic and linear fluxes were: ± 2215.7 and ± 641.5 µg C m−2 h−1, respectively, for CO2; ± 2.8 and ± 9.7 µg N m−2 h−1, respectively, for N2O; and ± 4.9 and ± 16.8 µg C m−2 h−1, respectively, for CH4. Fluxes below the minimum detectable flux were assigned a value of zero. Of the 387 fluxes measured, the QR method was used for 85, 81 and 55% of the CO2, N2O and CH4 fluxes, respectively, the LR method was used for 12, 19 and 44% of the CO2, N2O and CH4 fluxes, respectively, and 3, 1 and 1% of the CO2, N2O and CH4 fluxes were below detection, respectively.

Daily NH3 and GHG flux rates for the composted and stockpiled manures are presented by additive treatment as well as an average for all treatments together (“All”) to compare flux rates between storage methods. The daily gas emissions are expressed as fluxes per unit area (g m−2 d−1).

The cumulative emissions from each pile were determined by integrating (trapezoidal method) between daily measurements and summing the values, which were then averaged based on additive treatment and storage method.

The cumulative emissions for each treatment and storage method are presented on a per unit area basis (kg m−2), averaged over the initial surface area of the manure piles (kg pile−1), and determined as a proportion of the initial manure TC and TN content (kg C or N tonne−1).

The GHG and NH3 emissions were extrapolated from a per unit area to per surface area of the pile by multiplying the emissions (kg m−2) by the initial surface area of the piles (pile1 as m2).

The emissions as kg tonne−1 initial manure TC or TN were calculated by dividing the initial surface area of the piles (m2) by the initial surface area, multiplying the emissions by the kg tonne−1 initial DM manure, then multiplying by the fraction of initial TC or TN in the manure.

Data handling and statistics

Manure chemical concentrations, % losses of DM, TC and TN from mass balance estimates, and cumulative GHG and NH3 emissions were subject to statistical analyses. Data were analysed using R Statistics (version 3.4.3). Normality of each dataset was tested using a Kolmogorov–Smirnov test and homogeneity of variance assessed using Levene's test. None of the data sets were transformed. To test for differences in manure chemical concentrations by handling method (compost and stockpile), additive (control, 3-NOP and 3-NOP + Mon) and day (1 vs 202), a three-factor mixed model ANOVA was conducted (nlme package in R), and when appropriate, post-hoc analysis was completed using a Tukey’s HSD Test (lsmeans package in R). To test for differences in cumulative GHG and NH3 emissions and % mass losses of DM, TC and TN by handling method (compost and stockpile) and additive (control, 3-NOP and 3-NOP + Mon), a two-factor mixed model ANOVA was used. In the mixed models, replicate was treated as a random effect and all other factors included in the model were treated as fixed effects. Significance was evaluated at P ≤ 0.10.


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Funding for this research was provided by the Alberta and Livestock Agency Ltd. and Agriculture and Agri-Food Canada Growing Forward 3 Program. Thank you to Kui Lui, Courtney Soden, Haibo An, Colten Wolfe and Ann Marie Wolfe for sample collection and analyses, and Tony Curtis, Kyle Shade, Heather McGlynn and Todd Staffer for completing stockpile and compost windrow construction and compost turning.

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J.L.O. analysed the data, wrote the main manuscript text and prepared tables and figures. B.W.T. and J.L.S. carried out the experiment, and collected and processed data. F.J.L. and T.A.M. contributed to the design of the experiment. K.A.B. conducted the feeding trial that generated the cattle manure and contributed to the design of the experiment. X.H. conceived the idea, secured the funding and designed the experiment. All authors reviewed the manuscript, provided critical feedback and helped shape the research, analyses and manuscript.

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Correspondence to Jennifer L. Owens.

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Owens, J.L., Thomas, B.W., Stoeckli, J.L. et al. Greenhouse gas and ammonia emissions from stored manure from beef cattle supplemented 3-nitrooxypropanol and monensin to reduce enteric methane emissions. Sci Rep 10, 19310 (2020).

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