Adequate vegetative cover decreases nitrous oxide emissions from cattle urine deposited in grazed pastures under rainy season conditions

A decline in pasture productivity is often associated with a reduction in vegetative cover. We hypothesize that nitrogen (N) in urine deposited by grazing cattle on degraded pastures, with low vegetative cover, is highly susceptible to losses. Here, we quantified the magnitude of urine-based nitrous oxide (N2O) lost from soil under paired degraded (low vegetative cover) and non-degraded (adequate vegetative cover) pastures across five countries of the Latin America and the Caribbean (LAC) region and estimated urine-N emission factors. Soil N2O emissions from simulated cattle urine patches were quantified with closed static chambers and gas chromatography. At the regional level, rainy season cumulative N2O emissions (3.31 versus 1.91 kg N2O-N ha−1) and emission factors (0.42 versus 0.18%) were higher for low vegetative cover compared to adequate vegetative cover pastures. Findings indicate that under rainy season conditions, adequate vegetative cover through proper pasture management could help reduce urine-induced N2O emissions from grazed pastures.

Scientific RepoRts | (2019) 9:908 | https://doi.org/10.1038/s41598-018-37453-2 half of the 80 million ha of introduced tropical pastures are estimated to be in some state of degradation as they have, among other symptoms, low soil cover 4 . Cattle excreta deposited on grazed pastures is estimated to represent 16% of global anthropogenic nitrous oxide (N 2 O) emissions, a powerful greenhouse gas (GHG) 5 . About 75-95% of cattle ingested N is excreted in either urine or dung, which provides N-rich substrate for nitrification and denitrification 6,7 . Cattle urine patches can contain very high amounts of soluble N (equivalent to 500-1000 kg N ha −1 ), more than 2-3 times of the N uptake capacity of pastures 8 . Annually, about 1.5 Tg of total global anthropogenic N 2 O emissions (6.7 Tg N 2 O-N yr −1 ) are emitted from excreta produced by grazing cattle 9,10 through both direct and indirect (from leached and volatilized excreta nitrogen) emissions. About 2% (0.7-6% uncertainty) 11 of the nitrogen (N) in deposited urine is lost as N 2 O. Lower emission factors (EFs) (<0.7%), reported in other studies have been attributed to differences in climatic conditions, texture, soil moisture, and the N concentration in animal excreta 12 .
Pasture degradation may stimulate or constrain N losses. For example low vegetative cover, may reduce N sinks for deposited excreta and thus increase the vulnerability of N to loss through soil microbial processes and leaching. However, the low vegetative cover may also be associated with fewer plant root exudates and thus suppress microbial activity and N 2 O emissions 13 . On the other hand, overstocking and overgrazing without time for pasture recovery increases the risk of soil compaction -an indicator of pasture degradation. Soil compaction reduces soil porosity and pore continuity, decreases soil aeration, restricts plant growth and thus, consequently, increases soil N 2 O emissions from urine patches 14,15 . Soil acidification, which could also be an indicator of pasture degradation, has been shown to increase N 2 O emissions as acidic conditions generally reduce plant growth and inhibit N 2 O reductase enzyme activity which is responsible for transforming N 2 O to dinitrogen (N 2 ) 16,17 .
Clearly, the effect of pasture degradation on N 2 O emissions from urine deposition can influence emission through multiple, often interacting, mechanisms and thus has produced contradictory results in the literature. Previous studies suggest that variations in soil N 2 O emissions from deposited urine patches in grazed pastures are driven by differences in several factors including ambient temperature 18 , urine volume and urine-N content 15,19 , soil drainage 20,21 , and soil moisture 22,23 . No previous studies have systematically explored the variation in urine-based soil N 2 O emissions associated with low vegetative cover in pastures.
Here we tested the hypothesis that N 2 O emissions from cattle urine deposited on grazed pastures with adequate vegetative cover are less intense than those from pastures with lower vegetative cover by measuring soil N 2 O fluxes from urine patches deposited on different pastures located at seven contrasting sites, spread across five countries in the LAC region during rainy season.

Results
Soil texture at most of the study sites was similar in the low and adequate vegetation cover pastures with the exception of Balcarce (Argentina), Estelí (Nicaragua) and Taluma (Colombia) ( Table 1). Soil pH values at the study sites ranged between 5.0 and 8.9, with acidic soils (pH < 6) at Taluma (Colombia), Rio Grande do Sul (Brazil), St. Augustine (Trinidad and Tobago) and neutral to basic soils at other sites. Soil bulk density at the study sites ranged between 0.6 and 1.6 g cm −3 and was generally similar between the low and adequate vegetative cover pastures at each study location. The largest differences in bulk density, soil organic carbon and soil organic nitrogen between low and adequate vegetative cover pastures at the Estelí location in Nicaragua (Table 1).
Air temperature and rainfall data for one week before and during the sampling dates are shown in Supplementary Fig. S1 S1). N 2 O emission peaks observed in LVC pastures tended to be higher than those in AVC pastures in 5 out of the 7 sites ( Fig. 1; Table 2). However, the delayed N 2 O peaks observed at Rio Grande do Sul (Brazil) were higher in the AVC (  The N content in applied cattle urine ranged from 112-1,641 kg N ha −1 ( Table 2). Over a one-month period, the soil N 2 O emission factor of N in applied urine to soil ranged from 0.01 to 1.23%. The highest N 2 O emission factor values observed for the LVC pasture (1.23% of applied urine-N) and AVC (0.48% of applied urine-N) pasture were at Estelí (Nicaragua) and Rio Grande do Sul (Brazil), respectively. On the other hand, the lowest N 2 O emission factors for LVC (0.02% of applied urine-N) and AVC pasture (0.01% of applied urine-N) were both observed at the Taluma site in Colombia (Fig. 2). At the regional level, mean cumulative N 2 O emissions observed in the urine treatments ranged between 0.02 and 7.5 kg N 2 O-N ha −1 . The highest cumulative N 2 O emissions for both treatments (LVC and AVC pasture) were observed at St. Augustine (Trinidad and Tobago) and Estelí (Nicaragua) and the lowest were observed at Taluma (Colombia) ( Table 2). At the regional level, mean N 2 O emission factors were significantly, at most 2.5 times, higher (P < 0.0002) in LVC (0.42 ± 0.19% SEM) than AVC (0.18 ± 0.08% SEM) pastures. Also at the regional level mean cumulative N 2 O emissions in the LVC (3.31 ± 1.09 kg N 2 O-N ha −1 SEM) were higher than those observed in the AVC (1.91 ± 0.78 kg N 2 O-N ha −1 SEM) pasture at the 10% level of significance (P = 0.08), based on results presented in Table 2. The N 2 O emissions at each individual site tended to be higher in LVC pastures than in the AVC pastures, with t-test detecting significant differences at the Nicaragua (Estelí) and Colombia (Patia) sites (Table 2). Rainfall for the measurement period (including data for the period of one week before commencing the monitoring campaigns) explained less of the variation in N 2 O emission factors in the LVC (66%) pasture compared to the AVC (88%) pasture ( Supplementary  Fig. S2). No clear effects of air temperature were observed on N 2 O emission factors.

Discussion
The key finding of our study is that, at the regional level of LAC, N 2 O emission factors from cattle urine patches in grazed pastures are lower for AVC compared to LVC pastures during rainy season, suggesting the importance of adequate pasture improvement/management in mitigating soil N 2 O emissions. We did not have soil moisture data for all the sites to be able to compute the effects of water-filled pore space, which has been shown to be a major driver of N 2 O emissions 24,25 . The strong correlation observed between rainfall and the N 2 O emission factor suggests that soil moisture was possibly a key driver of N 2 O emissions in the current study. However, the fact that rainfall explained less of the variation in N 2 O emissions in the pasture with LVC compared to one with AVC may also imply that other factors, such as vegetative cover, are drivers of N 2 O emissions. Yet, interestingly, the steeper slope observed for the LVC (0.0086) compared to the AVC (0.0041) pastures suggests that urine deposited on LVC pastures is more vulnerable to high N 2 O losses when exposed to high rainfall. This may explain the significant differences between LVC and AVC pastures that were observed at Estelí (Nicaragua) and Patía (Colombia), where rainfall was high and the observed separation between the LVC and AVC pastures at the Rio Grande do Sul (Brazil) site where rainfall was also high. High peaks of N 2 O emissions observed in LVC pastures compared to AVC pastures were likely due to lower plant N uptake from soil. Moreover, this may in part explain why AVC pastures generally resulted in lower net cumulative N 2 O emissions compared to degraded pastures. Despite the significant difference (P < 0.10) in net cumulative N 2 O emissions between LVC and AVC pastures, at a regional level, site-level comparisons showed that cumulative N 2 O emissions from LVC pastures were only significantly higher than those of AVC pastures in two sites, at Estelí (Nicaragua) and Patía (Colombia). The fact that soils within the LVC pastures at the Estelí site were more clayey than those under AVC pastures may have also contributed to the high net cumulative N 2 O emission in the former. Previous studies have reported higher N 2 O emissions from urine deposited on fine textured soils 26 . However, at Patía, where both the LVC and AVC pastures were on a clay soil observed differences suggest that despite the obvious influence of soil texture, pasture condition, based on vegetative cover is a driver of N 2 O emissions.
Low emissions of N 2 O observed at the two study sites in Argentina could have been due to the lower mean air temperatures. At the Balcarce location, mean air temperatures and the amount of N in applied urine were higher than at the Manfredi location. Low temperatures are known to reduce microbial activity and thus the rate of N transformation processes such as nitrification and denitrification in soil and, consequently, N 2 O production 27 . We assume that low temperatures may also be the cause of low net emissions and emission factors at the site in Brazil. It is important to note that the high soil pH (>7) at the Balcarce site, which would be expected to further increase with urine application may have also resulted in the inhibition of nitrification and high ammonia volatilization [28][29][30] . In addition, at the Balcarce site, the high urine-N levels may have resulted in microbial stresses with possible impacts on soil N transformation 31

and thus contributed to the low N 2 O emissions and emission factors.
At the Taluma location in Colombia, the absence of a N 2 O emission peak following urine application may be because the frequency of measurements at this site was insufficient to capture the expected N 2 O emission spike. Several other studies using manual static chambers have reported having missed the N 2 O peak, due to the low temporal resolution [32][33][34] . This problem can be resolved by increasing the frequency of monitoring using manual static chambers or switching to automated chambers. Alternatively, the N content in applied urine was also the lowest at Taluma, which implies that N 2 O fluxes could have been limited by N substrate availability. In addition, the forage grass (Brachiaria humidicola) that was used at Taluma had high nitrification inhibition capacity [35][36][37] , which could have also contributed to the observed low N 2 O emissions.
Absence of significant differences, in N 2 O emissions, between LVC and AVC pastures at the St. Augustine (Trinidad and Tobago), Balcarce (Argentina), and Manfredi (Argentina) sites was possibly due to the fact that the spatial variation of vegetative cover of grass (soil cover) in the LVC (50-70%) pasture was high. As a result, local farmers based their classification on animal productivity differences which are influenced by both quantity (biomass) and quality (e.g. digestibility and crude protein content) of the forage on offer to animals. This further suggests that N 2 O emission differences between LVC pastures and AVC pastures are driven by differences in soil cover. With high plant density and greater plant vigour, we expect greater uptake of the urine-N by plants which could reduce the amount of N available for microbial transformations in soil such as nitrification and denitrification. It is therefore not surprising that when soil cover was high in the LVC pastures, there was no significant difference in N 2 O emissions with AVC pastures. This was however not the case for LVC pasture at the Patía (Colombia) site, which, though having similar soil cover (50-70%) showed significant differences between LVC and AVC pastures. This difference may be due to dissimilar vegetation types at the studied sites which would also affect N uptake and thus N availability for N 2 O emissions 38 .
The IPCC Tier 1 emission factor for urine deposited on grazed forages is 2% with an uncertainty range of 0.7-6% 11 . During this short-term study, several of the emission factors were below the uncertainty range of the IPCC Tier 1 emission factor. While this may be due to the short gas monitoring period (1 month), several other studies conducted under temperate conditions [39][40][41][42] , reported a similar range of emission factors (0.02-1.63%) as we observed under warm temperate or sub-tropical conditions in Argentina and Brazil (0.02-0.7%). Similarly, the range of emission factors reported from this study under the tropical conditions in Colombia, Nicaragua and Trinidad and Tobago (0.01-1.2%) are in agreement with the range of values that have been reported from studies conducted under tropical conditions 43,44 .
We conclude that in addition to the known effects of rainfall, temperature and the amount of urine-N, the pasture condition based on vegetative cover also influences N 2 O emissions from cattle urine patches. When pasture degradation is associated with a reduction in vegetative cover, N 2 O emissions are expected to increase. Therefore, better regional understanding of the state of pasture degradation is vital for a robust understanding of N 2 O emissions from cattle urine deposits. More importantly, these findings suggest that improving soil cover/pasture condition through adoption of appropriate grazing and nutrient management practices may contribute towards Scientific RepoRts | (2019) 9:908 | https://doi.org/10.1038/s41598-018-37453-2 mitigating excreta-based soil N 2 O emissions from grazed pastures during the rainy season. We expect findings from this regional study to contribute towards reducing uncertainties in future assessments on the importance of improving grassland management to achieve global commitments, such as the Bonn Challenge, 20 × 20 initiative 45 and the Paris Agreement 46 .

Methods
The experimental plots were located at seven different sites in five countries in LAC spanning diverse climatic conditions and soil types ( Supplementary Fig. S3 and Table S1). Rainfall is bimodal in Estelí (Nicaragua) with two seasons from May to June and August to November. In Patía (Colombia), the wet seasons occur during the period from March to May and September to November. Rainfall in St. Augustine (Trinidad and Tobago) is also bimodal with wet seasons occurring from June to August and October to November. At the rest of the study sites rainfall is unimodal with the main rains occurring during the period February to December and September to April at Taluma (Colombia) and the two Argentinean sites, respectively. During the monitoring period rainfall and air temperature data was collected at the nearest weather stations at each of the different study sites. At each of the study sites, paired experimental plots were set-up on fields with grazed pastures that were classified as having either low vegetative cover (LVC) or adequate vegetative cover (AVC). Pairs of LVC and AVC pastures were not always available at the very same location, but were always less than 1 km apart. We used a qualitative approach including expert knowledge, farmer perceptions and an arbitrary ranking system based on soil cover to define pasture conditions based on vegetative cover using criteria that combined those used by Hollman 47 , Brown 48 and McCormick and Lodge 49 . Specifically, through visual assessments by forage scientists 50 we broadly described soil cover as follows: low vegetation cover (<70%) and adequate vegetation cover (>70%) pastures where soil cover is simply the proportion of soil covered by vertical projection of a plant canopy and vegetative biomass (Supplementary Table S2). Aboveground biomass data for the study sites were also obtained from historical records when available. In addition, we also used local farmer assessments to differentiate low and adequate vegetation cover pastures.
On each pasture (low vegetative cover or adequate vegetative cover), experimental plots were organized following a systematic experimental design 51,52 with five replicates per treatment ( Supplementary Fig. S4). The two treatments were urine application and a control-without urine application. The urine was applied to individual independent plots and so there are five replicates for each of the control versus urine treatment within each site 53 . Individual replicate plots (2 m × 2 m) were demarcated within each pasture condition for making measurements of soil properties and N 2 O emissions. To simulate grazing, grass in each plot was cut to approximately 5 cm sward height, seven days prior to the beginning of the gas and soil sampling.
Prior to starting the experiment, ten soil subsamples (0-10 cm) were separately collected from LVC and AVC pastures, using augers with 5 cm diameter, and combined to give one composite soil sample for each pasture condition. Collected soil was characterized for texture, pH, total carbon (C) and organic and inorganic N as described by Gee and Bauder 54 , McLean 55 , and Vogel 56 , respectively. A total of 20 cylindrical PVC static chamber bases (10 per treatment) were inserted at the center of each subplot to a depth of 5 cm, five days prior to the start of gas and soil sampling. For each treatment, chamber bases with an internal diameter of 25 cm and a height of 10 cm were distributed in five replicate plots. At each site, cattle urine samples (about 7 L) that were collected from at least 10 local dairy cows were pooled and, immediately, following setting aside a subsample for N analysis, 500 ml of the collected urine was applied to soils to simulate a urination event on soil within each static chamber base at a rate of 1.27 L urine/m 2 . Nitrogen concentration in urine was characterized in each of the study countries using the direct distillation method described by Hoogendoorn et al. 57 . Unfortunately, we were unable to quantify urine-N in Trinidad and Tobago as there are currently no laboratories that quantify N in animal urine samples.
Gas measurements were conducted from a total of 20 non -vented PVC static chambers (10 cm height and 25 cm diameter) fitted with two rubber septa (one for gas sampling and another for inserting a thermometer). On each sampling day, PVC chambers were fitted to the chamber bases and sealed with an air-tight rubber belt. Syringes (15 ml) fitted with hypodermic needles were used to collect four gas samples from each of static chambers following chamber closure and at 15, 30 and 45 minutes after chamber closure. Collected gas samples were transferred to pre-evacuated 10 ml headspace glass vials fitted with rubber butyl septa crimp caps. At each site, at least eight gas samples were collected between the months of November to December in 2015 for the localities of Patía (Colombia) and Rio Grande do Sul (Brazil), Estelí (Nicaragua) and St. Augustine (Trinidad and Tobago). For Taluma (Colombia) and Balcarce (Argentina) measurements were conducted between February to March (2016) and at Manfredi, Argentina in the month of May (2016). Sampling months were chosen to coincide with the wet seasons at each of the study sites. The use of non-vented chambers has been reported to cause bias in the flux estimates 58 . Yet, Davidson et al. 59 reported the possibility of artefacts with both vented and non-vented chambers, making it difficult to know which chamber yielded the 'true' flux. Since similar non-vented chambers were used at all sites, the chambers did not affect observed differences between AVC and LVC pastures. However, the calculated N 2 O emissions factors may differ from those measured in other published studies that used vented chambers.
Gas sampling frequency was as follows: once before the application of urine, 1 hour after urine application, daily for the first three days following urine application, twice a week during the second and third week and once during last week of the experiment. Due to logistical challenges, less frequent measuring campaigns were done at the Taluma site in Colombia. Immediately after the gas sampling campaign, vials were sent to the Greenhouse Gas Laboratory at the International Center for Tropical Agriculture (CIAT) in Colombia, where N 2 O concentration was analyzed by gas chromatography (GC-2014 Shimadzu), within a month upon arrival. The daily N 2 O fluxes were calculated by regressing N 2 O emissions from each chamber on each sampling date against time in order to calculate the hourly flux which was then multiplied by 24 to determine the daily flux. Each calculated flux was where EF is the emission factor, N 2 O-N emitted and N 2 O-N Control are the cumulative N 2 O emissions from urine or control patches over the 18 to 24 days monitoring period. N applied represents the amount of N in the applied urine. Statistical analyses were conducted using the PROC MIXED procedure of SAS 62 . Cumulative N 2 O fluxes and emission factors were, correspondingly, log and square-root transformed to achieve normality and obtain homogenous variances. To determine effects at the region level, cumulative N 2 O fluxes were analyzed using a split-plot ANOVA where the main plot was the pasture condition (LVC, AVC) and split-plot was the nitrogen levels (with and without urine application) and the blocking factor was the location and the main plot error term was pasture condition nested within location. In addition, at the regional level the emission factor variable was analyzed using a one-way ANOVA where the treatment effect was the pasture condition and the blocking factor was the location (site). At the individual sites we used the t-test analysis for testing differences in emission factors as influenced by pasture condition.