Introduction

Finishing cattle in confinement feedlots enables the use of feed sources that are adequate for the animal’s requirements, which increases productivity and meat quality1. However, this system is responsible for a greater accumulation of manure, which contains several components such as N and organic materials2. These components may undergo transformation and serve as a source of emission of greenhouse gases (GHGs), such as nitrous oxide (N2O) and methane (CH4)3,4,5, as well as of ammonia (NH3)6,7. Greenhouse gas emissions contribute to global warming8, whereas NH3 volatilization harms human health7,9 and potentially increase GHG emissions as NH3 is a precursor for N2O generation10.

Nitrous oxide is emitted through the transformation of ammonium (NH4+) and nitrate (NO3–) in soil during nitrification, denitrification11, and nitrifier denitrification12 mediated by fungi, bacteria and archaea13. These processes are affected by precipitation, temperature and substrate availability14,15. The magnitude of gas emission from cattle manure depends on the form and concentration of N16. Therefore, the reduction of N loss via ruminant excreta, specifically of N in the form of urea, is relevant to mitigate N2O emission, since 70% of the N excreted by ruminants is in the form of urea, which releases NH4+ following hydrolysis17. In addition, microbial hydrolysis of urea results in NH3 emission18; thus, the reduction of N-urea from excreta might directly reduce NH3 emission19.

The amount of CH4 emitted from manure is small compared with the total amount of enteric CH4 produced by ruminants20. However, emission from manure in feedlots is relevant, because large volumes of manure can result in higher CH4 emission21. Nitrogen and C content22, moisture, and temperature23, are the major modulators of CH4 emissions. Strategies aimed at increasing the efficiency of N use, resulting in lower N excretion, can modify the CN ratio of manure, which is an important factor responsible for the reduction of CH4 emission24. The high CN ratio can promote the growth of populations of methanogenic archaea that are able to meet their protein requirements and therefore not react with the remaining carbon content of the substrate, resulting in low production of CH425. Thus, reducing nutrient excretion by animals may serve as a strategy to mitigate CH4 emission from manure.

Optimizing the use of N by ruminants can reduce N loss through urine and, therefore, minimize NH37, and N2O emission from manure26. Reducing the amount of rumen degradable protein (RDP) and increasing the amount of ruminal undegradable protein (RUP) in diets may increase overall N efficiency and enable adequate supply of metabolizable protein (PM) to reach the small intestine27. Thus, we hypothesized that different sources of RUP in the diets would reduce N loss via urine and contribute to decreased N2O, CH4, and NH3 emissions to the environment. By modulating the diet in order to reduce N excretion, there is a possibility of impacting the production of enteric CH428. However, in our study, the focus was intended to understand how the sources of RUP can affect the emission in the excreta, consequently, the emission of enteric CH4 was not measured. The evaluation in-situ will enable get more representative emissions from the feedlot environment. Therefore, the objective of the present study was to evaluate the effects of sources of RUP in diets on N2O, CH4 and NH3 emissions from manure of feedlot-finished Nellore and identify key driving variables that regulate the production of these gases.

Results

Characterization of animals’ excreta and N balance

There were no differences in the C and N content or C/N of the urine and fecal samples between the RUP and RDP sources (P > 0.05) (Table 1). Inclusion of CGM as a source of RUP in the diet increased N content (P = 0.012) but decreased the C/N in the fecal samples compared with the inclusion of BSM as a source of RUP (P = 0.009). However, there were no differences in the C/N of urine samples between the RUP and RDP sources (P = 0.632).

Table 1 Fecal and urinary N content and C and N balance of Nellore cattle fed with sources of rumen undegradable protein during the finishing phase in feedlots.
Full size table

None of the three evaluated protein sources affected N consumption, fecal and urinary N excretion, total N excretion and total N retention (P > 0.05). There were no differences in fecal and urinary N excretion, N retention (% intake) or fecal and urinary N excretion (% excreted) among the three protein sources (P > 0.05).

Gas emissions

Mean temperature during the N2O and CH4 emission sampling period was 20 °C; the lowest (3.3 °C) and highest (35.2 °C) temperatures were recorded close to sampling day 49 and on the last sampling day, respectively. Cumulative precipitation throughout the experimental period was 33.6 mm, occurring over 7 different days (Fig. 1).

Figure 1
figure 1

Daily rainfall and daily minimum (Tmin), daily, mean (Tmean) and daily maximum (Tmax) ambient temperature throughout the N2O and CH4 emission sampling period. Data were retrieved from the Agrometeorological Station, Department of Exact Sciences (FCAV/UNESP), located at 1 km from the experimental area.

Full size image

Daily mean N2O and CH4 fluxes varied from −62 to 318 µg N2O m2 h−1 and from −125 to 321 µg CH4 m2 h−1, respectively, during the experimental period (Fig. 2). Highest peak of N2O emission was observed in the 21st day, on all treatments. On the same day, an increase in CH4 fluxes was also observed. Differences in N2O and CH4 fluxes among treatments occurred in some days of evaluation and were not consistent along the studied period.

Figure 2
figure 2

source of rumen degradable protein (RDP), BSM = manure of animals fed by-pass soybean meal as a source of rumen undegradable protein (RUP), CGM = manure of animals fed gluten meal as a source of RUP. P-values for N2O (treatment = 0.003; time < 0.001; treatment × time interaction < 0.001) and CH4 (treatment = 0.165; time < 0.001; treatment × time interaction < 0.005). Chamber considered as an experimental unit (n = 9). The error bars representing standard error of the mean.

N2O and CH4 fluxes from the manure of Nellore cattle fed with sources of rumen undegradable protein during the finishing phase in feedlots. SM = manure of animals fed soybean meal as a

Full size image

Protein sources did not affect cumulative CH4 emission from animal manure (P > 0.05) (Table 2). However, the manure of animals fed RUP sources resulted in a higher cumulative N2O emission than that of animals fed the RDP source (P = 0.030). Emissions from manure of cattle fed CGM were almost double and emissions from manure from cattle fed BSM (P = 0.038) were quadrupled compared to SM-fed cattle.

Table 2 Cumulative CH4 and N2O emissions from the manure of Nellore cattle fed with sources of rumen undegradable protein during the finishing phase in feedlots.
Full size table

An interaction between sampling time and protein source was observed for DM, OM, N, C and NH4+ (Table 3, Fig. 3). The manure of animals fed CGM presented a lower N content and higher NH4+ than that of animals fed SM on day 42 (P < 0.001), while on day 63 higher values of N and NH4+ were observed for the manure of animals fed CGM in relation to BSM (P = 0.002 and P = 0.010 respectively) and SM (P = 0.004 and P < 0.001, respectively). The manure of animals fed SM showed a higher C content than that of animals fed source of RUP on day 42 (P = 0.001). The manure of animals fed SM showed a higher C/N than that of animals fed RUP (P = 0.001). Nitrate content of the analyzed samples was not detectable.

Table 3 Chemical composition of the manure, deposited in the soil, of Nellore cattle fed with sources of rumen undegradable protein during the finishing phase in feedlot.
Full size table
Figure 3
figure 3

source during the finishing phase in feedlot. SM = manure of animals fed soybean meal as a source of rumen degradable protein (RDP), BSM = manure of animals fed by-pass soybean meal as a source of rumen undegradable protein (RUP), CGM = manure of animals fed corn gluten meal as a source of RUP. Different letters represent significant differences by Tukey's Test (P ≤ 0.05) within the treatment vs time interaction. Chamber considered as an experimental unit (n = 9). The error bars representing standard error of the mean.

Dry matter, organic matter, N, C and NH4+ content of the manure, deposited in the soil, of Nellore cattle fed with rumen undegradable protein

Full size image

There were no correlations of manure gases (N2O and CH4) emissions with N, C, C/N ratio, DM, OM, and NH4+ (P > 0.05) (Table 4). Nitrogen was positively correlated with C (P < 0.001) and OM (P < 0.002). Carbon was positively correlated with C/N ratio (P < 0.001). Ammonium was positively correlated with OM (P = 0.045).

Table 4 Pearson’s correlation coefficients between explanatory variables during the evaluated period.
Full size table

A positive correlation was observed between CH4 and C/N ratio on day 42 (P = 0.025), and between CH4 and NH4+ on day 63 (P = 0.001). On day 105, N2O was positively correlated with DM (P = 0.018) and NH4+ (P = 0.008) (Table 5).

Table 5 Pearson’s correlation coefficients between explanatory variables on each sampling day.
Full size table

NH3 emission

Mean temperature during the NH3 emission sampling period was 25 °C. The lowest (15.2 °C) and highest (37.3 °C) temperatures were recorded on the first sampling day and on day 19, respectively. Cumulative precipitation throughout the experimental period was 320.5 mm, occurring on 30 different days (Fig. 4).

Figure 4
figure 4

Daily rainfall and daily minimum (Tmin), daily mean (Tmean) and daily maximum (Tmax) ambient temperature throughout the NH3 emission sampling period. Data were retrieved from the Agroclimatological Station, Department of Exact Sciences, (FCAV/UNESP), located at 1 km from the experimental area.

Full size image

Manure from all treatments showed the highest daily mean NH3 emission on the first day of evaluation (Fig. 5). Subsequently, NH3 emission decreased until the fourth day of evaluation under all treatments. From the 19th day, a new peak of NH3 emission was observed under all treatments. The SM treatment presented a small increase in NH3 emission on days 38 and 51, while the BSM and CGM treatments presented a decrease in emission. Ammonia emission under all treatments completely ceased on the 77th day. From day 13 to 25, cumulative NH3 emission under the SM treatment was higher than that under the BSM and CGM treatments. However, after this period, no differences were observed among the treatments.

Figure 5
figure 5

source of rumen degradable protein (RDP), BSM = manure of animals fed by-pass soybean meal as a source of rumen undegradable protein (RUP), CGM = manure of animals fed corn gluten meal as a source of RUP. Chamber considered as an experimental unit (n = 9). The error bars representing standard error of the mean.

Daily mean NH3 emission from the manure of Nellore cattle fed with sources of rumen undegradable protein during the finishing phase in feedlot. Evaluations started after the animals were removed from the feedlot. SM = manure of animals fed soybean meal as a

Full size image

There were no significant differences in cumulative NH3 emission from the manure during the evaluated period and manure content of DM, OM, N, and C (P > 0.05) among the three protein sources (Table 6). Likewise, there were no differences in the C/N ratio of the manure between the RDP and RUP sources (P = 0.491). However, the manure of animals fed BSM showed a higher C/N ratio than that of animals fed CGM (P < 0.001). The manure of animals fed RDP showed a higher NH4+ concentration than that of animals fed RUP (P < 0.001); however, there were no differences in NH4+ concentration between the manure of animals fed CGM and BSM (P = 0.670).

Table 6 Cumulative NH3 emission and manure characteristics of Nellore cattle fed with sources of rumen undegradable protein during the finishing phase in feedlot.
Full size table

Discussion

Gas emissions

The use of RUP sources in the diet did not reduce N loss via urine. Meanwhile, it increased N2O emission but did not affect CH4 emission from manure. Therefore, our hypothesis that RUP inclusion in the diet would reduce N loss and contribute to reduced N2O and CH4 emissions from the manure was rejected.

The manure deposited in the soil enhances its N and C content, thereby altering the N mineralization rate and stimulating N2O production29,30. Meanwhile, labile C released during material decomposition regulates the seasonality of N2O and N2 production30. Inorganic forms of N (NH4+ and NO3−) are determinants of N2O production. The manure of animals fed BSM and CGM presented a higher NH4+ concentration than that of animals fed SM in the 42nd day, and in 63rd only for the CGM treatment, evidencing a temporal variation of the manure characteristics in relation to treatments, probably resulting in higher urea hydrolysis at these moments (Table 3, Fig. 3). The higher NH4+ concentration under the RUP treatments may have promoted nitrification or denitrification, resulting in N2O production17 (Table 2).

A reduction in urinary N was expected with the inclusion of RUP in diets, since an increase in N use efficiency is observed when lower RDP amounts are used in the diet31. In other words, reduced NH3 concentration in the rumen was compensated via increased urea recycling to maintain an optimum ruminal NH3 concentration for maximum microbial growth, thus decreasing its N urinary excretion27,34,35. However, this did not occur because the content of RDP in diets with RUP likely met the microbial demand for N, thereby not achieving sufficient urea recycling and allowing urinary N excretion similar to the diet with RDP (Table 1).

Elevated amounts of amino acids reaching the small intestine is another factor contributing to a greater N loss. When absorbed in excess or in imbalance relative to the animal’s requirements, these amino acids can be oxidized for energy production, leading to urea production in the liver, which is then excreted via urine. This might occur when the diet offers adequate levels of NH3 to meet the ruminal demand34. Therefore, excess CP concentration in the diet, either as RDP or RUP, may contribute to urinary N excretion.

The greatest N2O emission from the manure of animals fed the RUP sources (Table 3). This indicates that these diets probably had a higher urea content of the manure, since N2O emission is particularly affected by the urinary urea content35.

When higher RUP levels are used in the diet, a change in the route of urine-to-feces excretion is expected due to a higher amount of intact protein that reaches the intestine, which contributes to fecal N excretion when not absorbed38,39. However, there were no differences in fecal N excretion between the RDP and RUP treatments (Table 1), although fecal N concentration differed between the two RUP sources. This might be attributed to the distinct amino acid composition or the different chemical structures of these sources. The processes through which corn (corn gluten, a by-product of wet corn milling) and soybean (thermally treated) have been subjected can make the protein undegradable in the rumen or unavailable38.

Despite different compositions of the manure among the treatments (Table 3), there were no differences in CH4 emission (Table 2). Nitrogen and OM contents and C/N ratio of manure are important factors associated with CH4 emission41,42. Nevertheless, differences in manure chemical composition among the treatments were observed in some sampling days (Fig. 3). This result can be related to variations in environmental conditions, such as temperature and precipitation, which can alter the chemical composition of manure. However, these differences among the treatments were not consistent throughout the experimental period, justifying the lack of differences in CH4 emission.

In manure, most of the N content comes from N excreted via urine in the form of urea, which is rapidly hydrolyzed to NH4+, and N losses from organic forms of feces also occur41. Organic N can promote CH4 emission, playing an important role in the transformation of acetate to CH442, whereas mineral N as NH4+ can inhibit CH4 production, breaking the link between acidification and methanogenesis in anaerobic processes43.

Nitrous oxide and CH4 fluxes varied from −62 to 318 µg N2O m2 h−1 and from −125 to 321 µg CH4 m2 h−1, respectively, during the experimental period (Fig. 2). These fluxes showed a great variation, which can be attributed to several factors, such as the temporal variation in the chemical composition of manure due to variations in climatic conditions, as explained above (Table 3, Fig. 3). Other researchers44 have reported a large variation in emissions, mainly associated with irregular fecal and urine deposition on the surface, which may also have occurred in the present study.

Frequent deposition and accumulation of feces and urine in the soil did not increase CH4 and N2O emissions over time under all treatments. Trampling by animals may have caused aeration of the surface material and have provided unfavorable environment for the action of methanogenic bacteria and nitrifying/denitrifying microorganisms. In addition, the humidity in the feedlot did not increase over time, based on the DM content of the manure, except on rainy days (Table 3). This is probably related to the dry climate at that time of year, associated with the compacted soil of the feedlot.

Precipitation and temperature changes strongly affect CH4 emission45. During the study period, CH4 flux was related to these variables. On the 21th day, increased emission peaks were observed under all treatments, probably due to precipitation in the previous week. Considering that CH4 emission occurs under anaerobic conditions, precipitation may have favored higher emissions due to increased moisture content of the manure46. On the 49th day, reduced CH4 emission was observed, possibly due to temperature drop on that day. Considering that CH4 emission is a biological and anaerobic process, temperature can act as a limiting factor by reducing methanogen activity47. After this period, CH4 emission tended to stabilize, probably due to the absence of high precipitation and little variation in temperature (Fig. 1).

The mean CH4 emission under all treatments during the finishing phase in the feedlot (SM = 53 µg C–CH4 m2 h−1; BSM = 33 µg C–CH4 m2 h−1; CGM = 16 µg C–CH4 m2 h−1; mean of 8.8 g C–CH4 day−1 pen−1) in the present study was lower than reported values by other researchers (mean of 110 g day−1 pen−1)48 under similar climatic conditions and a pen density of 6 m2 per animal, however, the floor was concreted and the excreta were removed every 15 days. The low moisture of the manure was possibly responsible for low CH4 emissions, because even under favorable chemical conditions, microbial activity is limited at low moisture levels. Of note, the density in each pen was 30 m2 per animal and the evaluations were performed near the feeders, in an area of 6.5 m × 10 m with higher excreta deposition. The density of animals is reflected in the condition of excreta deposition and accumulation on the surface, and it is a relevant factor to be considered when evaluating gas emissions in feedlots49.

On some sampling days, CH4 uptake occurred predominantly through the consumption of atmospheric CH4, which can occur in aerobic environments39. The environment is a CH4 source when the balance between methanogenic production and methanotrophic consumption is positive, leading to CH4 emission. In contrast, when this balance is negative, the environment is considered a CH4 sink39.

Considering that the feedlot system has emerged as a management strategy to minimize the impacts of lower forage production in the dry season, majority of feedlots in Brazil are managed from April to November, when rainfall is scarce and temperature is low. The climatic conditions during this period, when associated with feedlots of low animal density, can result in low CH4 emission. In an inventory to estimate GHG emission in Brazil50, it is clear that we do not have enough data to estimate emissions from Brazilian feedlots. Therefore, measurements must cover different systems, with different stockings, feedings and manure management to generate concrete data that allow the comparison between mitigation strategies.

The mean N2O fluxes (SM = 22 µg N–N2O m2 h−1; BSM = 59 µg N–N2O m2 h−1; CGM = 36 µg N–N2O m2 h−1; 12 g N–N2O day−1 pen−1) observed in the present study were high than to some report values reported ( 0.8 g N–N2O day−1 pen−1)48 even considering a higher density (6 m2 animal−1) and removal of excreta from the area every 15 days. A higher peak of N2O emission was observed on the 21st day under all treatments, possibly due to rainfall in the previous week. Other researchers51 in a controlled experiment simulating open feedlot, demonstrate increased emissions following precipitation events, with peaks that vary 2 h to 15 days after the rain.

Post-rainfall emissions and wetting of the area might be related to a combination of mineralization, nitrification, and/or denitrification, leading to the release of N2O absorbed in the dry soil52. Moisture is an important factor in N2O production, particularly when associated with temperature and a propitious chemical composition53, emission of N2O increases markedly with increasing temperature54. However, after reaching the peak, N2O emissions remained stable, with small variations across evaluation days; even in the presence of additional precipitation events, low temperature (minimum of 3.3 °C near the 49st day) may have hampered the occurrence of new emission peaks.

Nitrate was not detectable in the manure during the experiment. Nitrous oxide production is assumed to occur through nitrification, via the oxidation of NH4+ in hydroxylamine (NH2OH), with NOH as an intermediate and N2O as the product55. N2O can also be produced through denitrification by nitrifiers, wherein NH3 is nitrified and oxidized to nitrite (NO2−), which is then reduced to nitric oxide (NO), N2O, and molecular N (N2). Nitrous oxide is an intermediate in the reduction of NO2− to N256. During denitrification, NO3− is used as the primary substrate57. Denitrification may not have occurred in the present study.

Correlation analyses showed no significant linear associations of CH4 and N2O production with the tested variables related to the chemical composition of manure, which can be attributed to specific factors (Tables 4 and 5). The processes underlying the production of gases are complex and rely on the chemical composition of manure. In addition to the chemical composition, the emission of gases in the manure is dependent on other factors such as temperature, moisture, deposition conditions, and trampling by animals. The absence of significant correlations between gas production and manure composition may be related to the small variation in the characteristics analyzed during the sampling period, making it difficult to observe relationships among variables.

NH3 emission

The use of RUP in the diet did not reduce N loss via urine and did not influence NH3 emission from the manure. In this sense, our hypothesis that RUP inclusion in the diet would reduce N loss and contribute to decreased NH3 emission was rejected.

The manure of animals fed SM presented higher NH3 emissions than that of animals fed CGM and BSM from the 8th to 25th day of evaluation. This may be attributed to the higher NH4+ content of the manure of animals fed SM than that of animals fed CGM and BSM at the beginning of the sampling period (Table 6). Subsequently, the manure of animals fed CGM and BSM presented a new NH3 emission peak following the event of the highest precipitation (54.2 mm) throughout the experimental period. However, during this period, most of the NH4+ from the SM treatment had already been used, as reflected by the weak response to precipitation under this treatment. Urea present in the urine and feces is rapidly hydrolyzed, and the formed NH4+ is dissociated to aqueous NH3, depending on NH4+ concentration and pH of manure and environmental conditions. When precipitation occurs, urease activity is promoted, resulting in increased NH3 emission58. Of note, however, manure sampling for characterization was performed before implanting the chambers in the area. Thus, the chemical composition data presented herein do not represent the possible temporal variations during the NH3 evaluation period (Table 3).

Higher values of NH3 emission have been reported (49.1 kg NH3 animal−1) in beef cattle feedlots, which is mainly related to the fact that the majority of confinement feedlots are outdoors, given that wind speed in open environments increases emission59. According to others studies19, daily NH3 emission in feedlots rarely exceeds 2000 µg NH3 m−3; however, in the present study, higher values were observed. Importantly, as explained before, the evaluations were performed in an area of higher excreta deposition, with the objective of comparing the treatments in homogeneous conditions of excreta distribution. Therefore, the amount of emission by area of the total feedlot may have been overestimated in this study. Conversely, we did not account for emissions when the animals were present in the feedlots. Throughout the sampling period, the animals had already been removed from the area, and there was a large amount of accumulated manure. When the wet season starts, emission may have been favoured by increased moisture content due to the large amount of available substrate19. Therefore, the urea excreted by the animals was hydrolyzed and contributed to the stock of NH4+, which was emitted as NH3 when the moisture content increased as a function of precipitation.

Over time, as no new manure was deposited due to the absence of animals in the area, emission probably ceased when the substrate was consumed, which occurred around the 77th day in the present study. In experiments in which excreta from the animals is collected and then applied to the soil for evaluation in the absence of animals and new depositions, ammonia emission occurs for 3 weeks on average42,62,63. Therefore, further studies are warranted to investigate NH3 emission in open feedlots and to observe peaks occurrence in the presence of animals, maintaining the evaluations also after removing the animals, in the next rainy season.

Conclusions

The inclusion of RUP in the diet did not affect N excretion by animals. While the N2O emission from the manure was increased, CH4 emission and NH3 emission remained unaffected. Additional studies are warranted to investigate the effects of using different proportions of RDP and RUP in diets on NH3, N2O, and CH4 emissions from the manure of animals managed in feedlot systems under tropical conditions.

Material and methods

The experiment was approved by the Ethics, Bioethics, and Animal Welfare Committee of São Paulo State University (UNESP), Jaboticabal, under protocol numbered 16.668/16. All methods were carried out in accordance with relevant guidelines and regulations. Methods are reported in the manuscript following the recommendations in the ARRIVE guidelines.

Site description

The present study was conducted at the Campus of Jaboticabal of the São Paulo State University, Sao Paulo, Brazil (21°14′05″S, 48°17′09″W; altitude, 615.01 m). The region has a tropical climate, with a dry season from April to September and a wet season from October to March, during which over 80% of the annual precipitation occurs. The soil is Rhodic Ferralsol62 derived from basalt, with a sandy–clay–loam texture (10% silt and 61% sand) in the surface layer (0–10 cm). The soil pH in CaCl2 is 5.9, bulk density is 1.8 kg dm−3, and organic matter content is 16.6 g dm−3 at the same depth.

Meteorological data (daily precipitation and ambient temperature) were obtained from the dataset of the Agrometeorological Station of the Department of Exact Sciences, Universidade Estadual Paulista (UNESP), Campus of Jaboticabal, located 1 km from the experimental area.

Experimental design

The experiment was conducted for 210 days from May to December 2019. The first 21 days were dedicated to animal adaptation to the diet, followed by 112 days of confinement, during which weekly sampling of N2O and CH4 was performed. After removing the animals from the feedlots, NH3 was sampled for 77 days.

Fifty-four Nellore bulls with an initial body weight of approximately 360 kg were distributed in three treatments. The animals were divided into three treatments and allocated in collective pens (11 m × 50 m; one pen per treatment and 18 animals per pen). Each pen had a dirt floor with collective drinkers for every two pens. There were two covered automated feeders in each pen (INTERGADO®, Intergado Ltd., Contagem, Minas Gerais, Brazil). The feed system was equipped with an automated feeder monitor resting on load cells, allowing electronic registration of the amount of feed consumed by animal. The trough recognizes the animal from the electronic ear tag, automatically sends consumption data to a database, and stores the information.

Manure of animals fed with sources of protein (two sources of RUP and one source of RDP as a control) was collected, resulting in three treatments as follows:

  1. (1)

    Soybean meal (SM): source of RDP.

  2. (2)

    By-pass soybean meal (BSM): source of RUP

  3. (3)

    Corn gluten meal (CGM): source of RUP.

The experimental diets were composed of 30% roughage and 70% concentrate, formulated to meet the average daily gain (ADG) of 1.5 kg day−1, according to BR CORTE63. The diets were offered at 08:00 am and 04:00 pm. The amounts offered were sufficient to allow a daily leftover of 5–10% of the total offered.

The ingredients of the diets were analyz ed for chemical composition (Table 7). The AOAC64 method was used to determine dry matter (DM) (method 930.15), crude protein (CP) (method 990.03), organic matter (OM) (method 942.05), and ether extract (EE) (method 920.39) content. Neutral detergent fiber (NDF) content was determined according to the method described by65 using ANKOM® 2000 (Ankom Technologies, New York, USA) with thermostable α-amylase and without sodium sulfite, corrected for ashes and residual proteins. The RDP and RUP content was estimated based on the protein fraction66 and degradation rate of each fraction, considering a passage rate of 5% h−1.

Table 7 Ingredients and chemical composition of the diets.
Full size table

Gases (N2O, CH4 and NH3) were sampled using chambers (n = 9 per treatment) arranged in an area of 65 m2, near the feeders, where the manure (feces and urine) was deposited the most frequently. The chambers were placed on manure (feces and urine) that had been deposited on the feedlot surface by animals subjected to treatments. At the time of evaluation, the chambers were randomly placed in an area (6.5 m × 10 m) delimited near the feeders inside each confinement pen. Specifically, an area of higher excreta deposition was selected with the objective of treatment comparison, thus avoiding evaluation in places without homogenous excreta distribution (Fig. 6).

Figure 6
figure 6

Map of the experimental area.

Full size image

Evaluation of N2O and CH4 emissions

Nitrous oxide and CH4 emissions were determined using static closed chambers, according to the recommendations of the manual for GHG evaluation67. Plastic chambers (0.6 m × 0.4 m × 0.24 m) coated with a thermal insulator were positioned above the manure only at the time of gas collection, allowing the animals to trample, defecate and urinate freely around in the area. Sampling was performed once a week throughout the feedlot period (112 days), totaling 16 sampling events. Sampling was carried out between 4:00 pm and 04:00 pm. The chambers were closed for 20 min, and air samples were collected at 0, 10, and 20 min using a 50 mL polypropylene syringe and then transferred to previously evacuated chromatography flasks (20 mL). The temperature inside and outside the chamber was measured using a digital thermometer (Incoterm®) to correct gas fluxes.

Air samples were analyzed using gas chromatography (Shimadzu Greenhouse Gas Analyzer GC-2014; Kyoto, Japan) under the following conditions: (1) N2O: injector temperature, 250 °C; column temperature, 80 °C; N2 carrier gas (30 mL min−1); and electron capture detector temperature, 325 °C; and (2) CH4: H2 flame gas (30 mL min−1) and flame ingestion detector temperature, 280 °C.

Nitrous oxide (µg N–N2O m−2 h−1) and CH4 (µg C–CH4 m−2 h−1) fluxes were calculated considering a linear increase in gas concentration inside the chamber during the closed period and corrected for ambient temperature, ambient pressure, and chamber dimensions, as follows:

$$ {\text{Gas}}\;{\text{flow}} = ({\text{gas}} \times {\text{M}}\omega \times {\text{V}} \times {6}0 \times {1}0^{{ - {6}}} )/{\text{A}} \times {\text{VM}}_{{{\text{corr}}}} \times {1}0^{{ - {9}}} ) $$

where gas is the increment in the gas concentration inside the chamber during the closed period (ppb min−1); Mω is the molar mass of N–N2O (28 g mol−1) or C–CH4 (12 g mol−1); V is the chamber volume (m3); 60 is the conversion factor from minutes to hours; 10–6 represents the conversion factor from g to µg; A is the chamber area (m2); VMcorr is the molecular volume corrected by the normal conditions of temperature and pressure at the time of sampling; and 10–9 is the conversion factor from ppb to µL m−3.

Fluxes were multiplied by 24 to obtain daily emissions, and the daily values were integrated through linear interpolation to obtain cumulative emissions during the evaluated period. Negative fluxes were included in the calculations to avoid biased data68.

Evaluation of NH3 emission

After removing the animals from feedlots, NH3 volatilization was evaluated until the NH3 emission ceased by sampling on days 1, 2, 3, 4, 6, 8, 13, 19, 25, 31, 38, 44, 51, 59, 68 and 77 after positioning the chamber. The chambers were randomly placed above the manure (feces and urine) in the previously delimited areas. Quantification was performed according to the methodology of static chamber69, using semi-open chambers made of plastic bottles containing a foam soaked in 10 mL of 1.0 mol dm−3 H2SO4 solution + glycerin 2% (v/v) to capture N. The amount of N-NH3 retained in the foam was determined by distillation, following the Kjeldhal method (method 973.49)61 and a correction factor of 1.74 was used69.

Manure analysis

Manure samples composed of feces and urine deposited in the feedlot surface material soil, trampled by the animals, were collected on days 42, 63 and 105 after N2O and CH4 evaluations, directly above the ground surface at the places where the chambers were positioned. The samples were analyzed for DM (method 930.15)64, OM (method 942.05)64, total C, total N (dry combustion method, using Leco® CN-828, Leco Corporation, Michigan, USA), and soil inorganic N (NO3− and NH4+) (distillation using magnesium oxide and Devarda’s alloy, method 973.49)64 content.

Estimation of fecal and urinary production and N balance

Fecal production was estimated using the internal marker technique70 based on the indigestible NDF (NDFi) marker. Fecal sampling was performed from the 60th day after the animals entered the feedlots, for three consecutive days, directly from the rectum of the animals. Sampling was performed in the morning, middle of the day, and afternoon on the first, second, and third days, respectively. A composite fecal sample, by animal (9 animals/treatment), were made with the samples from these three days. The samples were mixed, homogenized, partially dried in a forced-air ventilation oven at 55 °C for 72 h, and milled in a mill with a 2 mm sieve. Samples of the ingredients of the animals’ diets were collected, and their consumption was determined using the INTERGADO®.

Fecal NDFi content was determined after incubating the samples in situ for 288 h71 followed by extraction with neutral detergent using an autoclave72. Fecal DM production was determined as the ratio of the concentration of the internal indicator ingested by the animal and its concentration in feces73.

Urine samples were collected simultaneously with fecal samples. In brief, 50 mL aliquots of urine were sampled (“spot” sample) during three consecutive days74. Creatinine concentration in the spot sample was determined with a colorimetric method using a commercial kit (Labtest®). Urinary excretion was estimated based on the association between creatinine excretion and body weight using the equation proposed by75:

$$ {\text{UCE}}\left( {{\text{g day}}^{{ - {1}}} } \right) = 0.0{345 } \times {\text{ BW}}^{{0.{9491}}} $$

where UCE = urinary creatinine excretion and BW = body weight in kg.

The fecal and urine samples were analyzed for total C and total N content using the dry combustion method with Leco® CN-828 (Leco Corporation). Nitrogen retention (NR) was expressed in grams per day and in percentage of NC, and fecal and urinary N excretion was expressed as the percentage of the total material excreted. The following equation was used to calculate NR:

$$ {\text{NR}} = {\text{NC}}\left( {{\text{g day}}^{{ - {1}}} } \right) - \left[ {{\text{EFN }}\left( {{\text{g day}}^{{ - {1}}} } \right) + {\text{EUN }}\left( {{\text{g day}}^{{ - {1}}} } \right)} \right] $$

where NC = N consumption, EFN = excretion of fecal N, and EUN = excretion of urinary N.

Statistics

All statistical analyses were performed using SAS 9.4 (SAS Inc., Cary, NC). Response variables were analyzed in a completely randomized design using the PROC MIXED procedure. There were nine experimental units per treatment. Mean values were compared using orthogonal contrasts (SM vs. RUP and BSM vs. CGM) at a 5% probability level.

Total N, total C, and C/N in feces and urine and N balance were analyzed considering a model including the treatments (SM, BSM, and CGM) as fixed effects, animals (experimental unit in the RANDOM SAS option) and residual random error (NIID) of (0, σ2) as random effects.

Cumulative N2O, CH4 and NH3 emissions, and manure characteristics (DM, OM, N, C, C/N, NH4+, and NO3− of manure, sampled on day 0, before the beginning of NH3 emissions measurements) were analyzed considering a model including the treatments (SM, BSM, and CGM) as fixed effects, chamber (experimental unit in the RANDOM SAS option) and residual random error (NIID) of (0, σ2) as random effects.

Nitrous oxide and CH4 daily fluxes and manure characteristics (DM, OM, N, C, C/N, NH4+, and NO3−, sampled on days 42, 63 and 105 of N2O and CH4 evaluation) were analyzed using a repeated measures mixed model over time including the treatments (SM, BSM, and CGM), collection period and interaction as fixed effects, chamber (experimental unit and RANDOM SAS option) and residual random error (NIID) of (0, σ2) as random effects. Distinct covariance matrices were evaluated and the best structure was selected according to the Akaike information criterion (AIC).

Pearson correlation analysis between gas emission (N2O and CH4) and chemical composition (N, C, C/N, DM, OM, and NH4+) of the manure was performed separately for each sampling day (days 42, 63 and 105 of manure evaluation), and also considering all data collected on these days.