Urease and nitrification inhibitors with pig slurry effects on ammonia and nitrous oxide emissions, nitrate leaching, and nitrogen use efficiency in perennial ryegrass sward

Objective The present study was conducted to assess the effect of urease inhibitor (hydroquinone [HQ]) and nitrification inhibitor (dicyandiamide [DCD]) on nitrogen (N) use efficiency of pig slurry for perennial ryegrass regrowth yield and its environmental impacts. Methods A micro-plot experiment was conducted using pig slurry-urea 15N treated with HQ and/or DCD and applied at a rate of 200 kg N/ha. The flows of N derived from the pig slurry urea to herbage regrowth and soils as well as soil N mineralization were estimated by tracing pig slurry-urea 15N, and the N losses via ammonia (NH3), nitrous oxide (N2O) emission, and nitrate (NO3−) leaching were quantified for a 56 d regrowth of perennial ryegrass (Lolium perenne) sward. Results Herbage dry matter at the final regrowth at 56 d was significantly higher in the HQ and/or DCD applied plots, with a 24.5% to 42.2% increase in 15N recovery by herbage compared with the control. Significant increases in soil 15N recovery were also observed in the plots applied with the inhibitors, accompanied by the increased N content converted to soil inorganic N (NH4++NO3−) (17.3% to 28.8% higher than that of the control). The estimated loss, which was not accounted for in the herbage-soil system, was lower in the plots applied with the inhibitors (25.6% on average) than that of control (38.0%). Positive effects of urease and/or nitrification inhibitors on reducing N losses to the environment were observed at the final regrowth (56 d), at which cumulative NH3 emission was reduced by 26.8% (on average 3 inhibitor treatments), N2O emission by 50.2% and NO3− leaching by 10.6% compared to those of the control. Conclusion The proper application of urease and nitrification inhibitors would be an efficient strategy to improve the N use efficiency of pig slurry while mitigating hazardous environmental impacts.


INTRODUCTION
Nitrogen (N) is an essential nutrient as a key limiting factor of the growth and develop ment of plants in agricultural ecosystems [1]. Incremental increases in global crop yields during the past several decades has mainly been dependent on the increasing application of synthetic N fertilizers. Animal manures have long been used as alternative organic N fertilizers. Most of the N in feces is present in organic form, while in urine, 65% to 90% of the N is present as urea [2]. In Korea, pig slurry is the most viable recycling option and represents more than 80% of all recycled animal manure [3] because pig farms usually have little or no arable land for forage production.
The amount of N supplied to agroecosystems is often higher than N uptake by crops. An excessive N input leads to N losses via volatilization of ammonia (NH 3 ), emission of N, which pose a significant threat to the environmental quality of the atmo sphere and aquatic systems [4]. Thus, management of N nutrition is important to increase crop productivity and control environmental pollution. The chemical or organic N applied to the soil, mainly in form of urea, hydrolyze into NH 4 + , hydroxyl, and carbonate ions by the microbial urease mediation. The NH 4 + produced then converts to NH 3 , which can be lost through volatilization under alkaline conditions. If soil condition does not favor volatilization, NH 4 + can either be held in the soil via cation exchange or converted to NO 3 -, leading to N losses through leaching or denitrification. A pro portion of volatilized and deposited NH 3 can generate N 2 O, which is a longlasting greenhouse gas, through both nitrifi cation, in which aerobic oxidation of NH 4 + to NO 2 and further NO 3 -, and denitrification, in which NO 3 is reduced to N 2 O [5].
Various management practices and technologies have at tempted to enhance N fertilizer use efficiency while minimizing N losses to the environment. One of the strategies is the use of inhibitors of urea hydrolysis (urease inhibitors) and of ammonia oxidation (nitrification inhibitors), which have been shown to be effective in enhancing N use efficiency by delaying nitrification/denitrification [6,7]. The efficacy of urease and/or nitrification inhibitors in mitigating NH 3 and N 2 O emissions varies with soil pH [8], type and level of ap plied N sources [9], the concentration of inhibitors [10], soil texture [11], as well as climatic factors such as rainfall [12]. Martins et al [7] showed that the urease and nitrification in hibitors enhanced urea 15 N recovery by maize and increased grain yield. In a metaanalysis with 111 datasets from 39 studies [5], nitrification inhibitors are effective in reducing N 2 O emissions with the highest inhibitory effect in grassland and followed by cropland, upland, and paddy. Li et al [13] reported that N 2 O emission was efficiently lower in urea to gether with urease and nitrification inhibitors than with either a single urease or nitrification inhibitor. However, the flux of N derived from animal manure to pasture plants and soil has not been fully elucidated. In addition, few studies have assessed the effects of inhibitors on gaseous emissions and nitrate leaching from animal manurebased N [14].
In the present study, we hypothesized that the synergistic effect of urease inhibitor (hydroquinone [HQ]) and nitrifi cation inhibitor (dicyandiamide [DCD]) may improve N use efficiency of pig slurry and minimize the N losses to the environment by regulating N mineralization processes in soil. To test this hypothesis, the turnover of pig slurryurea 15 N and its flow into the plant and soil inorganic N compo nents were directly quantified while accounting for N losses to the environment (NH 3 and N 2 O emissions and nitrate leaching). The resulting data were interpreted regarding the effectiveness of HQ and/or DCD.

Experimental design
The study was based on field experiments conducted on a permanent grass sward consisting mainly of perennial rye grass (Lolium perenne) on sandy loamy soil. The soil chemical properties of the experimental site are presented in Table 1. During the experimental period, the typical climate was temperate with high humidity, with an average temperature of 22.5°C and total precipitation of 420 mm. Four treatments of slurry application were compared: i) only pig slurry as a control, ii) HQ treatment (pig slurry + urease inhibitor [HQ, C 6 H 6 O 2 ]), iii) DCD treatment (pig slurry + nitrification in hibitor [DCD, NH 4 F]), and iv) HQ and DCD combination treatment. The experiment in a randomized complete block design consisted of four replications. Each treatment plot measured 2.5 m×10 length experiment and contained 12 microplots (0.5 m×0.5 m) for monitoring the fate of 15 Nla beled pig slurry. To prevent surface runoff and contamination by slurry application, there was a 2 m margin between plots with a 0.45 m metal retainer inserted 30 cm deep soil. The bottomless acrylic chambers (0.2 m diameter and 0.3 m length) were used for collecting gas samples and suction cups (P80, eco Tech, Bonn, Germany) for collecting leachate sam ples.

Pig slurry treatments and 15 N labeling
The pig slurry was obtained from pig livestock farm and stored in concrete tanks at ambient temperature for approxi mately 1 week. Four different 400 L plastic containers filled with pig slurry were mixed with 0.3% HQ or 5% DCD of the totalN in pig slurry, respectively. The slurry urea fraction of four treatments were labeled by thoroughly mixing with highly enriched 15 15 Nurea enrichment of 5.001±0.012 atom %. Treated pig slurry at a rate of 200 kg N/ha (e.g., 316 L per 25 m 2 plot, which contained 95.8 kg P/ha and 127 kg K/ha) was applied after herbage was cut at 50 mm above ground level [3].
Herbage, soil, gases, and leachate sampling The herbage sample was harvested from four randomly placed microplots by cutting manually, and the remained stubble was approximately 50 mm. About 500 g of collected herbage sample was chopped into 20 mm long segments, and then lyophilized, ground, and stored in a vacuum desic cator for chemical analysis. The soil samples were collected by soil cores (0 to 0.3 m depth) randomly using a 0.3 m diameter tube auger in the same microplots that herbage sampling place. The collected soil samples were airdried, ground, and sieved to <0.15 mm. The herbage and soil sampling were done at 7, 14, and 56 d after pig slurry application, respec tively.
Airtight acrylic chambers were located to 50 mm depth soil in each experimental plot for gas sampling. To collect NH 3 emission, the acid trap system method was used as de scribed by Ndegwa et al [15] with modifications. Each chamber was connected to NH 3 N trapping bottles contain ing 150 mL of 0.2 mol/L H 2 SO 4 and a vacuum system to pull air through the chambers. The NH 3 N traps a constant rate of 1.5 L per minute. Each chamber was closed with silicon sealing and clamped for 24 hours. The NH 3 sampling in each treatment block was done at the same time over 1 hour to avoid the impact of extraneous gases. The N 2 O gas was col lected by using a syringe before NH 3 emission sampling and then stored in 10 mL of vacutainer tuber. The gas of NH 3 and N 2 O was collected daily for the first 14 d, then at intervals 1 to 2 weeks. The leachate samples were obtained by suction cups in each plot at a depth of 0.5 m for NO 3 -N analysis. Soil water samples were obtained by applying a tension -250 hPa. A sampling of NO 3 -N was done weekly and stored at -20°C.

Measurements and chemical analysis
The herbage was harvested from each microplot and con verted to kg/ha. To calculate the N recovery in herbage (kg N/ha), the converted estimate was multiplied by the N con centration determined in the subsamples. The stable isotope ratio mass spectrometer (IRMS, IsoPrime, GV Instrument, Manchester, UK) was used for measuring the total N con tent and 15 N atom % of herbage, soil, and pig slurry samples. Inorganic nitrogen was extracted with 2 M KCl and the NH 4 + N was determined by distillation in an alkaline medium (MgO). The same procedure was used for NO 3 -N after re duction with Devarda's alloy. The N liberated from each distillation was collected in H 2 SO 4 and then evaporated to dryness to analyze the determination of 15 N atom % excess of each N fraction. The total N and inorganic N (NH 4 + N and NO 3 -N) concentration in soil samples were converted to kg N/ha using soil bulk density. To determination of NH 3 volatilization, the solution collected by acid traps in the form of (NH 4 ) 2 SO 4 was quantified by a colorimetric determination with ammonium color reagent (Nessler's reagent, Sigma, 72190; St. Louis, MO, USA) as described by Kim and Kim [16]. N 2 O concentration in gas samples collected was deter mined using a gas chromatograph (GC7890A, Agilent Technologies, Santa Clara, CA, USA) equipped with a ther mal conductivity detector (TCD) and with a HPPlot 5A column (30 m×0.53 mm×25 μm) under the following con ditions: column oven temperature 40°C; injector temperature 100°C; detector temperature 300°C; carrier gas helium (2 mL/min). The N 2 O fluxes were calculated as described by Guo et al [6]. The concentration of NO 3 -N leaching was de termined by ion chromatography DX 120 Dionex as described by Hamonts et al [17]. The total NH 3 , N 2 O emission, and NO 3 leaching over the entire experimental period were calculated by the sum of daily measurements.
The determined 15 N atom % excess abundances in the to tal N and inorganic N fractions in herbage and soil samples were converted to relative specific activity and the amount of N derived from pig slurry urea (NdfSU) in herbage samples was calculated as described by Park et al [18]. The ratio be tween the NdfSU and the quantity of applied N was applied for percentage of slurry ureaN recovery in the total N, NH 4 + , and NO 3 fractions in herbage and soil. Therefore, the por tion not recovered in herbage and soil indicate the percentage of loss.

Statistical analysis
Analysis of variance was conducted to assess the effects of urease and/or nitrification inhibitors with pig slurry at each sampling time on herbage yield, N uptake, gas emissions, leaching, and the fate of slurry ureaN. Statistical analysis were conducted using the SAS 9.1.3 software.

Dry matter, total N and N amount derived from slurry urea in herbage
Herbage dry matter (DM) was not influenced by the appli cation of urease and nitrification inhibitors during the first 14 d. However, at final regrowth at 56 d, combined application of HQ and DCD (HQ+DCD) induced the highest herbage DM yield (+30.8%), followed by DCD (+14.5%) and HQ (+9.6%) single applications, compared to that in the control (only pig slurry applied) ( Figure 1A). Total N content in herb age increased only in the HQ+DCD plot from 14 d, in which it was 21% to 33% higher than that in the control ( Figure  1B). The amount of NdfSU in herbage at the final regrowth at 56 d significantly increased only at 56 d by 24.5%, 33.0%, and 42.2% in the HQ, DCD, and HQ+DCD applied plots, respectively, compared to that of the control (p<0.001) ( Figure  1C). However, among the HQ, DCD, and HQ+DCD applied plots, there were no significant differences.

Soil N dynamics
The inhibitors (HQ and/or DCD) did not affect the total N pool size in soil throughout the regrowth period ( Figure 2A). However, the NdfSU in the soil at 56 d increased by 11.8%, 12.7%, and 20.3%, respectively, in the HQ, DCD, and HQ+ DCD plots compared with the control ( Figure 2B). The con tent of NH4+N in soil was significantly reduced by the application of the inhibitors during the first 14 d with a stronger effect of HQ, whereas it was higher than control in the DCD plot or recovered to the control level in the HQ and HQ+DCD plot at 56 d ( Figure 3A). The amount of N derived from slurry urea in the soil NH 4 + fraction (NdfSU NH 4 + ) during the first 14 d of regrowth showed a similar pattern, with a significant reduction following HQ and/or DCD application ( Figure 3B). The final NdfSUNH 4 + at 56 d was the highest in the HQ+DCD plot (4.9 kg N/ha) and fol lowed by the DCD (4.2 kg N/ha), HQ (3.0 kg N/ha), and control (1.8 kg N/ha) plot. The content of NO 3 -N in the soil was lower in all plots applied with the inhibitors than that in the control throughout whole the regrowth period ( Figure  3C). The amount of N derived from slurry urea in the soil    at 56 d significantly increased in the DCD (+14.5% compared to that of the control) and HQ+ DCD (+22.5%) plots ( Figure 3D).

Recovery of pig slurry-urea 15 N
The percentage of pig slurryurea 15 N recovered in herbage averaged over all treatments gradually increased from 3.9% (at 7 d) to 26.5% (at 56 d), whereas the soil 15 N recovery de creased from 67.3% to 44.8% over the same period (Table 2). Thus, at the end of regrowth (56 d after pig slurry applica tion), the herbage 15 N recovered was higher in the HQ and/ or DCD plots than in the control plots, with no significant difference among the three inhibitors treatments. The soil 15 N recovery was also significantly increased by the inhibitor treatments. The percentage of pig slurryurea 15 N recovered in the soil NH 4 + and NO 3 fractions were also increased by application of urease and nitrification inhibitors, with the combined application of HQ and DCD showing a stronger effect. The percentage of 15  ) leaching were quantified. On average, 58.8% of total NH 3 emission during a 56 d period of regrowth occurred within the first 14 d after application of pig slurry to the soil. The daily NH 3 emission during this period was relatively lower in the HQ and DCD +HQ plots than in the control and DCD plots ( Figure 4A). Cumulative NH 3 emission during 56 d of regrowth decreased by 30.0%, 16.3%, and 34.1% in the HQ, DCD, and DCD+HQ plots compared with the control plots ( Figure 5A). Consis tent with NH 3 emission, significant effects of inhibitors in reducing daily N 2 O emission was observed, with a stronger effect observed for DCD ( Figure 4B). N 2 O emission in all treatments decreased to near the background level after 56 d    Figure 5B). The weekly cumulative NO 3 leaching was lower in the plots applied with the inhibitors, especially prior to 21 d after pig slurry application. Overall DCD application (e.g., DCD and HQ+DCD treatment) was more effective in reducing NO 3 leaching ( Figure 4C). Cu mulative NO 3 leaching for the whole experimental period declined by 7.0%, 12.9%, and 11.8% in the HQ, DCD, and DCD+HQ plots, respectively, compared with the control plots ( Figure 5C).

Regrowth and pig slurry-urea 15 N recovery in herbage
The efficacy of different types of urease inhibitors [HQ, phenyl phosphorodiamidate (PPDA), and N(nbutyl) thiophos phoric triamide (NBPT)] and nitrification inhibitors (DCD, 3, 4dimethylpyrazole phosphate [DMPP], Nitrapyrin, and thiosulphate) have been tested to improve N use efficiency while minimizing N losses to the environment. For instance, a metaanalysis of 113 field experiments showed that the ef fectiveness of various urease and nitrification inhibitors was relatively consistent across land use types in both chemical and organic N fertilizers [19]. In this context, we focused on urease inhibitor HQ and nitrification inhibitor DCD be cause HQ is lower cost [20], DCD is less volatile, and easily blended with fertilizers [5]. In the present study, single or combined HQ and DCD treatments did not influence the amount of NdfSU in herbage during the first 14 d of re growth, whereas at the final regrowth (56 d) positive effects of HQ and/or DCD were observed, at which NdfSU was en hanced by 33.2% (on average 3 inhibitor treatments) compared with the control ( Figure 1C). Consistent with NdfSU, the final herbage DM at 56 d significantly increased in the HQ and/or DCD applied plots ( Figure 1A). This indicated that inorganic N might be more available during the later period of re growth due to delayed hydrolysis of urea in pig slurry by HQ, and reduced oxidation of NH 4 + to NO 3 by DCD. In ad dition, these results indicated that early regrowth might be less dependent on exogenous N uptake by plants [21]. At final regrowth (56 d), the recovery of pig slurryurea 15 N varied within the range of 26.4% to 30.2% in the HQ and/or DCD applied plots, which was higher than that of the control (21.2%) ( Table 2). By using 15 N tracing, Choi et al [22] revealed that N is produced from organic amendments and N uptake was more pronounced during the later growth period of Chinese cabbage.

Soil mineralization and pig slurry-urea 15 N recovery
Plant uptake of N released from animal manure gradually increases with progressing regrowth of perennial grasses [3,18,23]. In the present study, at the final regrowth (56 d), we found a significant increase in herbage N content in the HQ+DCD plot, and NdfSU in herbage of all plots applied with the inhibitors ( Figure 1B). However, the soil total N content was not affected by the inhibitors throughout the experimental period (Figure 2A). This indicates that enhanced N uptake and herbage growth in the HQ and/or DCD ap plied plots are due to inorganic N released from organic N rather than the N pool size in soil [3,23]. The NdfSU in the soil total N gradually decreased from 134.6 (at 7 d) to 89.2 kg N/ha (at 56 d) (based on average values of 4 treatments), corresponding to a decrease of 15 N recovery in soil from 67.0% to 44.8% (Figure 2). This implies that N released from the applied urea in pig slurry dilutes the soil inorganic N pool, which is available for herbage regrowth. However, the NdfSU in herbage was not significantly affected by HQ and/or DCD application during the first 14 d of regrowth, although the amount of N derived from the pig slurryurea in the soil NH 4 + (NdfSUNH 4 + ) or NO 3 fractions (NdfSUNO 3 -) de creased in the HQ and/or DCD treatments from 7 d ( Figure  3). This may reflect a common N utilization pattern during the early regrowth characterized by low exogenous N uptake because shoot regrowth during this period depends on a large portion of endogenous N rather than exogenous N up take [21]. In addition, during the first 7 d of regrowth, urea 15 N in pig slurry was mineralized mainly to NH 4 + N, which accounted for 63.6% to 88.6% of total NdfSU in the soil min   (Figure 3). The NdfSUNH 4 + was lower in the plots applied with the in hibitors, especially in the presence of HQ (e.g., HQ and HQ+ DCD treatments) during the first 14 d, suggesting that HQ delayed the hydrolysis of urea in pig slurry [7]. The NdfSU NH 4 + in soil slowed down with progressing regrowth with an opposite increase in the NdfSUNO 3 - (Figure 3B, D), re flecting nitrification of the NH 4 + released from pig slurry urea. The NdfSUNO 3 in the soil at 56 d of regrowth was significantly higher in the presence of the inhibitors, espe cially in the presence of DCD (e.g., DCD and HQ+DCD treatments), compared with the control (Figure 3). At the fi nal regrowth (56 d), the N content converted to soil inorganic N from pig slurryurea (NdfSUNH 4 + + NdfSUNO 3 -) was higher in the presence of DCD (70.4 to 77.3 kg N/ha) com pared to that of control (60.0 kg N/ha) ( Figure 3). Retention of higher NdfSUNH 4 + and NdfSUNO 3 in the soils amend ed the inhibitors may reflect the active onset of hydrolysis of urea and subsequent nitrification during the latter regrowth period when the uptake of exogenous N strongly occurs as a primary N source for the herbage regrowth [21]. Thus, en hanced final regrowth yield ( Figure 1A) and higher NdfSU in herbage at 56 d ( Figure 1C) in the HQ and/or DCD plots are certainly attributed to the higher availability of N re leased from pig slurry, as evidenced by higher percentages of urea 15 N recovered in the soil inorganic N, i.e., 38.6%, 33.6%, and 31.5% of the 15 N applied in the DCD, HQ, and HQ+DCD plots, respectively, compared with the control (22.4%). Many studies have shown positive effects of urease and/or nitrifica tion inhibitors on plant nutrient availability in soil, enhancing yields of annual crops [24,25] and herbage in perennial grass lands [26].

N losses via NH 3 , N 2 O emissions, and NO 3 leaching
Although the N in animal manure, especially for urine where urea makes up 65% to 90% of N, is economically attractive, it may also result in environmental pollution via N losses as odorous gases (e.g., NH 3 and H 2 S), greenhouse gases (e.g., N 2 O and CH 4 ) and NO 3 -N leaching when inefficiently used by plants. The options using inhibitors of the N cycle, such as urease and nitrification inhibitors, have been evaluated to mitigate N losses from chemical N fertilizers, mainly urea [25,27] and from animal manure [28]. The present 15 N re covery data has shown that 38.0%, 27.0%, 28.4%, and 21.6% of applied N were unaccounted in the control, HQ, DCD, and HQ+DCD plots, respectively (Table 2). In this study, these percentages were designated as the estimated N loss and the noxious N losses to NH 3 , N 2 O emission, and NO 3 leaching.
The application of animal manure causes NH 3 volatiliza tion via the N decomposition present in the feces and urea hydrolysis. Urea is hydrolyzed by urease and produces NH 3 and carbonic acid. Thus, significant enhancement of daily NH 3 emission after animal manure application has been ob served in various cropping systems [23,29]. In the present study, daily NH 3 emission significantly reduced in the pres ence of HQ (e.g., HQ and HQ+DCD plots) during the first 14 d (Figure 4A), when a large portion of NH 3 emission (58.8%, averaged over 4 treatments, of total NH 3 emission) occurred ( Figure 5A). This result indicates that the urease inhibitor HQ efficiently abates the pool of NH 4 + ( Figure 3A) by slowing the hydrolysis of urea, which alleviates the subse quent NH 3 emission, especially during the early period. Zhengping et al [20] estimated in the laboratory incubation that a urease inhibitor NBPT decreased NH 3 volatilization     [28]. In the pres ent study, daily N 2 O emissions ranged from 0.84 to 18.60 g N 2 ON/ha/d. The significant reduction of N 2 O emission by DCD treatments, as estimated by 59.8% of reduction by DCD alone and 50.0% by HQ+DCD compared with the control (Figure 6B), suggested that the nitrification inhibitor DCD deactivates the enzymes responsible for the oxidation of NH 4 + , reducing its conversion to NO 3 -, which limits the pool of denitrification for N 2 O emission [5] as well as susceptible leaching [6]. The present data showed that the urease inhibitor HQ also significantly reduced N 2 O emission by 40.7% com pared to the control, confirming that HQ plays an important role in reducing N 2 O emission by reducing the pool of NH 4 + released from urea hydrolysis ( Figure 3A), which is a primary source of nitrification and of following denitrifica tion [5,9]. The stronger effect of nitrification inhibitors, compared with that of the urease inhibitor, on reducing N 2 O emission has also been shown in several crop fields applied with urea [9]. Nitrification inhibitors have been shown to successfully reduce N 2 O emission from various cropping systems [24,27] and pastures [26].
In this study, positive effects of HQ and/or DCD in re ducing NO 3 leaching from the soil were observed, as demonstrated by 7.0%, 12.9%, and 11.8% reductions in NO 3 leaching in the soil in the HQ, DCD, and HQ+DCD plots, respectively. This result may reflect the priming effect of the inhibitors on delaying nitrification, as shown by the lower level of soil NO 3 -( Figure 3C) and slightly higher NH 4 + ( Figure 3A). Other studies have shown that nitrification inhibitors efficiently reduced NO 3 leaching from the soil amended with NH 4 + based N fertilizer (including urea based or other organic amendments, which subsequently convert to NH 4 + ) by retaining N in the soil NH 4 + form over a longer period, reducing the peak concentration of soil NO 3 and the potential for N losses through denitrification or NO 3 leaching from the soil [30]. In addition, Zaman and Blennerhassett [14] revealed that the addition of urease in hibitor NBPT reduces NO 3 leaching to a greater extent for synthetic fertilizer and animal excreta.
In conclusion, with progressing regrowth of perennial ryegrass pasture, the uptake of applied pig slurryurea 15 N by herbage gradually increases, whereas soil urea 15 N recovery decreased. The herbage urea 15 N recovery was not affected by the application of HQ and/or DCD during the first 14 d of regrowth. However, at the final regrowth (56 d), applica tion of HQ and/or DCD resulted in an increase in urea 15 N recovery in both the herbage and soil, with the strongest effect observed for HQ+DCD. The conversion of pig slurry urea derived N into soil NH 4 + and NO 3 fractions were reduced by the inhibitors, with a higher effect observed for HQ during the first 14 d. The conversion of pig slurryurea N into soil NH 4 + and NO 3 fractions was enhanced especially in the presence of DCD during the latter regrowth period. Higher retention of soil inorganic N derived from pig slurryurea at the final regrowth (56 d) in the HQ and/or DCD plots was in line with the enhanced herbage N recovery as well as the reduced N losses. The application of HQ and/or DCD re sulted in the efficient reduction of NH 3 , N 2 O emission, and NO 3 leaching. Application of HQ or DCD alone also signif icantly reduced N losses. Therefore, it can be concluded that HQ and DCD efficiently improve the N use efficiency of pig slurryurea, contributing a positive role in reducing N losses to the environment.

IMPLICATIONS
Management strategies of animal manure are necessary to improve nitrogen use efficiency while minimizing N losses to environmental pollution. The application of urease inhibitor (hydroquinone) and/or nitrification inhibitor (dicyandi amide) may enhance the nitrogen use efficiency of pig slurry by delaying the hydrolysis of urea and nitrification, thereby alleviating the nitrogen losses to nitrate leaching, ammonia, and nitrous oxide emission. Appropriate utilization of urease and nitrification inhibitors for pig slurry application to the grassland would be an efficient way to improve the nitrogen use efficiency, leading to a significant reduction of nitrate leaching and hazardous gases emission to the atmosphere.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.