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onment 125 (2008) 148–158

Agriculture, Ecosystems and Envir

A novel concept to reduce nitrogen losses from grazed pastures by

administering soil nitrogen process inhibitors to ruminant animals:

A study with sheep

S.F. Ledgard a,*, J.C. Menneer a, M.M. Dexter a, M.J. Kear a,S. Lindsey a, J.S. Peters b, D. Pacheco b

a AgResearch Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealandb AgResearch Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand

Received 1 March 2007; received in revised form 12 December 2007; accepted 13 December 2007

Available online 19 February 2008

Abstract

A cross-over sheep study examined the effects of infusion of nitrogen (N) process inhibitors into the gastrointestinal tract, on excretion in

urine and subsequent N transformations in soil. The aim of the study was to test a novel approach to reduce N losses from urine-N which is

recognised as the main source of N loss from grazed pastures. The study consisted of two experiments; in the first, the nitrification inhibitors

dicyandiamide (DCD) or 4-methylpyrazole (4MP) were continuously infused into the abomasum, and in the second, DCD was continuously

infused into the rumen. Administration of DCD or 4MP to the abomasum resulted in voided urine with a slowed rate of nitrification when

added to soil compared to that for urine from untreated sheep. DCD analyses revealed that over 90% of abomasum-infused DCD was excreted

in urine (99% after day 1) and markedly inhibited nitrification of urine-N in soil (>90% over 70 days; P < 0.001). Recovery of rumen-infused

DCD in urine was lower at 86%, but nitrification of urine-N in soil was still markedly inhibited (>90% over 69 days; P < 0.001). Less than 2%

of DCD was recovered in faeces after abomasum or rumen infusion. This study highlights the potential for using direct administration of N

process inhibitors to grazing animals to reduce environmental N emissions from urine patches in pasture systems.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Dicyandiamide; Nitrification; Nitrogen; Sheep; Urine

1. Introduction

Nitrogen (N) losses from agro-ecosystems are a major

contributor to global N emissions into the environment and

pastoral agriculture represents a significant component of

this (e.g. Galloway, 1998; Davidson and Mosier, 2004).

Intensively grazed pastures lose between 30 and

200 kg N ha�1 year�1 through nitrate leaching (e.g. Schole-

field et al., 1993; Simon et al., 1997; Ledgard, 2001).

Additionally, gaseous emissions of nitrous oxide (N2O) and

ammonia from grassland can be large and impact on the

environment (Jarvis et al., 1995). A large number of research

studies have identified animal urine, excreted onto pastoral

* Corresponding author. Tel.: +64 7 838 5133; fax: +64 7 838 5155.

E-mail address: stewart.ledgard@agresearch.co.nz (S.F. Ledgard).

0167-8809/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.agee.2007.12.006

soils at rates equivalent to c. 500–1000 kg N ha�1 (Haynes

and Williams, 1993), as the main source of these N

emissions (e.g. Ryden et al., 1984; Ledgard et al., 1999;

Davidson and Mosier, 2004).

Mitigation of N losses from grazed pastures must account

for urine as the dominant source of loss. Practices to reduce

loss of urinary N have targeted reduction of the amount of N

excreted in urine, such as through diet manipulation to avoid

protein levels in excess of animal requirements (e.g. Van

Vuuren et al., 1993), or avoidance of excreta deposition on

land by feeding animals on areas where excreta is collected

and applied evenly to land in spring/summer (e.g. de Klein

et al., 2000). An alternative option is the use of urease and/or

nitrification inhibitors to reduce N emissions from excreted

N. The urease inhibitor N-(n-butyl) thiophosphoric triamide

(NBPT) has been shown to be effective in reducing ammonia

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158 149

losses from urea in fertiliser or animal urine (e.g. Watson,

2000). Similarly, there are various products, including

dicyandiamide (DCD) and pyrazole compounds such as 4-

methylpyrazole (4MP), which are effective in inhibiting

nitrification in soil (McCarty and Bremner, 1989).

Akai et al. (2001) found that addition of the nitrification

inhibitor DCD to cow urine before application to pasture

reduced N2O emissions and increased uptake of urinary-N

by grass. This occurs by delaying the enzymatic conversion

of ammonium in soil to nitrite and nitrate, thereby retaining

N in the plant-available ammonium form which undergoes

little leaching and is not denitrified (Amberger, 1989). Di

and Cameron (2002) also showed in a lysimeter study that

DCD applied by spraying onto cow urine patches reduced

N2O emissions and nitrate leaching. While this represents a

potentially useful approach to reduce N emissions from

animal urine and increase N use efficiency in pasture

systems, a practical method is required that incorporates N

process inhibitors directly into the urine and thus targets

individual urine patches. Ideally such a concept would allow

inhibitors to be present in all urinations that occur during the

main period associated with N losses (e.g. late-autumn and

winter for nitrate leaching).

A novel approach to achieve this would be to supplement

animals with the inhibitor(s) during the main N loss period in

such a way that the inhibitor(s) would be excreted by the

animals, principally in the urine (Patent number NZPA

528648). However, a critical issue with this approach that

requires precursory investigation is the possible metabolic

degradation of the inhibitors within the animal rendering

them ineffective when excreted. In particular, the greatest

potential for degradation of the inhibitors is probably in the

rumen, given its biotransformation capacity resulting from

microbial enzymatic activity (e.g. Nebbia, 2001).

The objective of this study was to examine the

administration of inhibitors to sheep (as a test ruminant

animal), the excretion in urine and faeces of the inhibitors,

and their effectiveness in inhibition of urea hydrolysis and

nitrification in soil. The study consisted of two experiments.

The first experiment examined the potential for this

approach by infusion of inhibitors to the sheep abomasum,

thereby bypassing the rumen. The second experiment,

examined the fate and effectiveness of the nitrification

inhibitor DCD infused into the sheep rumen. By comparing

the excretion of DCD using post-ruminal and intra-ruminal

infusions we expected to identify if degradation occurred in

the rumen.

2. Methods

2.1. Experiment 1

2.1.1. Experimental design

A modified cross-over design was used with 4 (or 3)

treatments and 3 time periods. During the first period, the

following treatments, with 3 replicate sheep, were infused

via the abomasum continuously over 5 days:

(1) C

ontrol, 800 ml distilled water daily.

(2) D

CD solution with 15 g in 800 ml water daily.

(3) 4

MP solution with 0.8 g in 800 ml water daily.

(4) A

grotain (which contains the urease inhibitor NBPT)

solution with 80 g in 800 ml water daily.

The rates were calculated to achieve roughly 10 times the

per-hectare rates typically used in the field based on average

sheep excretion data (e.g. Haynes and Williams, 1993) and

no metabolism of compounds.

In the Agrotain treatment, infusion of Agrotain ceased

after day 3 due to reduced feed intake by animals and was

replaced with distilled water for the remaining 2 days. All

animals then had a 10-day non-treatment clearance/rest

period, before being allocated to one of three treatments (4

sheep per treatment): water infusion (Control), 15 g day�1

of DCD and 0.8 g day�1 of 4MP for periods 2 and 3.

Animals that received Agrotain in period 1 were re-allocated

to obtain a balanced design sequence of treatments over the 3

periods. In each period, infusion of treatments continued for

5 days, after which there was a 9-day non-treatment

clearance/rest period. The latter phase and treatments were

repeated for the third period. Thus, there were three infusion

and three rest periods. The cross-over design meant that 9

sheep received all treatments, thereby enabling within-sheep

comparisons.

2.1.2. Animal management and sampling

All manipulations described herein were approved by the

Palmerston North Crown Research Institutes Animal Ethics

Committee. Surgeries and experiments took place at

AgResearch Grasslands Research Centre in Palmerston

North, New Zealand. Before surgeries, sheep were given

preventive treatment against internal parasites (Leviben,

Novartis, New Zealand). Twelve Romney cryptorchid young

sheep (ca. 5-month-old; 38.7 (S.E., 1.27) kg live-weight at

the beginning of the experiment) were implanted with

custom-made Teflon cannulae in the abomasum and rumen

(for Experiment 2) under general anaesthesia as described

by Bermingham (2004). After a recovery period of 2 weeks,

they were placed in individual metabolism crates for three

experimental periods. Each experimental period comprised

3 days for acclimatisation to the metabolism crate and

collection of urine samples for estimation of basal status; 5

days for infusion of the treatments; and 6 days for collection

of ‘wash-out’ samples after infusion. Diets consisted of a

70:30 (as fed basis) mix of commercial sheep rearing pellets

(Denver Feeds, Palmerston North, New Zealand) and

molassed alfalfa hay (ChaffHageTM, The Great Hage

Company, Reporoa, New Zealand). The pellets contained

54% lucerne meal, 38.5% maize grain, 4% molasses and

3.5% of a mineral and vitamin premix. Diets were

formulated to contain, in dry basis, 15% crude protein,

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158150

26% neutral detergent fibre, 48% non-fibre carbohydrates

and 10.2 MJ metabolisable energy. Actual crude protein of

the diet was 15.5% of the dry matter. Diets were offered as

two equal meals at 0930 and 1630 h at a rate of 0.95 kg per

sheep per day. Any feed refusals were recorded and collected

for dry matter and nitrogen analyses. Animals had free

access to water at all times during the course of the

experiment.

All treatment solutions were continuously infused over

the treatment period using a peristaltic pump (Watson

Marlow; Watson Victor Ltd., Auckland, New Zealand).

Infusion lines were washed with warm de-ionised water

between each infusion period.

A custom-made urine collection device (200 ml capacity)

was tied around the middle of the sheep at the start of each

day. When urination had occurred, samples were placed in

sterile sample jars, frozen at �20 8C and retained for N

process measurements and analysis of urea and DCD. After

collection, the device was removed and subsequent urination

was funnelled from the metabolism crate into a container

with sufficient HCl (17%, w/v) to maintain the pH of the

urine below 2 to avoid ammonia volatilisation. At the end of

each 24 h period, urine volumes were recorded and samples

were frozen and retained for analysis. Total faecal output

over 24 h was collected into faecal collection bags harnessed

to the animals. Faecal samples were placed in sterile sample

jars, frozen at �20 8C and retained for DCD analysis. Urine

and faeces over the 5 days collection period were separately

pooled according to weight for each animal and analysed for

measurements of N balance according to the methods

described by Pinares-Patino et al. (2003). The N concentra-

tion of the faeces and urine samples was analysed in a

Nitrogen Analyser 1500 (Carlo Erba Instruments, Milan,

Italy) using an instrumental combustion method.

Blood samples were collected into evacuated tubes

(Vacutainer Serum Tubes, BD, Franklin Lakes, NJ, USA)

by jugular puncture at the beginning of the trial and at the end

of each infusion period. Serum samples were analysed to

ensure that treated animals maintained blood parameters

compatible with normal hepatic and renal function (phos-

phate, calcium, potassium, sodium, urea, creatinine, albumin,

globulin, total protein, bilirrubin, glutamate dehydrogenase,

gamma-glutamyl transferase; New Zealand Veterinary

Pathology Ltd., Palmerston North, New Zealand).

2.1.3. Nitrogen process measurements

Urea hydrolysis (i.e. transformation of urea to ammonium)

was determined in urine samples from the control and

Agrotain treatment sheep collected during the first 3 days in

period 1. Urine samples were first thawed to room

temperature, diluted 200 times and analysed for urea-N

concentration using the diacetyl monoxime method of

Wybenga et al. (1971) on a Jenway 6100 spectrophotometer

at 535 nm. Sub-samples of urine were then measured out and

made up to volume with distilled water in order to achieve the

same urea-N concentration in each sample, and therefore

compare relativity between treatments. In some samples with

very low urea-N concentrations, the required urea-N

concentration was achieved by adding urea solution. Urease

solution was then added and samples were incubated at 20 8Cfor 165 min (standardised to fit with laboratory function and

avoid complete hydrolysis) before analysis for urea-N

concentration. Four replicate standard samples with urine

from the control sheep, urease and different levels of Agrotain

(25% NBPT) addition were analysed for comparison. The rate

of urea hydrolysis was calculated from the urea concentration

after 165 min relative to the initial level. Preliminary studies

showed no effect of freezing/thawing urine samples on the

rate of urea hydrolysis (data not presented).

Nitrification rate in urine-amended soil was determined

using a soil incubation method. Urine samples were thawed

to room temperature, analysed for urea-N concentration and

sub-samples were applied to soil in plastic bags so that all

urine samples were applied at the same rate of urea-N

addition (40 mg per 100 g soil oven-dry weight equivalent).

In a few urine samples with low urea-N concentrations, urea

solution was added to the urine to achieve a constant

application rate. Additional distilled water was added so that

all samples received the same total volume of solution to

achieve soil moisture levels 70% of field capacity. The soil

was a Horotiu silt loam (Typic vitrandept, Singleton, 1991)

derived from volcanic ash, which had been taken from a

pastoral site (0–10 cm depth; sieved to remove pasture

residues and roots) that had been ungrazed for over 6 months

so that no residual animal urine was present. The urine-

treated soil samples (one sample for each sheep and each

daily urine collection for each period, i.e. 44 per treatment)

were incubated (aerobically) at 20 8C. Single sub-samples of

soil were then removed after 5, 8, 12, 15, 32 and 70 days for

extraction in 2 M KCl (soil:solution of 1:10) and analysis of

ammonium and nitrate using flow injection analysis, and for

analysis of DCD concentration.

2.1.4. DCD in urine, faeces and soil extracts

The DCD concentration in urine, faeces and soil KCl-

extracts was determined by High Performance Liquid

Chromatography (HPLC, Shimadzu HIC-6A) using a method

based on that of Schwarzer and Haselwandter (1996) but with

differences as follows: for the urine samples, solid phase

extraction was carried out before injection into the HPLC

which was equipped with a Rezex Organic Acid guard column

(50 mm � 7.8 mm I.D.) and a Aminex organic acid column

HPX-87H (300 mm � 7.80 mm I.D.) to resolve the DCD

peak from other interferences (e.g. urea) at a detection

wavelength of 220 nm. The same method was used for faeces

samples, except that they were first extracted with 2 M KCl,

centrifuged and filtered. DCD in soil KCl-extracts was also

determined but by direct injection of the sample into the

HPLC equipped with a guard column only. Initial studies

showed that there was no effect of freezing/thawing urine (or

other extracts) on their DCD concentration or on its

effectiveness in inhibiting nitrification (data not presented).

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158 151

2.2. Experiment 2

Methodologies were the same as those in Experiment 1

except for some minor differences described in this section.

Animal management, feeding and infusion apparatus and

methods were the same as for the abomasum experiment.

The same sampling and analysis methods for sheep urine,

faeces, blood and soil incubations were also used.

As in Experiment 1, a modified cross-over design was

used involving the same 12 sheep, but with only 2 treatments

and 2 time periods. The sheep had a 2-week non-treatment

clearance/rest period after the abomasum infusion experi-

ment, and before commencing this rumen infusion experi-

ment. The sheep were then placed in individual metabolism

crates for two experimental periods. During each period, the

following treatments, with 6 replicate sheep, were infused

via the rumen continuously over 5 days:

(1) C

ontrol, 500 ml distilled water daily.

(2) D

CD solution with 15 g in 500 ml water daily.

Fig. 1. Percentage of urea-N remaining in urine after addition of urease and

incubation for 165 min at 20 8C (Experiment 1). Data refer to urine

collected from sheep infused with water (Control) or Agrotain solution

into the abomasum during the first 3 days of infusion, and are compared with

that from Control urine which received different rates of Agrotain solution

at the start of the incubation period.

A lower daily solution volume was used than in

Experiment 1, based on sufficiency to fully dissolve the

DCD. DCD was selected as the test inhibitor for this

experiment because of its wider commercial availability and

the methodology developed to measure it directly.

After the first period, all animals had a 9-day non-

treatment clearance/rest period, before treatments were

reversed. A second 5-day infusion period then commenced,

followed by a 9-day non-treatment clearance/rest period.

The cross-over design meant that the 12 sheep received both

treatments, thereby enabling within-sheep comparisons.

Seventeen days after the final infusion period, the

sheep were slaughtered in a local abattoir and various

animal tissue samples were collected from each sheep,

including longissimus dorsi muscle, mesenteric and peri-

renal fat, liver, kidney and wool. Tissue samples were

immediately frozen after collection and stored at �20 8Cuntil analysed.

Animal tissue samples were first homogenised in

distilled water and centrifuged. The supernatant was then

mixed in 20% trichloroacetic acid, before further centri-

fugation, solid phase extraction of the resulting supernatent

and analysis for DCD by HPLC using the technique

described in Experiment 1.

2.3. Statistical analysis

Data for the sheep nitrogen balance variables was

analysed as a mixed model, with treatment being the fixed

effect, with period, sheep and their interaction being the

random effects. Estimates were calculated using the

restricted maximum likelihood (REML) directive in Genstat

(8th Edition, version 8.1.0.156). Significance of fixed effects

was assessed by means of Wald test statistics compared to a

chi-square distribution.

Data from the soil incubation studies were also analysed

using the REML procedure of Genstat for each incubation

time, using the same approach as for the nitrogen balance

data. The nitrate data was log transformed before analysis.

The means shown are those back-transformed from the log

scale, and then re-scaled so their mean is the same as the

mean of the treatments on the untransformed scale. The

ammonium data was not log transformed.

Data from the DCD analyses was analysed using the

Bayesian smoothing program Flexi (Upsdell, 1994). Only

the DCD treatment data was analysed as the control

treatment contained no DCD. The spline and its 95%

confidence interval are shown.

3. Results

3.1. Experiment 1

3.1.1. Urea hydrolysis

More (P < 0.01) urea remained after 165 min in the

incubated urine samples from Agrotain-treated sheep than

from control sheep (Fig. 1). There was no significant effect

of day of infusion on urea concentrations in control or

Agrotain treatments and therefore data in Fig. 1 are the

means for the infusion period. Associated urine samples

spiked with increasing rates of Agrotain showed increased

concentrations of urea, and implies increased inhibition of

urea hydrolysis. The proportion of initial urea present after

165 min of incubation in urine from the Agrotain-treated

sheep was above that measured for the highest rate of

Agrotain in the standardised urine samples. This indicates

that the concentration of the inhibitor NBPT in urine from

the Agrotain-treated sheep was above our Agrotain–urine

standard of 500 mg l�1.

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158152

Fig. 2. Temporal changes in DCD concentration in urine (mg l�1) collected

from DCD-treated and Control (untreated) sheep covering 1-day pre-treat-

ment (day 0), a 5-day abomasum infusion period, and 6 days after ceasing

infusion (Experiment 1). Bars are standard errors for the +DCD treatment.

Fig. 4. Variation over the abomasum infusion period in (A) sample DCD

concentration in urine, and (B) daily urination volume for different indi-

vidual sheep (Experiment 1).

3.1.2. DCD in urine and faeces

In all sheep treated with DCD, the concentration of DCD

in urine increased markedly within the 1st day of infusion

and remained high throughout the infusion period (samples

were not collected at the end of day 5), after which it

declined to near basal concentrations (Fig. 2). Concentra-

tions of DCD in urine from all DCD-treated sheep were

significantly higher (P < 0.001) than in the control sheep.

There was no significant difference in DCD concentration

with period of infusion.

There was a positive correlation (P < 0.01) between the

DCD and corresponding urea-N concentrations in urine

within animal (Fig. 3). Individual sheep generally showed

similar DCD concentrations in urine over the period of

infusion (Fig. 4A), but there was a 12-fold difference

between sheep in average DCD concentration for the

infusion period. This was inversely related (R2 = 0.68;

Fig. 5) to the average daily volume of urination, which also

showed little variation over time (Fig. 4B). Urine volume

associated with the spot-sample collection used for DCD

analysis at the start of each day was not recorded and

Fig. 3. Relationship between the DCD and urea-N concentrations in the

urine excreted by sheep after infusion of DCD into the abomasum (Experi-

ment 1).

therefore it is not possible to directly relate individual

urination volume and DCD concentration to calculate the

amount of DCD excreted. However, the apparent daily

amount of DCD excreted by sheep (estimated using sample

DCD concentration and daily volume) showed much less

variation between sheep (a threefold range) than either the

DCD concentration or urination volume.

Unfortunately, total DCD recovery in urine could not be

accurately estimated using the bulked urine samples due to a

measured degradation of DCD in these samples caused by

the addition of acid to reduce ammonia volatilisation after

collection. Thus, an estimate of apparent overall recovery of

DCD in urine was obtained by using data for the individual

urine spot-sample (not acidified) analyses, in combination

with the average daily urination volumes (overall daily

mean = 1650 ml). This calculation resulted in an average

apparent recovery of 91% (S.E. = 8) across all urine samples

collected over the infusion period. The corresponding value

for samples excluding the first collection day was 99%

(S.E. = 10) and may better reflect the average since the first

sampling may have represented insufficient time for

complete assimilation through to urine from the 1st day

of infusion.

The average weight of faeces excreted over a 5-day

period after commencing infusion was 1860 g wet-weight.

Faeces samples were analysed for DCD and associated

measurements in control-faeces spiked with DCD indicated

that our method recovered 78% of the added DCD. The net

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158 153

Fig. 5. Correlation between the DCD concentration in urine (from a single

sampling) and the daily urination volume (Experiment 1). Data is the

average for individual sheep for the abomasum infusion period.

result of these analyses, corrected for incomplete DCD

recovery, was an apparent average recovery of DCD in

faeces over the 5-day period of 1.35% (S.E. = 0.24).

3.1.3. DCD in soil KCl-extracts

Concentrations of DCD measured in the KCl-extracts of

the incubated soil were high during the first two samplings at

over 90% of that added (Fig. 6). Levels declined slowly in

subsequent samplings to approximately one-third of that

added in the urine at day 70.

3.1.4. Nitrification of urine

There was no significant effect of time period or day of

infusion on ammonium-N or nitrate-N concentration in soil.

However, there was a highly significant (P < 0.001) effect of

treatment on ammonium-N and nitrate-N concentrations at

all soil incubation dates (Fig. 7).

In the control treatment, ammonium-N concentrations in

the incubated soil declined rapidly over time to negligible

Fig. 6. Temporal decrease in DCD concentration in soil KCl-extracts after

incubation (20 8C) of soil which received urine from sheep infused with

DCD into the abomasum (Experiment 1). Confidence Interval (95%) for

data was based on use of FLEXI.

Fig. 7. Temporal changes in (A) ammonium and (B) nitrate concentrations

in soil KCl-extracts and C) inhibition of nitrification after incubation

(20 8C) of soil with urine from sheep infused with water (Nil), DCD or

4-methylpyrazole (4MP) into the abomasum (Experiment 1). Data are

means for urine samples from days 1 to 4 of infusions. Error bars for

(A) represent LSD (P < 0.05) values. Nitrate data are geometric means and

the average Least Significant Ratio (P < 0.05) for treatment comparisons is

1.5 for each incubation time.

levels at days 32 and 70 (Fig. 7A). There was a

corresponding increase in nitrate-N concentrations over

time due to nitrification. Nitrate-N concentrations in soil not

amended with urine were <20 mg kg�1 during the first 15

days and increased to approximately 50 and 100 mg kg�1 at

days 32 and 70, respectively (units all in oven-dry equivalent

basis). Thus, the apparent increase in nitrate-N concentra-

tion in the control soil between days 32 and 70 was probably

largely due to nitrification of indigenous soil N.

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158154

Fig. 8. Temporal changes in DCD concentration in urine voided from DCD-

treated and Control (untreated) sheep covering 1-day pre-treatment (day 0),

a 5-day rumen infusion period, and 6 days after ceasing infusion (Experi-

ment 2). Each data point represents the mean of 6 sheep. Bars are standard

errors for +DCD.

Table 1

Concentrations and apparent recovery of DCD in urine, faeces and various

tissues of sheep (Experiment 2)

Wet-weight (g) DCD concentration Recovery (%)

mg kg�1 S.E.

Urinea 4500 8580 750 86

Faecesa 1840 248 35 1.3

Kidneyb 80 0.7 0.13 0.0001

Liverb 620 0.4 0.07 0.0002

Muscleb 9900 0 0

Fatb 6600 0.2 0.16 0.002

Wool 1500 55 20 0.007

a Weights are for a 3- or 5-day period for urine and dung, respectively.b Weights based on assumption of 0.2, 1.6, 24 and 16% of live-weight for

kidneys, liver, muscle and fat, respectively (Pittroff et al., 2006: control

group).

There was a highly significant effect (P < 0.001) of

treatment on ammonium-N and nitrate-N concentrations in

soil at all incubation sampling times (Fig. 7). In the DCD

treatment, ammonium-N concentrations in soil remained

constant over time, except for a small increase at day 70,

presumably due to mineralisation of indigenous soil N.

Conversely, nitrate-N concentrations were minimal through-

out the incubation period, indicating almost complete

inhibition of nitrification.

The 4MP treatment also showed significant (P < 0.01)

inhibition of nitrate production during the first 15 days. This

equated to 40–51% inhibition in nitrification during days 5–

15, reducing to 10% at day 32 and nil at day 70 when nitrate-

N concentrations were the same for the 4MP and control

treatments.

3.1.5. Sheep N balance and health

Mean dry matter intake were not significantly different

(P > 0.10) between treatments (0.91, 0.92 and 0.83 kg per

sheep per day for Control, 4MP and DCD, respectively: SED

0.053). There was no treatment effect on the partitioning of

excreta nitrogen between faeces and urine (30.0, 31.0 and

32.2 g faecal N per 100 g excreta N for Control, 4MP and

DCD, respectively: SED 3.78), after correction to account

for the treatment contribution to the amounts of N measured

during the balance period.

Over the 3 periods, sheep live-weight, and N balance

(difference of N input and N excreta) were unaffected by the

treatments (data not presented) and tests for hepatic and

renal function showed no evidence of deleterious effects

caused by the treatments (results not shown).

3.2. Experiment 2

3.2.1. DCD in urine and faeces

In all sheep treated with DCD, the concentration of DCD

in urine showed a similar temporal pattern to Experiment 1

except that the concentration at day 1 was only half that for

subsequent days of infusion (Fig. 8). Concentrations of DCD

in urine from all DCD-treated sheep were significantly

higher (P < 0.001) than in the control sheep during the

infusion period and than declined rapidly to background

concentration.

As in Experiment 1, an estimate of apparent overall

recovery of DCD in urine was obtained by using data for the

individual urine spot-sample analyses, in combination with

the average daily urination volumes. This calculation

resulted in an average apparent recovery of 86%

(S.E. = 7; Table 1) across all urine samples from days 2

to 4 of the infusion period.

The corresponding estimate of DCD recovery in faeces

excreted over a 5-day period after commencing infusion was

1.33% (S.E. = 0.18). This estimate includes a correction for

incomplete recovery based on associated measurements in

control-faeces spiked with DCD which indicated that our

method recovered only 78% of the added DCD.

3.2.2. DCD in animal tissues

Concentrations of DCD in tissues of sheep slaughtered at

the end of the experimental period were very low relative to

those in urine and dung (Table 1). All sheep had detectable

levels of DCD in kidney, liver and wool, but only 3 sheep had

detectable levels in fat and none had detectable levels in

muscle tissue.

3.2.3. DCD in soil KCl-extracts

Concentrations of DCD measured in the KCl-extracts of

the incubated soil were high at the first sampling at about

85% of that added (Fig. 9). Levels declined slowly in

subsequent samplings to approximately one-third of that

added in the urine at day 69.

3.2.4. Nitrification of urine

As in Experiment 1, there was no significant effect of

time period or day of infusion on ammonium-N or nitrate-N

concentration in soil. The excreted DCD-urine had a highly

significant (P < 0.001) effect on ammonium-N and nitrate-

N concentrations at all soil incubation dates (Fig. 10).

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158 155

Fig. 9. Temporal decrease in DCD concentration in soil KCl-extracts after

incubation (20 8C) of soil which received urine from sheep infused with

DCD into the rumen (Experiment 2). Confidence Interval (95%) for data

was based on use of FLEXI.

Fig. 10. Changes in (A) ammonium and (B) nitrate concentrations and (C)

In the control treatment, ammonium-N concentrations in

the incubated soil declined rapidly over time to minimal

levels at days 32 and 69 (Fig. 10A) and there was a

corresponding increase in nitrate-N concentrations over time

(Fig. 10B) due to nitrification.

In the DCD treatment, ammonium-N concentrations in

soil remained constantly high over time. Conversely, nitrate-

N concentrations were low throughout the incubation period

and showed a small increase at days 32 and 69 only. This

indicates almost complete inhibition of nitrification in soils

receiving DCD-treated urine (over 90% inhibition for days

8–69; Fig. 10C), which is similar to that achieved by

infusing DCD into the abomasum (Experiment 1).

3.2.5. Sheep health

Mean dry matter intake were not significantly different

(P > 0.10) between treatments (0.86 kg vs. 0.88 kg per

sheep per day for Control and DCD, respectively: SED

0.067). There was no significant deleterious effect of DCD

treatment on general sheep health, live-weight, N balance, or

hepatic and renal function (results not shown).

inhibition of nitrification after incubation of soil treated with urine from

sheep that had infusion of water (control) or DCD into the rumen (Experi-

ment 2). Data are means for urine samples from days 2 to 4 of infusions.

Error bars for (A) represent LSD (P < 0.05) values. Nitrate data are

geometric means and the average Least Significant Ratio (P < 0.05) for

treatment comparisons is 1.5 for each incubation time. Treatment compar-

isons for (B) and (C) showed highly significant differences (P < 0.001) at

all times.

4. Discussion

4.1. General implications

This study has demonstrated a novel concept involving

animal supplementation of soil N process inhibitors, with

minimal metabolic degradation and excretion predomi-

nantly in urine. This represents a potential novel mitigation

option for reducing N losses by leaching and N2O emission

from intensively grazed pastures since urine excreted by

grazing animals is the main source of the N losses (e.g.

Ryden et al., 1984).

Rapid throughput of the inhibitors occurred in both

experiments, as evidenced by the sharp decline in DCD

concentration in urine to background levels within a few

days of ceasing administration. This rapid throughput

indicates that regular administration (e.g. in feed supple-

ments or daily oral drenching) would be required for

sustained delivery over the main period of N loss (e.g. winter

drainage for nitrate leaching; Jarvis et al., 1995). Alter-

natively, this could be achieved using controlled-release

delivery systems in the rumen (e.g. Grace et al., 1997; Wu

and Papas, 1997) and this is the main focus of our current

research.

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158156

The apparent recovery in urine of 86% of the DCD

infused into the rumen (Experiment 2) was less than

the corresponding value of 99% from DCD infused into the

abomasum (Experiment 1), but still represented the

majority of that administered. This suggested that some

(c, 13%) degradation of DCD had occurred in the rumen.

Similarly, Peter et al. (1986) measured reduced selenium

availability from selenite and selenomethionine infused

into the rumen of sheep and attributed it to rumen

metabolism.

It is not possible to determine how much of the DCD

infused in the rumen was absorbed across the rumen wall

compared to being absorbed in other parts of the digestive

system. Other research has shown that the sites of

absorption of compounds vary. For example, Field and

Munro (1977) found that the main site of absorption of

magnesium was in the rumen of sheep and that there was no

absorption when infused into the abomasum. In contrast,

Swan et al. (1999) measured greater absorption by sheep of

the anthelmintic rafoxanide from the abomasum than from

the rumen. Detailed studies with isotopes (e.g. Chiu et al.,

1990; Afzal et al., 1994) are required to accurately define

the absorption, transformation, and excretion of N process

inhibitors.

Almost all of the DCD that could be recovered in this

study was in the urine, with less than 2% in the faeces. Van

Montfoort et al. (2003) noted that water-soluble compounds

with relatively low molecular weights are predominantly

excreted in urine. Very low concentrations of DCD were

measured in the various animal tissues, with none in the

muscle tissue and only 3 sheep with DCD concentrations in

fat which were just above the minimum detectable level.

Additionally, the sheep in Experiment 2 were slaughtered at

17 days after ceasing DCD infusion and it is possible that a

longer withholding period would have reduced DCD

concentrations in animal tissues. Conversely, increased

DCD retention in tissues as a result of prolonged

administration periods, as required for practical application,

cannot be ruled out. Further research in this area is planned

and one study is currently underway with dairy cows to

examine the potential for DCD to enter milk when it is

administered to lactating cows.

There were no adverse effects of DCD infusion on sheep

health or on kidney and liver functioning. Clearly, the use of

animal supplementation of N process inhibitors for

mitigation of environmental impacts requires compounds

which are non-toxic to animals. Studies outlined in the

patent files and publications for DCD (e.g. Amberger, 1989)

revealed very low ecotoxicity, e.g. oral LD50 values of

10 g kg�1. This indicates that strategic supplementation

could be used to achieve environmental benefits while

remaining well below this value. In the present study,

0.4 g DCD kg�1 live-weight day�1 was used but calcula-

tions on the amounts needed to achieve the rate of

DCD commonly applied in the field indicate that

<0.1 g DCD kg�1 live-weight day�1 would be effective.

4.2. Variation between individual sheep

This study showed high levels of inhibition of nitrifica-

tion of urine-N produced by all sheep and was supported by

high DCD concentrations in urine from all DCD-treated

sheep. A cross-over design was used for the study to increase

experimental precision and enable examination of the

within-animal variability, such that each sheep was subject

to the main treatments (control, +4MP and/or +DCD).

Analysis of Experiment 1 data for individual sheep

showed large differences in the volume of urine excreted and

in the associated urea-N and DCD concentrations. However,

the urea-N and DCD concentrations were negatively

correlated with urine volumes and therefore the amounts

of urea-N and DCD in urinations showed much less

variability than that of the volumes or concentrations. The

high positive correlation between the urea-N and DCD

concentrations means that urine patches with high rates of N

have correspondingly higher amounts of DCD for inhibition

of nitrification.

4.3. Specific N process inhibitors

Infusion of Agrotain (active ingredient NBPT) into the

abomasum resulted in the excreted urine having a

significantly lower rate of urea hydrolysis in incubated

samples than in urine from untreated sheep. Direct addition

of Agrotain to urine from the untreated sheep also displayed

inhibition of urea hydrolysis. This indicates that NBPT in the

Agrotain was absorbed and excreted in the urine in an

unaltered form (or an associated metabolite of NBPT)

thereby resulting in inhibition of urea hydrolysis. However,

by 3 days after infusion the sheep were showing signs of

gastrointestinal irritation and the infusion with Agrotain was

ceased. Ludden et al. (2000) administered the specific

compound NBPT to sheep at rates of 20% or less than in the

current study to improve ruminal uptake of consumed urea

and measured no clear deleterious effects on sheep health,

although some post-absorptive effects on N metabolism

were observed. This suggests that the high rate of NBPT or

other ingredients in Agrotain may have affected the sheep in

our study. Thus, further research is required specifically with

NBPT to determine the feasibility of administration to

animals for inhibition of urease activity and reduced

ammonia losses from urine on deposition to soil.

Urine from sheep infused with 4MP showed significantly

lower rates of nitrification in soil, indicating that it, or an

associated metabolite, was absorbed in the abomasum,

passed through to the bladder and excreted in urine. McCarty

and Bremner (1989) examined a wide range of compounds

for nitrification in soils and showed that 4MP was an

effective inhibitor. In the present study, the magnitude and

longevity of inhibition of nitrification was less than that for

DCD. However, the level and duration of inhibition is likely

to be rate-dependent and the amount of 4MP infused in our

experiment was low at only 7% of the DCD dosage.

S.F. Ledgard et al. / Agriculture, Ecosystems and Environment 125 (2008) 148–158 157

The high recovery of DCD in urine (>85%) in both

experiments resulted in almost complete inhibition of

nitrification of urine-N in soil throughout the 70-day

incubation period. The high degree of inhibition will have

been due to the high rate of DCD excreted in urine. A relatively

high rate of DCD was used for infusion because of the

uncertainty of its fate. Based on these results a much lower rate

could have been used to achieve effective inhibition of

nitrification. For example, based on a 10 cm soil depth, the

average rate of DCD application in the soil incubations would

equate to approximately 70 kg ha�1, whereas most field

research has used rates of 10–30 kg DCD ha�1 (e.g.

Amberger, 1989; Akai et al., 2001; Di and Cameron, 2002).

Broadcast application of DCD in suspension at 10 kg ha�1

onto urine patches reduced N2O emissions by over 60% on the

same soil type as used in the present study and in three other

New Zealand soils (Di et al., 2007), indicating potential

effectiveness across a range of soils. The novel approach of the

animal delivering N process inhibitors directly in urine means

that it is more intimately mixed with the urine and that a much

lesser amount is required compared to that for broadcast

applications (which also covers inter-urine areas).

5. Conclusions

The present study has shown that N process inhibitors

administered into the abomasum or rumen of sheep were

excreted in urine and retained their activity. DCD analyses

revealed that 86% or more of the infused DCD was absorbed

and passed through into urine where it was effective in

inhibiting the nitrification of urine-N. The administration of

nitrification inhibitors had no deleterious effects on animal

health or performance. Further research is required and is

ongoing to examine simple methods, such as slow-release

delivery systems, to achieve strategic sustained delivery and to

determine the longer-term implications of using this approach

in grazed pasture systems. Nevertheless, this study highlights

the potential for using direct administration of DCD to animals

to inhibit nitrification of N excreted in animal urine and reduce

environmental N emissions from grazed pastoral systems.

Acknowledgements

The research was supported by the New Zealand

Foundation for Research, Science and Technology. The

authors thank John Waller and Fred Potter for statistical

analyses; Matthew Deighton and Peter Schreurs for

assistance in running the Animal Research Unit.

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