The Effects of Dietary Crude Protein Level on Ammonia Emissions from Slurry from Lactating Holstein-Friesian Cows as Measured in Open-Circuit Respiration Chambers

Abstract

Simple Summary
Farmed livestock, particularly dairy cows, are the largest source of ammonia (NH3) emissions to the atmosphere in Europe and other parts of the developed world. Generally, more than 80% of the total agricultural NH3 emissions in Europe come from manure slurries (mixtures of urine and faeces) with hydrolysis of urea nitrogen (N) in urine, and ammonification of the organic N fraction in faeces as the two main sources of the NH3. It is also worth noting that the concentration of these two main sources of NH3 emissions from manure slurries (particularly urea N in urine) is positively associated with dietary protein content.

Abstract
The effect of dietary crude protein (CP) level on ammonia (NH3) emissions from slurry from lactating Holstein-Friesian cows was studied. Twenty-four-hour total collections of faeces and urine were made from 24 lactating Holstein-Friesian cows fed four total mixed rations containing 141, 151, 177, and 201 g CP/kg DM (6 cows/diet). The collected urine and faeces from each cow were combined to form 2 kg duplicate slurry samples (weight/weight; fresh basis) according to the proportions in which they were excreted. NH3 emissions from the slurry samples were measured, during 0–24 and 24–48 h intervals in six open-circuit respiration chambers maintained at two temperatures (8 or 18 °C). NH3 emissions for the 0–24 and 0–48 h intervals, as well as the average daily emissions, increased linearly with increasing dietary CP level. Increasing the temperature from 8 to 18 °C positively affected NH3 emissions, but only for the 0–24 h interval. In situations where direct measurements are impossible, NH3 emissions from slurry can be predicted accurately using equations based on dietary CP level supported by either urinary nitrogen, faeces nitrogen, or both. In summary, increasing dietary CP level linearly increased average daily NH3 emissions from slurry, with a 5.4 g increase for each 10 g increase in dietary CP.

Full text

Citation: Katongole, C.B.; Yan, T. The
Effects of Dietary Crude Protein
Level on Ammonia Emissions from
Slurry from Lactating
Holstein-Friesian Cows as Measured
in Open-Circuit Respiration
Chambers. Animals 2022,12, 1243.
https://doi.org/10.3390/
ani12101243
Academic Editor: Marian
Czauderna
Received: 27 March 2022
Accepted: 10 May 2022
Published: 12 May 2022
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4.0/).
animals
Article
The Effects of Dietary Crude Protein Level on Ammonia
Emissions from Slurry from Lactating Holstein-Friesian Cows
as Measured in Open-Circuit Respiration Chambers
Constantine Bakyusa Katongole and Tianhai Yan *
Sustainable Agri-Food Sciences Division, Agri-Food and Biosciences Institute, Large Park, Hillsborough,
Belfast BT26 6DR, UK; constantine.katongole@gmail.com
*Correspondence: tianhai.yan@afbini.gov.uk
Simple Summary:
Farmed livestock, particularly dairy cows, are the largest source of ammonia
(NH
3
) emissions to the atmosphere in Europe and other parts of the developed world. Generally,
more than 80% of the total agricultural NH
3
emissions in Europe come from manure slurries (mixtures
of urine and faeces) with hydrolysis of urea nitrogen (N) in urine, and ammonification of the organic
N fraction in faeces as the two main sources of the NH
3
. It is also worth noting that the concentration
of these two main sources of NH
3
emissions from manure slurries (particularly urea N in urine) is
positively associated with dietary protein content.
Abstract:
The effect of dietary crude protein (CP) level on ammonia (NH
3
) emissions from slurry from
lactating Holstein-Friesian cows was studied. Twenty-four-hour total collections of faeces and urine
were made from 24 lactating Holstein-Friesian cows fed four total mixed rations containing 141, 151,
177, and 201 g CP/kg DM (6 cows/diet). The collected urine and faeces from each cow were combined
to form 2 kg duplicate slurry samples (weight/weight; fresh basis) according to the proportions in
which they were excreted. NH
3
emissions from the slurry samples were measured, during 0–24 and
24–48 h intervals in six open-circuit respiration chambers maintained at two temperatures (8 or 18
◦
C).
NH
3
emissions for the 0–24 and 0–48 h intervals, as well as the average daily emissions, increased
linearly with increasing dietary CP level. Increasing the temperature from 8 to 18
◦
C positively
affected NH
3
emissions, but only for the 0–24 h interval. In situations where direct measurements are
impossible, NH
3
emissions from slurry can be predicted accurately using equations based on dietary
CP level supported by either urinary nitrogen, faeces nitrogen, or both. In summary, increasing
dietary CP level linearly increased average daily NH
3
emissions from slurry, with a 5.4 g increase for
each 10 g increase in dietary CP.
Keywords: ammonia emission; dietary crude protein; respiration chamber; slurry
1. Introduction
Manure slurries (mixtures of urine and faeces) are a significant source of anthropogenic
ammonia (NH
3
) emissions to the atmosphere in Europe and other parts of the developed
world. In the UK, for example, in 2018, an average of 86% of the total NH
3
emissions from
agriculture came from slurry-related emissions, with 78% for England, 85% for Scotland,
90% for Wales, and 92% for Northern Ireland [
1
]. The ruminant livestock sector, particularly
cattle farming, is the dominant source of NH
3
emissions from agriculture in Northern
Ireland and throughout the UK. The “Making Ammonia Visible Report 2017” produced by
the Expert Working Group on Sustainable Agricultural Land Management for Northern
Ireland showed that over 70% of NH
3
emissions in Northern Ireland emanate from cattle
production, with the pig and poultry sectors responsible for only 20% of agricultural NH
3
.
NH
3
is strongly implicated in the damage of ecosystems and biodiversity through its
deposition in terrestrial and aquatic environments [
2
,
3
], human health respiratory problems
Animals 2022,12, 1243. https://doi.org/10.3390/ani12101243 https://www.mdpi.com/journal/animals

Animals 2022,12, 1243 2 of 14
through formation of secondary products that contribute to fine particulate matter [
4
],
groundwater contamination with nitrate-N [
5
], and emissions of the greenhouse gas nitrous
oxide (N
2
O) through nitrification–denitrification processes [
6
,
7
]. Although NH
3
is not
considered a direct greenhouse gas (because of its short lifetime in the atmosphere), NH
3
deposition induces N
2
O (a potent greenhouse gas) formation, hence indirectly contributing
to the greenhouse effect [
7
]. Consequently, monitoring and reduction of NH
3
emissions
from livestock farming is a legal requirement in the UK and Europe (revised National
Emission Ceilings Directive or Directive 2016/2284/EU; UK National Emissions Ceilings
Regulations 2018). Furthermore, storage of manure slurry (a significant source of NH
3
emissions from livestock) is increasingly becoming necessary due to restrictions of its land
application period to only during crop germination and vegetative growth [8].
There are two main sources of NH
3
emissions from manure slurries: the hydrolysis
of urea nitrogen (N) in urine to NH
3
, and the NH
3
resulting from the ammonification
(mineralization) of the organic-N fraction in faeces [
9
]. Under favourable conditions of
warm temperatures, large emission surface areas, and high pH and air velocity, the urinary
urea-N is relatively quickly hydrolysed by urease enzyme (which is readily present in
faeces [
10
]) to ammonium-N, resulting in NH
3
volatilization to the atmosphere (leading to
environmental pollution). The hydrolysis starts as soon as urine comes into contact with
faeces, and it is completed within 1–2 days [11,12].
Most efforts to reduce release of NH
3
from manure slurries have focused on improved
methods for their storage, treatment, and processing options, as well as their application
under field conditions. Reducing the excretion of N in manure through manipulation
of animal diets represents another opportunity where effective and economic reductions
in subsequent manure NH
3
emissions could be made [
13
]. Substantial evidence in the
literature supports higher NH
3
emissions from cattle manure slurries with increasing
dietary crude protein (CP) level. For instance, Burgos et al. [
14
] and Edouard et al. [
15
]
have reported from 2.3- to 4.6-fold increases in NH
3
emissions associated with manure
slurries of dairy cows when dietary CP contents were increased from 15 to 21% DM and
120 to 180 g/kg DM, respectively. The magnitude of N excretion (particularly urinary N),
and hence the likelihood of NH
3
emissions from slurry is highly dependent on dietary CP
concentration [16–18].
However, most of the studies on NH
3
volatilization losses from manure slurries
completed in different parts of the world have been largely based on the use of micromete-
orological and mass N balance techniques. The mathematical solutions for such techniques
include some assumptions that must be fulfilled to ensure the results are reliable [
19
]. These
solutions can be quite complex, thus requiring expert users and dedicated software tools.
In addition, besides the urinary urea-N concentration and the slurry pH, the hydrolysis of
the urinary urea-N content is controlled to the largest extent by climatic factors, particularly
air temperature and rainfall [
5
,
20
,
21
]. Thus, there is a need for NH
3
emission measure-
ments that separate the effect of diet from the effects of geographical and climatic factors.
The objective of this study was therefore to determine the effect of increasing dietary CP
level on NH3emissions from slurry from lactating Holstein-Friesian cows as measured in
open-circuit respiration chambers.
2. Materials and Methods
This study was conducted at the Agri-Food and Biosciences Institute (AFBI), Hills-
borough (Northern Ireland, UK) under Project Licence Number PPL 2824. All procedures
performed in this study involving animals were approved by the Ethical Review Committee
of AFBI, and were in accordance with the Animals (Scientific Procedures) Act 1986.
2.1. Diets and Feeding of Animals for Slurry Production
The different slurries used in this study were collected from 24 lactating Holstein-
Friesian cows of the sister study that evaluated the effect of feeding total mixed ration
(TMR) diets containing varying levels of CP on feed intake, nutrient digestibility, milk

Animals 2022,12, 1243 3 of 14
production, and nitrogen use efficiency [
22
]. In brief, the 24 cows (between 146 and 200 days
in milk, averaging 2.4 parity, 645 kg live weight (SD 57), and 32 kg of milk/cow/day (SD 6)
at the time of enrolment in the study) were randomly assigned to four TMR diets containing
141, 151, 177, and 201 g CP/kg DM (Table 1). The TMR diets were prepared from perennial
ryegrass (Lolium perenne L.) silage and a concentrate mix. The formulation was targeted to
produce TMR diets containing 130, 150, 170, and 190 g CP/kg DM in a silage:concentrate
ratio of 50:50 (DM basis). However, the actual CP levels were 141, 151, 177, and 201 g
CP/kg DM, respectively, in a silage:concentrate ratio of about 48:52 (DM basis).
Table 1. Composition of the TMR treatment diets.
Dietary CP Content (Formulated), g/kg DM
141 151 177 201
Ingredient, % of TMR DM
Ryegrass silage 47.3 47.9 47.7 47.5
Concentrate mix 52.7 52.1 52.3 52.5
Chemical composition 1
Dry matter (DM), g/kg 507 513 510 508
Crude protein (CP), g/kg DM 141 151 177 201
Rumen-degradable protein (RDP) 2, g/kg CP 103 109 127 144
Rumen-undegradable protein (RUP) 2,
g/kg CP 39 41 49 56
Neutral detergent fibre (NDF), g/kg DM 411 408 399 388
Acid detergent fibre (ADF), g/kg DM 218 218 214 209
Water-soluble carbohydrates) WSC, g/kg DM 81 82 86 90
Ash, g/kg DM 78 78 80 81
1
Calculated as formulated from the analysed chemical compositions of the individual ingredients;
2
Feed into
milk (FiM) system according to Thomas [23].
The cows were offered feed once daily at 09:00 h, and the amounts of feed offered were
adjusted daily to obtain approximately 10% orts (as-fed basis) daily throughout 25-day
experimental periods. The first 20 days were for adaptation to the TMR treatment diets,
followed by a 5-day period of individual feeding (for milk yield, feed intake, and total faeces
and urine collection data). The feeding management, feed sampling and laboratory analyses
were as described in the sister study [
22
]. During the 20-day adaptation period, the cows
were group-fed their TMR treatment diets. The respective TMR diets were mixed separately
(for each treatment) using a feeder wagon (Vari-Cut 12, Redrock, Armagh, Northern Ireland)
and tipped into a series of feeders mounted on weigh scales. Individual cow access to the
feeders was programmed (cows were able to only access feeders containing their rightful
treatment diets) by means of an electronic “neck-tag” identification system (Controlling
and Recording Feed Intake (CRFI), BioControl, Rakkestad, Norway).
During the 5-day individual feeding period, the cows were housed in individual
tie-stalls (with continuous access to fresh water), and the head of each cow was loosely tied
at a halter in the individual tie-stall. The respective TMR diets were mixed separately (for
each cow) using a power concrete mixer. The cows were fed their TMR treatment diets once
daily at 9:00 a.m., and then any orts (refusals) and spilled feed were collected and weighed
on the following day at about 8:00 a.m. (before new feed was offered). The amounts fed
were adjusted daily on the basis of the previous day’s intake to obtain approximately 10%
orts. The daily TMR intakes were manually recorded.
2.2. Faeces and Urine Collection
On Day 1 of each of the 5-day individual feeding periods, each individually confined
cow was fitted with a system to facilitate collection of urine separately from faeces. The
separation system was comprised of a Velcro patch (vinyl fabric, about 15 cm
×
15 cm),
which was designed with a hole corresponding to the vulva opening. The patch was glued
(EVO-STIK 528 Instant Contact Adhesive, Bostik Ltd., Stafford, UK) to each cow around

Animals 2022,12, 1243 4 of 14
the perineal region (area under the tail), leaving the vulva opening and anus open. The
Velcro patch was supported by two straps (one on either side) that were glued to the rump.
For each patch, Velcro tape (loop side) was sewn around the hole corresponding to the
vulva, which helped to hold in position a urine collection unit (with hook-side Velcro tape)
connected to an 80 mm polyvinyl chloride (PVC) Layflat Hose (for channelling the urine
into a plastic collection container).
On the following day, 24 h total collections of faeces and urine were made. To prevent
NH
3
from volatilizing, urine was collected on ice (i.e., after inserting each plastic urine
collection container in an ice box). The faeces of each cow were collected in a large plastic
collection tray that was placed behind each tie-stall. The 24 h faeces and urine collections
were weighed at 08:00 a.m. About 4 and 2 kg aliquots of the faeces and urine, respectively,
of each cow were taken and stored in a refrigerator (at 5
◦
C) for later assessment of NH
3
emissions from the ensuing slurry. The faeces samples were collected in plastic bags and
tied tightly, while urine samples were collected in plastic screw-capped containers.
2.3. Slurry Sample Preparation
The refrigerated urine and faeces of each cow were combined to form 2 kg duplicate
samples of slurry (weight/weight; fresh basis) according to the proportions in which they
were excreted by the respective cows (simulating urine and faeces deposition in concrete
floor cattle housing systems). The urine:faeces production ratios averaged 1:2.27, 1:2.15,
1:2.21, and 1:1.65 (weight/weight; fresh basis) for the 141, 151, 177, and 201 g CP/kg DM
diets, respectively.
2.4. Slurry Allocation to Respiration Chambers and NH3Emission Measurements
Considering that there were only six open-circuit respiration chambers (RC) at AFBI,
NH
3
emission measurements were conducted in eight batches (chamber runs) at intervals
of 2–3 days. The RCs were numbered from 1 to 6, and arranged in two rows facing one
another (with RC1 facing RC6, RC2 facing RC5, and RC3 facing RC6). On a random basis,
RCs 1, 2, and 3 were maintained at 8
◦
C, while RCs 4, 5, and 6 were maintained at 18
◦
C.
For each of the eight chamber runs, slurry samples of only three randomly selected cows
were studied. The duplicate slurry samples from the same cow were randomly assigned to
one of two chamber temperatures (8 and 18
◦
C), while ensuring that slurry samples from
the same cow were not assigned to RCs directly facing each other (Figure 1).
To constitute the 2 kg slurry samples, the corresponding urine and faeces quantities
were weighed out separately. While inside their randomly assigned RCs, the weighed
out quantities of urine and faeces were quickly poured into a plastic bucket (with the
RC door closed), stirred for 1 min using a Mac Allister paddle mixer, and thereafter the
entire mixture was poured off on a plastic tray (8
×
44
×
75 cm height, width and length,
respectively), spreading it in a thin layer to cover the entire surface of the tray.
The six indirect open-circuit RCs used were made with double Perspex (Lucite Inter-
national, Darwen, UK) walls fitted in aluminium frames and mounted on a profiled plastic
floor, with a single unplasticised polyvinyl chloride (uPVC) door. The total volume of
4.86 m
3
(1.98 m long, 1.46 m wide, and 1.68 m high) for each chamber was ventilated by suc-
tion pumps set at 10 Normal Cubic Metres per Hour (NCM/hour), giving approximately
1.6 air changes/hour. Temperature and relative humidity control were achieved with
Hitachi air-conditioning units set at either 8
±
1
◦
C or 18
±
1
◦
C and 50
±
10%, respectively.
The chambers were operated under negative pressure (30 N/m
2
), with exhaust air removed
at one position (through a 50 mm (internal diameter) PVC duct) for volume measurement
and gas analysis.

Animals 2022,12, 1243 5 of 14
Animals 2022, 12, x 5 of 15
Figure 1.
Layout of the allocation of slurry sub-samples of the 24 cows (from cow 1 to cow 24) to
the 6 open-circuit respiration chambers (from RC1 to RC6).
Figure 1.
Layout of the allocation of slurry sub-samples of the 24 cows (from cow 1 to cow 24) to the
6 open-circuit respiration chambers (from RC1 to RC6).

Animals 2022,12, 1243 6 of 14
Chamber air samples were taken from the 50 mm exhaust ducts through 6 mm
polytetrafluoroethylene (PTFE) Teflon tubing by an ADC MGA 3500 gas sampling unit
(Analytical Development Co. Ltd., Hoddesdon, UK). NH
3
concentrations were measured
using a LumaSense, INNOVA 1512i Photoacoustic Gas Monitor (Lumasense Technologies
A/S, Denmark). Temperature and humidity were measured using a TREND HT/D/2%
combined temperature/humidity sensor (TREND Control Systems, Horsham, UK), and
atmospheric pressure was measured using a Vaisala PTA 427 digital barometer (Delta-T
Devices, Cambridge, UK). Results were collected via TREND IQ4 Controllers, (TREND
Control Systems, Horsham, UK) for 2 min from each chamber, and the average values
of the final minute were logged into an SQL Database on the secure server on site for
post-run analysis, giving 4.3 measurements/chamber per hour. The analysis train was
designed to run automatically. Prior to the beginning and at the end of each experimental
period, the chambers were tested by comparing the amount of NH
3
recorded by the ADC
MGA3500 gas analyser 3 h after the release of a known quantity of 800 ppm NH
3
by Mass
Flow Controllers (4 L/min) (Cole Palmer, www.colepalmer.co.uk, accessed on 9 May 2022).
The recovery was found to be almost 75% in all periods. Samples remained in the chamber
for 72 h, with all NH
3
measurements (in litres) for the final 48 h (in two time intervals of
0–24 and 0–48 h). These measurements accounted for NH
3
released by 1 m
2
of slurry spread
on the plastic tray placed on the floor of the chambers. NH
3
emissions from the 2 kg slurry
samples were converted to grams of NH
3
per kg of fresh slurry, and then extrapolated to
grams of NH
3
per cow per day after factoring in the total amount of slurry for each cow
per day.
2.5. Laboratory Analysises
For each of the 24 refrigerated faeces and urine samples, about 500 g and 200 mL,
respectively, were taken for laboratory analysis. A subsample of about 100 g was taken
from each of the refrigerated faeces samples and analysed for total N content using wet
chemistry analysis. The rest of the faeces sample was oven-dried at 60
◦
C for 144 h
(for DM content determination). After oven-drying, the samples were ground through a
1 mm sieve and analysed for neutral detergent fibre (NDF) and acid detergent fibre (ADF)
concentrations [
24
]. The urine samples were analysed for total N content. The total N
concentration in faeces and urine was analysed using a Tecator Kjeldahl Auto 1030 Analyser
(Foss Tecator AB, Höganäs, Sweden).
2.6. Statistical Analysis
Data were analysed using SAS (version 9.1, SAS Institute, Cary, NC, USA, 2003).
Differences in NH
3
emissions were considered significant when p
≤
0.05, and tendencies
towards significant differences when 0.05
≤
p
≤
0.10. Data were analysed using the PROC
MIXED procedure. The interaction of dietary CP level
×
chamber temperature was initially
included in the model as a fixed effect, but was removed because it was not significant in
all cases. Therefore, statistical models containing only the main effects of dietary CP level
or chamber temperature were fitted. The statistical models had the following forms:
Yijk =µ+ Pi+ cow(cr)j(i) + PNik + eijk
where Y
ijk
= NH
3
emissions per cow,
µ
= overall mean effect, P
i
= fixed effect of either
dietary CP level or chamber temperature, cow(cr)
j(i)
= random effect of the cow from which
the slurry was collected within the respiration chamber run (order of investigation in the
respiration chambers), PN
ik
= fixed effect of the dietary CP level
×
respiration chamber
number interaction, and eijk = effect of the residual error.
Selection of the fixed and random effects depended on whether their inclusion in the
model resulted in a smaller Akaike information criterion (AIC) and/or made a meaningful
improvement in treatment means and standard errors. Polynomial contrasts were used to
detect linear and quadratic responses to dietary CP level.

Animals 2022,12, 1243 7 of 14
The two-sample t-test was used to compare (within dietary CP level or chamber
temperature groups) the average NH3emissions measured for the 24–48 and 0–48 h inter-
vals, as well as the average daily NH
3
emissions with the 0–24 h average NH
3
emissions.
Linear regressions of NH
3
emissions with dietary CP level, total urinary nitrogen (UN),
and faeces nitrogen (FN) were performed using the PROC REG procedure of SAS. Re-
gression coefficients of the regression equations for 8 and 18
◦
C chamber temperatures
were compared.
3. Results and Discussion
3.1. Urine and Faeces Characteristics
Table 2provides a summary of the weights and chemical characteristics of the urine
and faeces combined to form the slurry samples. The amount of urine increased linearly
(p< 0.05
) with increasing dietary CP level, being higher with the 177 or 201 diets (22.9 and
23.2 kg/day, respectively). As dietary CP level was increased from 141 to 151, 177, and
then 201 g/kg DM, the amount of urine increased by 6.4, 45.9, and 47.8%, respectively.
Rumen-degradable protein (RDP) concentrations were relatively high in the high-CP diets
(Table 1), which might have resulted in excess rumen NH
3
-N being excreted as urea in
urine and reduced blood urea-N transfer to the rumen for recycling [
25
]. When dietary RDP
is in excess of the amount required by ruminal microorganisms, the protein is degraded
to NH
3
-N, absorbed, metabolized to urea in the liver, and lost in the urine [
26
] rather
than being salvaged and recycled to the rumen. However, the excretion of urea in urine
requires water [
27
], which inevitably leads to higher water intake and, hence, increased
urine amount. Others studies [
14
,
28
] also reported linear increases in urine amount with
increasing dietary CP level.
Table 2.
Weight and chemical characteristics of the urine and faeces combined to form the slurry samples.
Dietary CP Level, g/kg DM p-Value
141 151 177 201 SEM Diet Linear Quadratic
Urine
Weight, kg/cow/day 16.7 b16.8 b21.6 ab 23.2 a1.85 0.039 0.006 0.805
Total urine N, g/L 8.0 b8.5 b9.5 b12.0 a0.53 0.0001 <0.0001 0.179
Faeces
Weight, kg/cow/day 34.9 36.3 46.3 39.4 4.66 0.343 0.275 0.180
Total faeces N, g/kg
(fresh) 4.9 4.8 5.1 5.2 0.36 0.812 0.393 0.942
DM, g/kg 149.5 145.7 140.8 152.9 7.24 0.654 0.770 0.242
NDF, g/kg DM 545.7 559.6 570.2 534.3 10.1 0.108 0.412 0.082
ADF, g/kg DM 286.3 299.1 298.7 279.9 6.50 0.111 0.303 0.094
Total slurry 1
Weight, kg/cow/day 51.6 b53.1 b67.9 a62.6 ab 5.48 0.043 0.038 0.070
SEM—standard error of least square means;
ab
Least square means in the same row with different superscript
letters differ (p< 0.05); 1Calculated as sum of weight of fresh faeces plus urine (per cow/day).
The amount of faeces voided and the N, DM, NDF, and ADF contents of the faeces did
not differ (p> 0.05) across dietary CP levels, and averaged 39.2 kg/cow/day and 5.0 g/kg
(fresh), 147.2 g/kg, 552.5 g/kg, and 291.0 g/kg DM, respectively (Table 2). The lack of
dietary CP level effect on faecal N content is consistent with Van Vliet et al. [
29
], who
observed no significant differences in the faecal N content of cows fed high-protein diets
compared with those fed low-protein diets. According to the nutrient digestibility results of
the sister study [
22
], the low-protein diets had the lowest apparent total-tract digestibility
of CP, which obviously resulted in increased faecal N excretion for the low-protein diets,
thus causing the lack of difference observed.
The amount of total slurry (mixture of urine and faeces) was affected in a linear
(
p< 0.05
) manner, showing a tendency (p= 0.070) for a quadratic response to increasing
dietary CP level. This effect was attributed more to the higher urine amount (23.2 kg/day

Animals 2022,12, 1243 8 of 14
for the 201 diet compared with 15.7 kg/day for the 141 diet), particularly since the effect
of dietary CP level on amount of faeces was not significant (p> 0.05). Similarly, Burgos
et al. [
14
] also reported a linear increase in the amount of slurry as dietary CP level was
increased from 15 to 17, 19, and then 21% DM.
3.2. Dietary CP Level and NH3Emissions from Slurry
The effect of dietary CP level on NH
3
emissions from slurry from lactating cows by
measurement time interval is presented in Table 3. The interaction between dietary CP level
and chamber temperature was not significant (p> 0.05); thus, only tests on main effects
are presented. The average NH
3
emissions for the 0–24 h interval increased in a linear
(p< 0.05
) manner with increasing dietary CP level, being higher (p< 0.05) with the 177 or
201 diets and not different between the 141 and 151 diets. The average NH
3
emissions
for the 24–48 h interval increased in a linear (p< 0.05) manner with increasing dietary CP
level, being highest (p< 0.05) with the 177 or 201 diets. The average NH
3
emissions for the
0–48 h interval also increased in a linear (p< 0.05) manner with increasing dietary CP level,
being highest with the 201 diet, intermediate with the 177 diet, and lowest with the 141 and
151 diets. The same pattern was observed for the average daily NH3emissions.
Table 3.
Effect of dietary CP level on NH
3
emissions from slurry from lactating cows by measurement
time interval.
Dietary CP Level, g/kg DM p-Value
141 151 177 201 SEM Diet Linear Quadratic
0–24 h
NH3, g/cow 21.3 c29.5 bc 42.2 b63.4 a4.97 0.0002 <0.0001 0.538
24–48 h
NH3, g/cow 9.4 c13.8 bc 23.5 ab 33.1 a3.80 0.003 0.0003 0.992
0–48 h 1
NH3, g/cow 30.6 c43.4 bc 65.8 b96.6 a7.91 0.0002 <0.0001 0.712
Average daily emissions
NH3, g/cow/day 15.3 c21.7 bc 32.9 b48.3 a3.96 0.0002 <0.0001 0.712
SEM—standard error of least square means;
abc
Least square means in the same row with different superscript
letters differ (p< 0.05); 1Total cumulative NH3emissions during the entire study period.
The average NH
3
emissions for the 0–24, 24–48, and 0–48 h intervals and the average
daily NH
3
emissions were increased by 2.9- to 3.5-fold as dietary CP level was increased
from 141 to 201 g/kg DM. This result was expected and corroborates the observation that
the principal driver of NH
3
emission from slurries is dietary CP level or N intake [
17
,
18
].
Accordingly, previous studies [
13
–
15
] reported higher NH
3
emissions from manure slurries
as dietary CP level was increased. Most NH
3
emissions from slurries are produced from
the hydrolysis of urinary urea-N to ammonium (NH
4+
) by urease enzyme [
30
] that is
present in faeces [
10
] from rumen bacteria [
31
]. High dietary CP levels result in increased
rumen NH
3
-N concentration relative to rumen microbial requirements [
32
], which has
been reported to be inversely related to urea-N transfer to the rumen [
25
] for recycling.
Consequently, the total urea-N salvaged and recycled to the rumen decreases as dietary
CP level increases, which ends up being excreted in urine rather than being salvaged and
reutilized. Therefore, because increasing dietary CP level increases urinary urea-N content,
an increase in dietary CP level leads to an increased potential for NH
3
volatilisation [
33
,
34
].
For this reason, Arriaga et al. [
35
] observed a 36.5% reduction in NH
3
concentration on
tie-stall floors, and without compromising milk nitrogen use efficiency when dietary CP
content was reduced from 17 to 14% DM in mid- to late-lactating cows.
Unsurprisingly, the difference between diet 177 and diet 201 for the 0–24 h average
NH
3
emissions was significant, but not significant for the 24–48 h interval (Table 3). Pre-
vious studies [
36
,
37
] have shown that the urease hydrolysis of urea-N in slurry to NH
4+
(and thus NH
3
volatilization) is complete within approximately 20 h after the urine and

Animals 2022,12, 1243 9 of 14
faeces come into contact. This rapid hydrolysis of urea-N in slurry to NH
4+
decreases
the available NH
4+
to be converted to NH
3
[
38
], hence causing the levelling off of NH
3
emissions for those diets.
Results of the two-sample t-tests (within each dietary CP level group; Figure 2) indi-
cated that the average NH
3
emissions for the 0–24 h intervals were higher (p< 0.05) than for
the 24–48 h intervals across the four dietary CP levels. Chantigny et al. [
39
] reported higher
NH
3
emission rates from slurry within the first 12 h following surface application in the
field. Moal et al. [
40
] reported that an average of 75% of the total NH
3
emissions occurred
within the first 15 h following the surface spreading of pig and cattle slurry, while Carozzi
et al. [
41
] reported that the last high peak of NH
3
emissions following the surface spreading
of dairy slurry occurred after 24 h before gradually decreasing. According to Sommer and
Hutchings [
20
] and Sommer et al. [
42
], NH
3
emission rate is usually highest immediately
after slurry application, partly due to the high initial concentration of ammonium-N (NH
4+
-
N). Christensen et al. [
43
] observed that approximately 70% of the N in slurry is present as
NH
4+
, which is lost as NH
3
due to volatilization. However, the rate normally falls rapidly
as the concentration of NH
4+
decreases after the high initial NH
3
emission [
44
], levelling
off within the first 30 h [
39
]. This explains why the average NH
3
emissions for the 0–24 h
interval was significantly higher (p< 0.05) than for the 24–48 h interval.
Animals 2022, 12, x 10 of 15
Figure 2.
NH
3
emissions from slurry from lactating cows fed at 4 dietary CP levels (141, 151, 177,
and 201 g CP/kg DM) measured at different time intervals (0–24, 24–48, and 0–48 h) in open-
circuit respiration chambers. The figure presents 24–48 and 0–48 h averages, as well as daily
averages compared with the 0–24 h averages (within dietary CP levels) using the two-sample
t
-
test.
Vertical bars represent standard errors of the means.
The two-sample
t
-tests between the 0–24 and 0–48 h intervals showed that the
average NH
3
emissions for the 0–48 h intervals (i.e., total cumulative NH
3
emissions for
the entire study period) were higher (
p
< 0.05) than for the 0–24 h intervals for the 151,
177, and 201 diets. For the 141 diet, the average NH
3
emissions for the 0–48 h interval
tended (
p
= 0.092) to be higher. The average NH
3
emissions observed in this study for
the 0–24 h interval represented 68, 68, 67, and 67% of the total cumulative NH
3
emissions (0–48 h average emissions) from the slurries from cows fed diets containing
141, 151, 177, and 201 g CP/kg DM, respectively. Sommer and Hutchings [20] and
Sommer et al. [42] reported a value of close to 50% following the field application of
slurry. We attributed much of this disparity to the different methodologies and
conditions in which these studies were conducted with respect to measurement
technique (open-circuit respiration chambers vs. micrometeorological methods),
instruments used, location (enclosure vs. open air), slurry characteristics, and climatic
conditions (controlled vs. uncontrolled environment). Each of these factors can
influence NH
3
emission estimates [19,30].
3.3. Chamber Temperature and NH3 Emissions from Slurry
The fffect of chamber temperature on NH3 emissions from slurry from lactating cows
by measurement time interval is presented in Table 4. The average NH3 emissions for the
0–24 h interval were greater (p < 0.05) for 18 °C than for 8 °C. Maintaining the chamber
temperature at 18 °C resulted in about 35% greater average NH3 emissions for the 0–24 h
interval, compared with 8 °C. The effect of chamber temperature on the average daily NH3
emissions was not significant (p > 0.05), and averaged 31 g of NH3/cow/day. The same
pattern was observed for the average NH3 emissions for the 0–48 h interval, averaging 62
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
110.00
120.00
130.00
141 151 177 201
NH3(g/cow)
Dietary CP level (g/kg DM)
0-24 hours 24-48 hours 0-48 hours Average daily
P = 0.006
P = 0.092
P = 0.130
P = 0.002
P = 0.025
P = 0.047
P = 0.007
P = 0.012
P = 0.047
P = 0.0007
P = 0.002
P = 0.030
Figure 2.
NH
3
emissions from slurry from lactating cows fed at 4 dietary CP levels (141, 151, 177,
and 201 g CP/kg DM) measured at different time intervals (0–24, 24–48, and 0–48 h) in open-circuit
respiration chambers. The figure presents 24–48 and 0–48 h averages, as well as daily averages
compared with the 0–24 h averages (within dietary CP levels) using the two-sample t-test. Vertical
bars represent standard errors of the means.
The two-sample t-tests between the 0–24 and 0–48 h intervals showed that the average
NH
3
emissions for the 0–48 h intervals (i.e., total cumulative NH
3
emissions for the entire
study period) were higher (p< 0.05) than for the 0–24 h intervals for the 151, 177, and
201 diets. For the 141 diet, the average NH
3
emissions for the 0–48 h interval tended

Animals 2022,12, 1243 10 of 14
(p= 0.092)
to be higher. The average NH
3
emissions observed in this study for the
0–24 h
interval represented 68, 68, 67, and 67% of the total cumulative NH
3
emissions (0–48 h
average emissions) from the slurries from cows fed diets containing 141, 151, 177, and
201 g CP/kg DM, respectively. Sommer and Hutchings [
20
] and Sommer et al. [
42
] reported
a value of close to 50% following the field application of slurry. We attributed much of
this disparity to the different methodologies and conditions in which these studies were
conducted with respect to measurement technique (open-circuit respiration chambers vs.
micrometeorological methods), instruments used, location (enclosure vs. open air), slurry
characteristics, and climatic conditions (controlled vs. uncontrolled environment). Each of
these factors can influence NH3emission estimates [19,30].
3.3. Chamber Temperature and NH3Emissions from Slurry
The fffect of chamber temperature on NH
3
emissions from slurry from lactating cows
by measurement time interval is presented in Table 4. The average NH
3
emissions for the
0–24 h interval were greater (p< 0.05) for 18
◦
C than for 8
◦
C. Maintaining the chamber
temperature at 18
◦
C resulted in about 35% greater average NH
3
emissions for the 0–24 h
interval, compared with 8
◦
C. The effect of chamber temperature on the average daily NH
3
emissions was not significant (p> 0.05), and averaged 31 g of NH
3
/cow/day. The same
pattern was observed for the average NH
3
emissions for the 0–48 h interval, averaging 62 g
of NH
3
/cow. According to Sommer et al. [
42
], the relationship between NH
3
emissions
from slurry and measurement temperature is often simply a reflection of the warming effect
(increased temperature), which displaces the NH
3
and NH
4+
equilibrium to NH
3
[
45
], and
also increases urea activity.
Table 4.
Effect of chamber temperature on NH
3
emissions from slurry from lactating cows by
measurement time interval.
Chamber Temperature, ◦C
8 18 SEM p-Value
Measurement time interval
0–24 h
NH3, g/cow 33.9 b45.4 a4.78 0.009
24–48 h
NH3, g/cow 29.4 a14.3 b3.20 0.001
0–48 h
NH3, g/cow 63.4 59.7 6.56 0.475
Average daily emissions
NH3,
g/cow/day 31.7 29.8 3.28 0.475
SEM—standard error of least square means;
ab
Least square means in the same row with different superscript
letters differ (p< 0.05).
The absence of differences in average NH
3
emissions between the 8 and 18
◦
C for the
0–48 h interval and the average daily NH
3
emissions could be attributed to the fact that in
general, NH
3
emissions from manure slurries depend on the concentration of free NH
3
in
aqueous solutions [
46
]. As described earlier in this paper, the rate of NH
3
production from
manure slurries is determined by the availability of NH
4+
, which dissociates to form NH
3
and H
+
[
46
]. The rate normally falls rapidly as the concentration of NH
4+
decreases [
44
],
levelling off within the first 30 h [
39
]. This also explains why 8
◦
C yielded significantly
higher (p< 0.05) average NH3emissions for the 24–48 h interval compared with 18 ◦C.
The two-sample t-tests (within each chamber temperature group; Figure 3) comparing
the average NH
3
emissions for the 0–24 h interval with the 24–48 h interval and the average
daily NH
3
emissions produced mixed results. Within the 8
◦
C chamber temperature group,
the average NH
3
emissions for the 0–24 and 24–48 h intervals as well as the average daily
NH
3
emissions were similar (p> 0.05). However, a different pattern was observed for the
18
◦
C chamber temperature group, in which the average NH
3
emissions for the 0–24 h

Animals 2022,12, 1243 11 of 14
interval were higher (p< 0.05) than for the 24–48 h interval and the average daily NH
3
emissions. Similarly, the two-sample t-tests between the 0–24 and 0–48 h intervals also
produced mixed results. Within the 8
◦
C chamber temperature group, the average NH
3
emissions for the 0–24 h interval were lower (p< 0.05) than for the 0–48 h interval. However,
a different pattern was observed for the 18
◦
C chamber temperature group, in which the
average NH3emissions for the 0–24 and 0–48 h intervals were not different (p> 0.05).
Animals 2022, 12, x 12 of 15
Figure 3.
NH
3
emissions from slurry from lactating cows measured at 2 temperatures (8 and 18
°C) at different time intervals (0–24, 24–48, and 0–48 h) in open-circuit respiration chambers. The
figure presents 24–48 and 0–48 h averages, as well as daily averages compared with the 0–24 h
averages (within each chamber temperature) using the two-sample
t
-test.
Vertical bars represent
standard errors of the means.
These mixed results might be attributable to the effect of the interaction between pH
and temperature on urease activity and the dissociation of NH4+. As described earlier in
this paper, the dissociation of NH4+ is dependent on manure properties, including
temperature and pH among others [46]. Increases in temperature and pH of the liquid
manure increase the NH4+ dissociation and thus NH3 volatilization [46,47]. However, Liu
et al. [45] observed that temperature within a range of 10–25 °C had little if any effect on
NH4+ dissociation for a normal pH range of 6.5–9. This could explain why the average NH3
emissions for the 0–24 and 0–48 h intervals were not different for the 18 °C chamber
temperature group.
3.4. Relationships of NH3 Emissions from Slurry and Dietary CP Level, Total Urinary N, and
Faeces N
The linear regression equations for predicting NH3 emissions from slurry from
lactating cows based on dietary CP level, total urinary nitrogen (UN), and faeces N (FN)
are presented in Table 5. The numbers in parentheses are the standard errors of the
coefficient estimates. Slopes of the regression equations for predicting NH3 emissions
were not different (p > 0.05) between the two chamber temperatures, so overall
relationships (regardless of chamber temperature) are presented. Using dietary CP level,
total UN, or FN contents as the only predictors of NH3 emissions from slurry indicated
positive relationships (Equations (1)–(3)). The best single independent variable was
dietary CP level (R2 = 0.67), closely followed by total UN content (R2 = 0.62). The
relationship between NH3 emissions and FN was weak, which was consistent with the
fact that N in faeces is less susceptible to NH3 volatilization losses than urea-N in urine.
The addition of either total UN or FN as supporting predictors to dietary CP considerably
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
818
NH3(g/cow)
Chamber temperature (oC)
0-24 hours 24-48 hours 0-48 hours Average daily
P = 0.117
P = 0.004
P = 0.380
P = <0.0001
P = 0.132
P = 0.029
Figure 3.
NH
3
emissions from slurry from lactating cows measured at 2 temperatures (8 and 18
◦
C)
at different time intervals (0–24, 24–48, and 0–48 h) in open-circuit respiration chambers. The figure
presents 24–48 and 0–48 h averages, as well as daily averages compared with the 0–24 h averages
(within each chamber temperature) using the two-sample t-test. Vertical bars represent standard
errors of the means.
These mixed results might be attributable to the effect of the interaction between pH
and temperature on urease activity and the dissociation of NH
4+
. As described earlier
in this paper, the dissociation of NH
4+
is dependent on manure properties, including
temperature and pH among others [
46
]. Increases in temperature and pH of the liquid
manure increase the NH
4+
dissociation and thus NH
3
volatilization [
46
,
47
]. However, Liu
et al. [
45
] observed that temperature within a range of 10–25
◦
C had little if any effect on
NH
4+
dissociation for a normal pH range of 6.5–9. This could explain why the average
NH
3
emissions for the 0–24 and 0–48 h intervals were not different for the 18
◦
C chamber
temperature group.
3.4. Relationships of NH3Emissions from Slurry and Dietary CP Level, Total Urinary N, and
Faeces N
The linear regression equations for predicting NH
3
emissions from slurry from lac-
tating cows based on dietary CP level, total urinary nitrogen (UN), and faeces N (FN) are
presented in Table 5. The numbers in parentheses are the standard errors of the coefficient
estimates. Slopes of the regression equations for predicting NH
3
emissions were not differ-

Animals 2022,12, 1243 12 of 14
ent (p> 0.05) between the two chamber temperatures, so overall relationships (regardless
of chamber temperature) are presented. Using dietary CP level, total UN, or FN con-
tents as the only predictors of NH
3
emissions from slurry indicated positive relationships
(
Equations (1)–(3))
. The best single independent variable was dietary CP level (R
2
= 0.67),
closely followed by total UN content (R
2
= 0.62). The relationship between NH
3
emissions
and FN was weak, which was consistent with the fact that N in faeces is less susceptible to
NH
3
volatilization losses than urea-N in urine. The addition of either total UN or FN as
supporting predictors to dietary CP considerably increased the prediction accuracy to 0.72
and 0.71, respectively (Equations (4) and (5)). Combining the three variables (dietary CP
level, total UN, and FN) only marginally increased the prediction accuracy to 0.73.
Table 5.
Linear regression of NH
3
emissions from slurry from lactating cows with dietary CP level,
total urinary nitrogen (UN), and faeces N (FN).
Linear Equation R2
NH3, g/Cow/Day =
(1) −60.09 (11.1) + 0.54 (0.07) ×CP a0.67
(2) −30.98 (8.86) + 6.55 (0.91) ×UN b0.62
(3) +30.04 (19.4) + 0.32 (3.77) ×FN c0.0002
(4) −56.07 (10.9) + 0.37 (0.11) ×CP a+
2.66 (1.44)
×
UN
b0.72
(5) −47.2 (14.0) + 0.56 (0.06) ×CP a−3.13 (2.12) ×FN c0.71
(6) −31.7 (15.0) + 6.55 (0.92) ×UN b+ 0.14 (2.36) ×FN c0.62
(7) −47.4 (13.7) + 0.41 (0.12) ×CP a+
2.24 (1.49)
×
UN
b−2.25 (2.15) ×FN c0.73
aCP in g/kg DM; bUN in g/L; cFN in g/kg fresh weight.
4. Conclusions
The results showed that increasing dietary CP level linearly increased the average daily
NH
3
emissions from slurry and emissions for the 0–24, 24–48, and 0–48 h intervals. Each
10 g increase in dietary CP level was associated with a 5.4 g increase in the average daily
NH
3
emissions. Regardless of dietary CP level, over 65% of the total NH
3
emissions from
slurry occurred within the first 24 h of urine coming into contact with faeces. Increasing
the chamber temperature from 8 to 18
◦
C also increased NH
3
emissions from slurry, but
only for the 0–24 h interval. There were strong relationships of NH
3
emissions from slurry
with dietary CP level and with total urinary N content. The most accurate equations for
predicting NH
3
emissions from slurry were those based on dietary CP level supported by
either the contents of urinary nitrogen, faeces nitrogen, or both.
Author Contributions:
Conceptualization, T.Y.; data curation, C.B.K.; formal analysis, C.B.K.; fund-
ing acquisition, T.Y.; methodology, C.B.K. and T.Y.; project administration, T.Y.; software, C.B.K.;
writing–original draft, C.B.K. and T.Y. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research project was funded by the Department of Agriculture Environment and Rural
Affairs (DAERA) in Northern Ireland.
Institutional Review Board Statement:
All procedures performed in this study involving ani-
mals were approved by the Ethical Review Committee of the Agri-Food and Biosciences Institute
(PPL 2824), and were in accordance with the Animals (Scientific Procedures) Act 1986.
Data Availability Statement: All data used in this paper were stored in the server of the Agri-Food
and Biosciences Institute which can be accessed upon the request.
Conflicts of Interest: The authors declare no conflict of interest.

Animals 2022,12, 1243 13 of 14
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