Published November 24, 2014
Technical note: Nitrogen isotopic fractionation can be
used to predict nitrogen-use efficiency in dairy cows fed temperate pasture1
L. Cheng,*2 A. J. Sheahan,† S. J. Gibbs,* A. G. Rius,†3
J. K. Kay,† S. Meier,† G. R. Edwards,* R. J. Dewhurst,‡ and J. R. Roche†
*Faculty of Agriculture and Life Sciences, P.O. Box 84, Lincoln University, New Zealand; †DairyNZ, Private Bag 3221, Hamilton,
New Zealand; and ‡Teagasc, Animal and Grassland Research and Innovation Centre, Grange, Dunsany, County Meath, Ireland.
ABSTRACT: The objective of this study was to
investigate the relationship between nitrogen isotopic
fractionation (δ15N) and nitrogen-use efficiency (milk
nitrogen/nitrogen intake; NUE) in pasture-fed dairy
cows supplemented with increasing levels of urea to
mimic high rumen degradable protein pastures in spring.
Fifteen cows were randomly assigned to freshly cut
pasture and either supplemented with 0, 250, or 336 g
urea/d. Feed, milk, and plasma were analyzed for δ15N,
milk and plasma for urea nitrogen concentration, and
plasma for ammonia concentration. Treatment effects
were tested using ANOVA and relationships between
variables were established by linear regression. Lower
dry matter intake (P = 0.002) and milk yield (P = 0.002)
occurred with the highest urea supplementation (336 g
urea/d) compared with the other two treatments. There
was a strong linear relationship between milk δ15N –
feed δ15N and NUE: [NUE (%) = 58.9 – 10.17 × milk
δ15N – feed δ15N (‰) (r2 = 0.83, P < 0.001, SE = 1.67)]
and between plasma δ15N – feed δ15N and NUE: [NUE
(%) = 52.4 – 8.61 × plasma δ15N – feed δ15N (‰) (r2 =
0.85, P < 0.001, SE = 1.56)] . This study confirmed the
potential use of δ15N to predict NUE in cows consuming
different levels of rumen degradable protein.
Key words: nitrogen-15, rumen degradable protein, sustainability, urea utilization
© 2013 American Society of Animal Science. All rights reserved. INTRODUCTION
The N content of perennial ryegrass-based pasture
frequently exceeds 4% of DM (Roche et al., 2009).
This surplus N results in low N-use efficiency (milk
N/N intake; NUE) in pasture-based systems and large
amounts of N are excreted to the environment (Litherland and Lambert, 2007). Direct measurement of
NUE requires knowledge of pasture DMI, which is dif1The project was supported by New Zealand dairy farmers through
DairyNZ Inc. (Project AN803), New Zealand Ministry of Primary
Industries through Sustainable Farming Fund (Project 08/012), and
New Zealand Ministry of Business, Innovation, and Employment
(Project DRCX 0802). Dr. A. M. Nicol and Dr. R. H. Bryant from
Lincoln University are thanked for providing useful discussion. Thanks
also go to DairyNZ Lye Farm technicians and Lincoln University
Analytical Laboratory technicians.
2Corresponding author: [email protected]
3Current address: Department of Animal Science, The University
of Tennessee, Knoxville, TN.
Received April 14, 2012.
Accepted September 6, 2013.
J. Anim. Sci. 2013.91:5785–5788
doi:10.2527/jas2012-5378
ficult to quantify under grazing. Although milk urea N
(MUN) and plasma urea N (PUN) concentrations generally correlate with each other and have been used to
predict NUE (Oltner et al., 1985), Reynolds and Kristensen (2008) suggested that urea N excretion reaches
a maximum when dietary N exceeds 3.2% of DM, reducing the sensitivity of using urea N to predict NUE
in grazing systems with high dietary N.
An alternative approach based on N isotopic fractionation is proposed to predict NUE. Nitrogen isotopic fractionation has been used to reflect dietary
changes and N metabolism in various animal species (Parker et al., 1995; Halley et al., 2010). Studies
with rats indicated a relationship between the level of
protein supplied and nitrogen isotopic fractionation
(δ15N) measured in plasma (Sick et al., 1997), with
isotopic fractionation during hepatic deamination
or transamination responsible for the relationship
(Macko et al., 1987). However, the situation is likely
to be more complex with ruminants compared with
monogastrics, as the rumen is an additional site for
5785
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Cheng et al.
isotopic fractionation through amino acid deamination
and protein synthesis by microbes (Wattiaux and Reed,
1995). The current study was conducted to determine if
NUE can be predicted in dairy cows fed pasture with
different levels of RDP using δ15N measures.
MATERIALS AND METHODS
This study was undertaken at DairyNZ, Lye Farm,
Hamilton, New Zealand, under the authority of the
Ruakura Animal Ethics Committee (Approval No.
11896). Fifteen multiparous, rumen-cannulated, Holstein-Friesian cows (103 ± 9.4 DIM and 505 ± 20.9 kg
BW, mean ± SD) were used. All cows were grazed on
a perennial ryegrass-based pasture and 10 cows were
gradually acclimated to full urea supplementation over
8 d before the beginning of the study. After the acclimation period, all cows stayed on full urea supplementation levels and were housed in metabolism stalls
for 4 d to adapt to the facilities before commencing a
5-d collection period. Freshly cut pasture was offered
twice daily at 0900 and 1600 h, throughout the study, to
allow at least 5% (DM based) refusal per day for each
cow. The study was a completely randomized design (5
cows/treatment) and treatments included supplementing with 3 levels of granular urea [0 g/d (U0), 250 g/d
(U250), and 336 g/d (U336); Biolab, Australia; 46.7%
N]. Equal amounts of urea were placed in the rumen
via the cannula and mixed with rumen digesta 3 times
daily at 0930, 1300, and 1630 h.
Prefeeding and postfeeding pasture samples were
collected from individual cows daily and pooled at
the end of the collection period. Pasture samples were
freeze dried, ground to pass through a 1.0-mm sieve
(Christy Lab Mill, Suffolk, UK), and analyzed for
CP, OM digestibility, and NDF by near-infrared spectroscopy (Corson et al., 1999). Metabolizable energy
was derived directly from predicted OM digestibility
on the basis of an in vitro cellulase digestibility assay (Roughan and Hollan, 1977; Dowman and Collins,
1982), which had been calibrated against in vivo standards (Corson et al., 1999).
Cows were milked twice daily. Milk yield (MY)
was recorded (Tru-Test milk meters; Tru-Test Ltd;
New Zealand) and milk was sampled for compositional
analysis. A 10-mL blood sample using a Li-heparinized
evacuated tube was collected from the jugular vein of
each cow at 1130 h daily. Plasma was harvested following centrifugation at 1,200 × g for 12 min at 4°C.
At the end of the collection period, all samples (urea,
milk, and plasma) were pooled per cow and stored at
–20°C until analyses were conducted.
Milk samples were analyzed for fat, N, and lactose
concentrations, using Fourier-transform infrared spec-
troscopy (FT120; Hillerød, Denmark). Milk CP concentration was calculated from milk N: CP = N × 6.38.
Urea N in plasma and milk, and plasma ammonia concentrations were quantified using a kinetic UV and colorimetric assay, respectively (Modular P800; Germany).
Pooled freeze-dried samples of urea, pasture, milk, and
plasma were ground through a 1-mm sieve and weighed
before 15N analysis, using an isotope ratio mass spectrometer (PDZ Europa Ltd.; Rudheath, Northwich,
Cheshire, UK). All results were expressed in delta units
(δ15N; ‰); this is the 15N/14N ratio in the sample relative to the 15N/14N ratio in the standard (air). Content of
δ15N in pasture and urea were analyzed separately and
total δ15N from feed (i.e., pasture plus feed urea) was
calculated using the formula: Feed δ15N = [(N intake of
pasture) × (δ15N of pasture) + (N intake of urea) × (δ15N
of urea)]/[N intake of pasture + N intake of urea].
GenStat statistical package (Version 13.3) was
used for ANOVA, multiple range test (Student–Newman–Keuls Test), and linear regression analysis. The
statistical model included the fixed effect of treatment
and the random effect of cow. Analyses of NUE and
N isotopic fractionation were conducted using a single
mean value for each cow. The significance of treatment
effect was declared at P < 0.05.
RESULTS AND DISCUSSION
Lower DMI (P = 0.002) and MY (P = 0.002) were
recorded with the highest urea supplementation (336 g
urea/d; Table 1) compared with the other two treatments
(0 and 250 g urea/d). This effect was not expected, as
previous studies reported no adverse effect of urea supTable 1. Effect of urea supplementation in addition to
fresh pasture1 on DMI, milk yield and composition,
N-use efficiency (milk N/N intake; NUE), plasma and
milk urea nitrogen concentration, and plasma ammonia
concentration of dairy cows
Item
DMI, kg/d
N intake, g/d
Milk yield, kg/d
Milk fat concentration, %
Milk N concentration, %
Milk lactose concentration, %
Milk N output, g/d
NUE, %
Plasma urea N, mmol/L
Milk urea N, mmol/L
Plasma ammonia, μmol/L
U0
U250
U336
SED
19.1b
18.7b
15.1a
0.93
510a
616b
543a
24.6
24.7b
22.7b
16.6a
1.79
4.17
4.33
4.24
0.454
0.52
0.56
0.53
0.033
5.04b
4.94b
4.67a 0.117
129b
126b
89a
7.0
25b
20b
16a
1.0
8.68a 11.90b 11.06b 0.590
8.20a 11.48b 12.30b 0.666
59.5a
64.4a 101.1b 12.17
P-value
0.002
0.003
0.002
0.940
0.477
0.020
<0.001
<0.001
<0.001
<0.001
0.01
a–cMeans with different superscripts are significantly different at the 5%
confidence level.
1Pasture plus urea supplementation: U0 (0 g urea/d); U250 (250 g urea/d);
U336 (336 g urea/d).
Nitrogen-use efficiency and isotopic fractionation in dairy cows
plementation on cow DMI up to 54% of total N intake
(NIT; 379 g urea/d; Mugerwa and Conrad, 1971). In this
study, U250 and U336 treatments represented urea supplementation of 18% and 29% of total NIT, respectively
(Table 1). The reason for the decline in DMI in the current study is not known. The possibility of ammonia toxicity was closely monitored, but cows did not show any
clinical signs and plasma ammonia results (Table 1) did
not indicate toxicity (Zhu et al., 2000). The lower DMI
may reflect an inherent safety mechanism to avoid toxicity. For example, Cocimano and Leng (1967) reported a
drop in DMI when cows reached a physiological limit
for renal excretion of urea. More research is needed to
quantify this physiological limit to enable better management of high RDP diets.
The average plasma and milk across 3 treatments
were enriched in δ15N by 3.68% and 3.76% compared
with the feed (Table 2), probably because the majority
of N in milk and plasma exists as true protein, which
is naturally enriched in δ15N (Sick et al., 1997; Cheng
et al., 2010). As feed δ15N decreased with urea supplementation, little change was detected in plasma
and milk δ15N across the dietary treatments (Table 2).
This resulted in an increased enrichment of plasma and
milk δ15N from U0 to U336 relative to feed (Table 2),
whereas NUE declined from 25 to 16% (Table 1).
There were strong negative relationships between
plasma δ15N – feed δ15N and NUE, and between milk
δ15N – feed δ15N and NUE in the current study (Fig. 1).
These results in dairy cows are consistent with earlier
reports in rats (Sick et al. (1997). Under the feeding regime investigated here, the relationships presented in
Fig. 1 are most plausibly due to isotopic fractionation
within the liver through deamination and/or transamination processes (Parker et al., 1995; Sick et al., 1997). It
is also worth noting that the relationships in Fig. 1 were
stronger than those between MUN and NUE (r2 = 0.63;
P < 0.001; SE = 0.65), and PUN and NUE (r2 = 0.31; P <
Table 2. Effect of urea supplementation in addition to
fresh pasture1 on δ15N content of plasma and milk, as
well as the differences in δ15N between milk and feed,
and plasma and feed for cows
Item
U0
U250
U336
SED P-value
Feed δ15N, ‰2
3.25c
2.83b
2.55a 0.018 <0.001
Milk δ15N, ‰
6.59
6.63
6.68
0.095
0.682
Plasma δ15N, ‰
6.44
6.57
6.67
0.120
0.197
Milk δ15N – feed δ15N, ‰
3.34a
3.80b
4.13c 0.096 <0.001
15
15
a
b
Plasma δ N – feed δ N, ‰
3.19
3.73
4.13c 0.121 <0.001
a–cMeans with different superscripts are significantly different at the 5%
confidence level.
1Pasture plus urea supplementation: U0 (0 g urea/d); U250 (250 g urea/d);
U336 (336 g urea/d).
2Feed δ15N = [(N intake of pasture) × (δ15N of pasture) + (N intake of
urea) × (δ15N of urea)]/(N intake of pasture + N intake of urea).
5787
Figure 1. Relationship between N-use efficiency (NUE) and N isotopic
fractionation in cows fed on fresh pasture and supplemented with urea (milk
δ15N – feed δ15N, closed symbols; plasma δ15N – feed δ15N, open symbols;
U0, ▲▲; U250,
G; U336,
).
■□
●●
NUE (%) = 58.9 – 10.17 × milk δ15N – feed δ15N (‰)
(n = 15, r2 = 0.83, P < 0.001, SE = 1.67)
NUE (%) = 52.4 – 8.61 × plasma δ15N – feed δ15N (‰)
(n = 15, r2 = 0.85, P < 0.001, SE = 1.56)
0.05; SE = 2.00). This is probably because urea N excretion reaches a maximum when dietary N exceeds 3.2%
of DM, reducing the sensitivity of using urea N to predict NUE in U250 and U336 treatments (Reynolds and
Kristensen, 2008). In conclusion, this study confirms
that isotopic fractionation has the potential for accurately predicting NUE for cows fed on pasture, without the
need to measure DMI. Further research is needed to field
test this technology in grazing situations.
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