Metabolomics (2010) 6:583–594
DOI 10.1007/s11306-010-0227-6
ORIGINAL ARTICLE
Metabolomics reveals unhealthy alterations in rumen metabolism
with increased proportion of cereal grain in the diet of dairy cows
Burim N. Ametaj • Qendrim Zebeli • Fozia Saleem •
Nikolaos Psychogios • Michael J. Lewis •
Suzanna M. Dunn • Jianguo Xia • David S. Wishart
Received: 4 February 2010 / Accepted: 25 June 2010 / Published online: 14 July 2010
Ó Springer Science+Business Media, LLC 2010
Abstract This study presents the first application of
metabolomics to evaluate changes in rumen metabolites of
dairy cows fed increasing proportions of barley grain (i.e.,
0, 15, 30, and 45% of diet dry matter). 1H-NMR spec-
troscopy was used to analyze rumen fluid samples repre-
senting 4 different diets. Results showed that for cows fed
30 and 45% grain, increases were observed in the con-
centration of rumen methylamine as well as glucose, ala-
nine, maltose, propionate, uracil, valerate, xanthine,
ethanol, and phenylacetate. These studies also revealed
lower rumen 3-phenylpropionate in cows fed greater
amounts of cereal grain. Furthermore, ANOVA tests
showed noteworthy increases in rumen concentrations of
N-nitrosodimethylamine, dimethylamine, lysine, leucine,
phenylacetylglycine, nicotinate, glycerol, fumarate, buty-
rate, and valine with an enriched grain diet. Using principal
component analysis it was also found that each of the 4
diets could be distinguished on the basis of the measured
rumen metabolites. The two closest clusters corresponded
to the 0 and 15% grain diets, whereas the 45% barley grain
diet was significantly separated from the other clusters.
Unhealthly levels of a number of potentially toxic metab-
olites were found in the rumen of cattle fed 30 and 45%
Electronic supplementary material The online version of this
article (doi:10.1007/s11306-010-0227-6) contains supplementary
material, which is available to authorized users.
B. N. Ametaj (&)  Q. Zebeli  S. M. Dunn
Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, AB T6G 2P5, Canada
e-mail: burim.ametaj@ualberta.ca
F. Saleem  N. Psychogios  M. J. Lewis  J. Xia  D. S. Wishart
Departments of Biological Sciences and Computing Science,
University of Alberta, Edmonton, AB T6G 2E9, Canada
e-mail: david.wishart@ualberta.ca
grain diets. These results may have a number of implica-
tions regarding the influence of grain on the overall health
of dairy cows.
Keywords Rumen metabolic profile  NMR  Dairy cow 
Principal component analysis  Hierarchical clustering
analysis  Barley grain
1 Introduction
Dairy cows have evolved through a close symbiotic rela-
tionship with a vast ensemble of rumen microbes (i.e., the
microbiota). These microbes give dairy cattle important
metabolic capabilities including the ability to digest cel-
lulose-rich feedstuffs and to convert them into a wide range
of compounds important for body maintenance and milk
production. Keeping a stable and healthy rumen microbiota
is also critical for maintaining healthy cows (Nocek 1997;
Emmanuel et al. 2008) and high milk quality (Jenkins and
McGuire 2006).
Today’s intensive dairy management systems encourage
the inclusion of large amounts of cereal grains in the diets
of dairy cattle to support high milk yields and enhance cost
efficiency. Barley grain, a cereal characterized by rapid
degradation of its starch in the rumen, is widely used in
Western Canada and elsewhere as one of the main energy
components in the diets of dairy cows (15–40% in diet dry
matter) and beef cattle (40–85%). Yet, these feeding
practices often lead to the accumulation of large amounts
of short-chained fatty acids (SCFA) and to the acidification
of rumen fluid. This often leads to a pathological condition
commonly referred to as acute or sub-acute rumen acidosis
(ARA or SARA; Nocek 1997). Moreover, feeding diets
rich in readily available carbohydrates is associated with
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major alterations to the rumen microbiota, which are
accompanied by significant changes to the rumen meta-
bolic pathways (Tajima et al. 2000; Khafipour et al. 2009).
Several lines of evidence support a role for altered
rumen metabolism, (due to the feeding of high grain/low
fiber diets) in the development of several pathologic con-
ditions including laminitis (Nocek 1997), fatty liver
(Ametaj et al. 2005), systemic inflammation (Emmanuel
et al. 2008), and milk fat depression syndrome (Jenkins and
McGuire 2006; Zebeli and Ametaj 2009). Although the
interrelationships of rumen metabolism and host health
status are widely accepted, it is not yet clear which of the
compounds generated by the activity of rumen microbiota,
under conditions of diet-induced stress (i.e., high grain
feeding), are significant to a cow’s health. Moreover, little
is known with respect to microbial-mediated changes in
rumen metabolism during the feeding of graded propor-
tions of cereal grain in the diet. So far, most conventional
studies addressing rumen metabolism, have focused on
dietary effects on a single class of rumen compounds, such
as SCFA, and have not attempted a comprehensive meta-
bolomic characterization of the rumen of dairy cattle.
Metabolomics is an emerging field of ‘‘omics’’ science
that uses high-throughput approaches, such as 1H-NMR
spectroscopy, coupled with multivariate analysis to extract
comprehensive metabolic information and measure meta-
bolic phenotypes in mammals, plants, and microbes
(Vinayavekhin et al. 2010). Metabolomics has opened new
perspectives in the field of nutrition research, allowing
scientists to explore the complex metabolic pathways in
response to diet (Wishart 2008a). To our knowledge, only a
few studies have used modern metabolomics techniques to
address nutritional issues in ruminant animals. One study
looked at the effects of intra-ruminal infusion of SCFA in
non-lactating heifers (Bertram et al. 2005), another study
investigated the metabolism of rumen epithelial tissue of
newborn calves (Bertram et al. 2009), and a third study
used metabolomics to look at the quality of marketed milk
(Boudonck et al. 2010). Recent research has also shown
that metabolomics can be used to characterize metabolic
profiles at the gut level. These studies allow one to explore
the symbiotic complexity between the host and its micro-
biota as affected by diet, and to unravel the effects of diet
on the development of metabolic diseases (Dumas et al.
2006). Given this potential we hypothesized that the use of
metabolomics would provide a more comprehensive view
on how rumen metabolism changes during the feeding of
graded proportions of cereal grain. Such a study could
improve our understanding of diet-related rumen metabo-
lism and give further insight into metabolite-disease asso-
ciations in dairy cattle. For this investigation we used
1
H-NMR spectroscopic profiling along with multivariate
statistics to determine the influence of feeding graded
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B. N. Ametaj et al.
amounts of barley grain on rumen metabolite patterns in
lactating dairy cows.
2 Materials and methods
All experimental procedures were approved by the Uni-
versity of Alberta Animal Care and Use Committee for
Livestock, and animals were cared for in accordance with
the guidelines of the Canadian Council on Animal Care
(1993). Data related to concentration of endotoxin and pH
of the rumen fluid, plasma acute phase proteins, and milk
production for this experiment have been reported previ-
ously (Emmanuel et al. 2008; Zebeli and Ametaj 2009). In
this manuscript, we reinterpret previous data collected on
rumen pH and endotoxin in the context of the metabolomic
profile changes evaluated in this research.
2.1 Animals and diets
Eight clinically healthy, rumen-fistulated primiparous
Holstein cows (60 ± 15 days in milk) at the University of
Alberta’s Dairy Research and Technology Centre were
used in this study. The original experimental design of this
research was a replicated 4 9 4 Latin square design with
four 21-day periods (Emmanuel et al. 2008; Zebeli and
Ametaj 2009). However, because of the complexity of the
study only the rumen samples collected during the first
experimental period were used in this report. The first
11 days were used for adaptation to the experimental diets,
and the last 10 days for collection of rumen fluid samples.
Based on previous similar feeding experiments, an adap-
tation period of 11 days was considered sufficient to allow
wash-in of the experimental diets before the measurements.
Cows were fed one of the total mixed rations (TMR)
containing increasing proportions of rolled barley grain
(i.e., 0, 15, 30, or 45% on a dry matter basis). Thus, 2
different cows were randomly allocated to each diet. All
diets were formulated to meet the nutrient requirements of
a 680 kg lactating cow as per NRC (2001) guidelines.
Ingredients and nutrient composition of the 4 TMR are
presented in the Supplementary Table 1.
2.2 Rumen fluid sample collection
For this study, samples from rumen fluid (100 mL) were
obtained on days 1, 3, 5, 7, and 10 of the measurement
period (i.e., days 12, 14, 16, 18 and 21 of the experiment) at
0800 h. All rumen fluid samples were collected through the
cannula using a tube fitted with a strainer and a syringe into
a 140-mL plastic container. The pH of the rumen fluid was
determined immediately after collection by a mobile pH
meter (Accumet AP61, Fischer Scientific, Ottawa, Ontario,
Rumen metabolomics
Canada). Subsequently, rumen fluid samples were centri-
fuged at 6,0009g for 15 min (Rotanta 460 R, Hettich
Zentrifugan, Tuttlingen, Germany), and the supernatant
was stored at -20°C until it was analyzed for metabolite
and endotoxin content.
2.2.1 Rumen fluid sample preparation for NMR
spectroscopy
Rumen samples were thawed at room temperature and
centrifuged at 10,000 rpm for 140 min to remove macro-
molecules (fine feed particles and microbiota). The super-
natant was collected and passed through 0.2 lM syringe
filters (Fischer Scientific, Fairlawn, NJ). Subsequently,
35 lL of D2O and 15 lL of a standard buffer solution
(0.16 mM DSS [disodium-2,2-dimethyl-2-silapentane-5-
sulphonate], 10 mM imidazole, and 0.02% NaN3 in H2O)
were added to 300 lL of filtered rumen sample. Subse-
quently, these samples (350 lL) were transferred to a
standard Shigemi microcell NMR tube for subsequent
NMR spectral analysis.
2.3 1H-NMR spectrum acquisition
All 1H-NMR spectra were collected on a 500 MHz Inova
(Varian Inc., Palo Alto, CA) spectrometer equipped with
either a 5 mm HCN Z-gradient pulsed-field gradient (PFG)
room-temperature probe or a Z-gradient PFG Varian cold-
probe. 1H-NMR spectra were acquired at 25°C using the
first transient of the tnnoesy-presaturation pulse sequence,
which was chosen for its high degree of quantitative
accuracy (Saude et al. 2006). Spectra were collected with
64 transients and 4 steady state scans using a 4 s acquisi-
tion time and a 990 ms solvent presaturation. A spectral
width of 5,997 Hz was used for collecting 24 k data
points. Spectra were processed using the Processor mod-
ule of Chenomx NMR Suite 6.0. The free induction
decays (FIDs) were zero filled to 64 k in the frequency
domain, and multiplied by an exponential weighting
function corresponding to a line broadening (0.5 Hz)
before Fourier transformation. Spectra were referenced to
DSS (0.0 ppm) and manually corrected for phase and
baseline distortions.
2.4 Sample fitting
Processed spectra were imported into the Chenomx
NMRSuite 6.0 software for quantification (Weljie et al.
2006; Wishart 2008b). In total, 46 compounds were
quantified from each spectrum. Briefly, test spectra were
chosen and concentrations for each compound were aver-
aged over the test profiles. These averages were used as the
starting concentration vector for fitting or refitting all the
585
acquired spectra. All spectra were randomly ordered
(within acquisition batches) for spectral fitting using
Chenomx Profiler. For each spectrum, the compounds
identified were sorted by decreasing concentration and then
fit for concentration and translation in that order. The three
test profiles were compared with the corresponding profiles
to test for consistency. Each compound concentration was
then normalized by dividing the measured concentration
into the total concentration of all metabolites in that sample
(Weljie et al. 2006). We also used sample spiking to con-
firm the identity of several spectral signatures seen in our
NMR spectra. This was conducted by adding 50–500 lM
of the presumptive compound to selected rumen samples to
test if the corresponding 1H-NMR signals changed as
expected.
2.5 GC-MS compound identification
An additional step for compound identification was per-
formed, by selecting 2 rumen spectra from 0% grain diet for
Gas Chromatography-Mass Spectrometry (GC-MS) analy-
sis. The samples were extracted separately to obtain separate
pools of polar and lipophilic metabolites using previously
reported protocols (Wishart et al. 2008). Derivatized polar
extracts and lipophilic extracts were analyzed using an
Agilent 5890 Series II GC-MS instrument operating in an
Electron Impact (EI) ionization mode. For GC-MS analysis,
2 lL of extract were injected using a split/splitless injector
with a split ratio 5:1 onto a DB-5 capillary column (Agilent
J&W Scientific, 30 m 9 250 lm 9 0.25 lm). The injector
port temperature was held at 250°C and the helium carrier
gas flow rate was set to 1 mL/min at the initial oven tem-
perature of 50°C. The oven temperature was increased at
10°C/min to 310°C for a final run time of 26 min. The full
scan mode of the quadrupole MS was used at a mass range of
50–500 m/z, with a solvent delay of 6 min. The MS ion
source temperature was set to 200°C.
The AMDIS spectral deconvolution software (Version
2.62) from NIST (National Institute of Standards and
Technology) was used to process the total ion chromato-
gram and the EI-MS spectra of each GC peak. After
deconvolution, the purified mass spectrum of each of the
trimethylsilylated metabolites was identified using the
NIST MS Search program (version 2.0d) which was linked
to the NIST mass spectral library (2008). In AMDIS, each
search produces a list of library spectra (‘‘hits’’), which is
ranked by the similarity to the target spectrum according to
a mathematically computed ‘‘match factor’’. The match
factor indicates the likelihood that our spectrum and the
reference NIST spectrum arose from the same compound.
In the current case, we considered hits with a match factor
of[60% and a probability[20%. These cutoff values have
been shown to yield consistent results with synthetic
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mixtures containing known compounds and with samples
independently analyzed by NMR. Retention Indices (RIs)
were calculated using a C8-C20 alkane standard solution
(Fluka, Sigma-Aldrich).
2.6 Rumen endotoxin
Concentration of cell-free endotoxin in the rumen fluid
supernatant was determined by the pyrochrome Limulus
amebocyte lysate assay (Associates of Cape Cod Inc., East
Falmouth, MA), as described previously (Emmanuel et al.
2008). Briefly, 10 mL of rumen fluid samples were cen-
trifuged at 6,0009g for 15 min and the supernatant stored
at -20°C until analysis. For the assay, the supernatant was
thawed and 1.5 mL centrifuged at 10,0009g for 30 min.
All samples were tested in duplicate, and the optical den-
sity values were read on a microplate spectrophotometer
(Spectramax 190, Molecular Devices Corporation, Sunny-
vale, CA) at a wavelength of 405 nm. The inter- and intra-
assay CV were controlled to limits of \10%.
2.7 Statistical analyses
Parametric analysis of the data was conducted using the
MIXED procedure provided by SAS (SAS Institute Inc.,
Cary, NC, Version 9.1.3). The model included the fixed
effect of treatment and the ‘‘random’’ effect of the individual
cow. The measurement day was considered as a repeated
measure with a first-order autoregressive covariance struc-
ture, which assumes that the covariance of measurements for
the same animal decays with time (Littell et al. 1998).
Instead of raw P values, the Bonferroni correction and the
false discovery rate (FDR) were applied to counter the effect
of multiple hypothesis testing, since they are considered
more appropriate for analyzing the multivariate results
found in metabolomic data (Broadhurst and Kell 2006).
For multivariate analysis, data were imported into the
MetaboAnalyst software (http://www.metaboanalyst.ca;
Xia et al. 2009). Principal component analysis (PCA),
which is an unsupervised pattern recognition method, was
performed to examine the intrinsic variation in the NMR
data set (39 in total; n = 9 for diet 0% grain, and n = 10
for the other dietary treatments), and to reduce the
dimensionality of the data. A score plot was used to show
the similarities and differences among the data sets. In a
score plot the data sets exhibiting similarities are clustered
together and those that are different are placed further
apart. The loadings plot shows the variables responsible for
the variation within the dataset, and the correlations among
individual rumen fluid metabolites corresponding to the
first two eigenvalues (i.e., PC 1 and PC 2). Sample nor-
malization against baseline concentration at 0% grain diet
was done by first creating a dummy baseline sample by
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B. N. Ametaj et al.
averaging all the nine samples collected at 0%. After this
normalization step, all samples were normalized against
this reference sample using probabilistic quotient normal-
ization procedures (Dieterle et al. 2006; Xia et al. 2009).
The compound normalization was conducted using auto-
scaling, which made all compound variance equal to 1.
Hierarchical clustering analysis (HCA) with Euclidean
distance measures and an average linkage method was
performed to explore the presence of clustering patterns
among the rumen fluid metabolites. Pretreatment of the
data in HCA included data normalization (x - m/std),
where x denotes given values, m is the mean, and std
indicates the standard deviation of a certain variable, as
well as conversion to log base 2 ratios. The expression
patterns and a heat map of each variable were categorized
using an average linkage hierarchical clustering program
(Seo 2005).
3 Results
3.1 Outcome of ANOVA tests
Results of the ANOVA tests with Bonferroni and FDR
corrections with regard to metabolites measured in the
rumen fluid are presented in Table 1. Our data showed that
feeding dairy cows increasing proportions of barley grain
was associated with statistically significant increases of
several rumen metabolites including methylamine (MA),
glucose, alanine, maltose, uracil, xanthine, and phenylac-
etate. Moreover, the analysis of variance indicated greater
levels of rumen endotoxin and acetate. ANOVA also
indicated potential increases in the concentrations of pro-
pionate, ethanol (EtOH), valerate, N-nitrosodimethylamine
(NDMA), dimethylamine (DMA), lysine, leucine, pheny-
lacetylglycine (PAG), nicotinate, glycerol, fumarate,
butyrate, and valine, whereas rumen fluid pH was lower
with increasing dietary grain (Table 1). On the other hand,
greater proportions of barley grain in the diet resulted in
lower concentrations of 3-phenylpropionate (3-PP) in the
rumen fluid.
3.2 Multivariate analysis of rumen metabolites
Typical 1H-NMR spectra of rumen fluid corresponding to
diets with 0, 15, 30, and 45% barley grain are shown in
Fig. 1. All NMR spectra were collected on samples
obtained on day 18 of the experiment (day 7 of the mea-
surement period). Although there were some day-to-day
differences in the responses of rumen metabolomic profile,
the day 18 spectra were considered as typical responses of
the cows to the different diets due to relatively long time
that the cows were under the feeding regimen.
Rumen metabolomics 587

Table 1 Concentrations of rumen metabolites and endotoxin measured in rumen fluid of lactating dairy cows fed graded amounts of barley grain
Itemb Barley grain proportion (% of diet dry matter) SEMc Statisticsa
0 15 30 45 BON FDR

Metabolite concentrations (lM, unless otherwise stated)

1,3-Dihydroxyacetone (1,3-D) 15.6 7.2 7.9 6.4 5.64 1.00 0.731
3-Hydroxybutyrate (3-HB) 44.8 60.6 46.7 77.1 11.8 0.96 0.517
3-Hydroxyphenylacetate (3-HP) 36.1 56.7 45.7 42.9 6.88 0.51 0.499
3-Phenylpropionate (3-PP) 446 396 322 328 29.2 0.04 0.031
Acetate (mM) 47.6 43.8 41.4 56.5 2.04 \0.01 0.003
Acetoacetate 49.5 47.1 54.1 58.0 10.1 1.00 0.914
Alanine 197 212 397 415 26.8 0.10 0.040
Aspartate 157 164 201 123 23.1 0.65 0.522
Benzoate 36.9 37.4 53.9 38.2 6.22 0.69 0.396
Butyrate (mM) 7.97 6.89 6.42 19.3 1.22 0.40 0.280
Cadaverine 73.2 56.2 93.6 110 19.7 1.00 0.639
Caffeine 15.3 10.8 15.7 13.7 3.36 1.00 0.928
Choline 17.2 14.7 16.9 19.8 5.49 1.00 0.943
Dimethylamine 4.36 7.59 29.5 33.7 10.8 0.77 0.344
Ethanol 131 176 203 341 50.9 0.49 0.357
Ferulate 4.41 11.8 3.91 11.9 2.88 0.71 0.439
Formate 321 337 320 258 36.7 1.00 0.950
Fumarate 1.64 5.82 1.42 6.44 0.57 0.33 0.255
Glucose 400 580 720 2090 120 \0.01 0.002
Glutamate 317 342 442 425 62.3 1.00 0.589
Glycerol 93.9 99.1 148 170 26.7 0.77 0.410
Glycine 104 105 158 150 24.9 1.00 0.470
Histidine 31.8 29.7 35.2 24.8 4.35 1.00 0.641
Hypoxanthine 87.9 134 219 220 59.9 1.00 0.538
Isobutyrate 1280 1283 1216 1430 121.8 1.00 0.723
Isoleucine 144 124 222 148 29.9 0.47 0.307
Isovalerate 891 720 932 998 98.1 0.82 0.539
Lactate 124 122 228 133 28.5 0.34 0.149
Leucine 109 99.2 164 155 19.8 0.92 0.442
Lysine 116 105 125 197 24.3 0.41 0.246
Maltose 49.9 53.7 51.7 124 21.1 0.19 0.072
Methanol 24.1 22.9 28.0 71.9 23.8 1.00 0.512
Methylamine 34.3 136 202 608 45.8 \0.01 0.001
N-nitrosodimethylamine (NDMA) 67.9 49.3 86.8 97.0 9.87 0.22 0.207
Nicotinate 21.5 24.7 35.7 32.2 3.60 0.32 0.220
Phenylacetate 241 363 371 443 33.6 0.01 0.010
Phenylacetylglycine (PAG) 62.1 55.0 80.1 88.7 11.4 0.38 0.305
Proline 130 117 165 141 30.2 1.00 0.808
Propionate (mM) 15.0 17.9 18.0 27.6 2.45 0.12 0.100
Ribose 237 238 298 219 46.4 1.00 0.846
Succinate 82.0 84.5 103.6 80.7 15.4 1.00 0.827
Tyrosine 46.5 41.3 52.2 50.6 8.90 1.00 0.902
Uracil 92.6 149 193 224 16.2 \0.01 0.026
Valerate (mM) 1.41 2.03 2.51 2.70 0.30 0.26 0.355
Valine 109 109 166 173 25.2 0.91 0.291
Xanthine 68.7 101 137 155 8.79 \0.01 0.002

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Table 1 continued
Itemb Barley grain proportion (% of diet dry matter) SEMc Statisticsa
0 15 30 45 BON FDR

Endotoxin (mg/L) 0.64 0.81 6.31 7.90 0.80 \0.01 0.001

Rumen pH 6.81 6.70 6.85 6.52 0.08 0.32 0.210
a
BON Bonferroni correction, FDR false discovery rate (Broadhurst and Kell 2006); Significances were considered at p = 0.05
b
Rumen fluid for the analysis was collected shortly before the morning feeding, and the cows were fed once daily at 0800 h
c
SEM standard error of the mean

Fig. 1 Typical 500 MHz 1H-NMR rumen fluid spectra (0–9 ppm)
from dairy cows fed 0% (a), 15% (b), 30% (c), and 45% (d) barley
grain. Numbers indicate metabolites, as follows: 1, acetate; 2,
propionate; 3, butyrate; 4, valerate; 5, isovalerate; 6, DSS; 7,
3-phenylpropionate; 8, alanine; 9, isoleucine; 10, glucose; 11, proline;
12, isobutyrate; 13, phenylacetate; 14, glycine; 15, cadaverine; 16,
aspartate; 17, ethanol; 18, leucine; 19, succinate; 20, glutamate; 21,
methylamine; 22, lactate; 23, choline; 24, fumarate; 25, acetone; 26,
isopropanol; 27, 3-hydroxybutyrate; 28, acetoacetate; 29, 3-hydroxy-
phenylacetate; 30, N-nitrosodimethylamine; 31, methanol; 32, valine;
33, lysine; 34, dimethylamine; 35, glycerol; 36, maltose; 37,
imidazole; 38, formate; 39, uracil; 40, nicotinate; 41, tyrosine; 42,
benzoate; 43, histidine; 44, hypoxanthine; 45, phenylacetylglycine;
46, xanthine; 47, ribose. The intensity of the aromatic region
(5.1–9.0 ppm) is 4 times higher than that of the aliphatic one
(0.0–4.2 ppm)

Figure 2a shows the score plot of the first two principal
components (PCs), extracted in this study. The 4 ellipses
indicate 95% bivariate normal ellipses that summarize the
distribution of the principal component scores for the 4
different diets. Inspection of this figure shows clear clusters
corresponding to cows fed different amounts of barley
grain, which appeared to be well separated by both PC 1
and PC 2. The ellipses corresponding to the responses of
diets with 0 and 15% grain strongly overlapped with each
other. However the responses of cows fed 45% grain are
clustered further apart from those corresponding to diets 0
and 15%, whereas the cluster of cows with 30% grain in
their diet is located between the clusters corresponding to
diets with 0, 15, and 45% grain (Fig. 2a). As shown in
the responses of specific cows at 5 different sampling days,
the PCA revealed certain inter-day variation, whereby the
responses of the last day (day 10) of the measurement
period were clustered closer to each other, near the lower
central region of the graph (Fig. 2a).
Loading plots, showing the individual rumen fluid
metabolites responsible for the variation of the first two
eigenvalues (PC 1 and PC 2), are given in Fig. 2b. This

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Fig. 2 Principal component analysis (PCA) of the rumen metabolo-
mic profile. a The PCA score plot distinguishes the metabolic profiles
of rumen fluid in cows fed 0% (open triangle), 15% (plus), 30%
(times), and 45% (open diamond) rolled barely grain in the diet. The
first number in the data points represents diet (0, 15, 30, and 45), the
second is about the cow number (1–8 cows), and the third indicates
sampling day (1, 3, 5, 7, and 10). b Loading plot showing the
correlations among individual rumen fluid metabolites of the first two
eigenvalues (PC 1 and PC 2). This gives a graphical representation of
the extent to which each factor accounts for the variance in the data
and shows the relationship between the different metabolites.
Variables with the same distance from 0 with similar positions are
positively correlated. Those in the opposite direction are negatively
correlated. For abbreviations of rumen fluid metabolites see Table 1
589
gives a graphical representation of the extent to which each
factor accounts for the variance in the data and shows the
relationship between the different rumen variables. Deter-
minants with the same distance from 0 and with similar
directions are positively correlated. Those in the opposite
direction are negatively correlated. The first PC essentially
shows a contrast between amino acids and glucose degra-
dation products as well as rumen pH. The second PC
highlights the contrast between the primary amines and
branched chain fatty acids and aspartate (Fig. 2b). The
metabolite methylamine (MA) appears to play an important
role in the separation along the PC 2.
The results of hierarchical clustering analysis (HCA) are
presented in Fig. 3. This dendrogram shows the presence of
different sub-clusters having different numbers of metab-
olites with various degrees of similarity. The responses of
each variable, to four different diets, are indicated with
changes in the color intensity on the heatmap. The vari-
ables with higher similarity are positioned near the top of
the dendrogram. Interestingly, the HCA revealed the
presence of one cluster consisting of the amino acids gly-
cine, valine, leucine, glutamate, and isoleucine as well as
lactate and a semi-volatile organic compound such as
NDMA. Another cluster included the branched chain fatty
acids isobutyrate and isovalerate as well as tyrosine and
phenylacetate. A distinct feature of the HCA was that
rumen metabolites positioned on the top (i.e., from glycine
to succinate) of the heat map showed a much more variable
response, whereas the cluster that included metabolites
from alanine to butyrate showed a clear increase with
increased grain in the diet. Except for 3-PP that decreased
with increased dietary grain, the heat map for the remain-
ing metabolites (i.e., from ferulate to rumen pH) seemed to
be independent of the amount of dietary grain (Fig. 3).
4 Discussion
In this study 1H-NMR-based metabolomics was used to
evaluate whether feeding different proportions of grain in
the diet would affect rumen metabolism. We also wanted to
investigate whether the observed metabolite changes could
give further insight into the role of grain feeding on the
etiopathology of diet-related diseases in dairy cows.
Our results clearly showed that these four different grain
diets could be easily distinguished on the basis of rumen
metabolites. Our data demonstrated that cows fed on 45%
barley grain had greater concentrations of MA, endotoxin,
propionate, glucose, and EtOH in their rumen fluid. On the
other hand, cows fed 0 and 15% barley grain fell into two
similar clusters characterized by lower concentrations of
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590 B. N. Ametaj et al.

Fig. 3 Hierarchical clustering

analysis for different rumen
fluid metabolites measured
before morning feeding in dairy
cows fed four different diets
(0, 15, 30, and 45% barley grain
inclusion in dry matter). The
expression patterns of each
parameter (shown in each row)
were categorized by an average
linkage hierarchical clustering
program (Seo 2005). The bars
indicate the normalized fold
change levels, with lighter grey
boxes denoting an expression
ratio greater than the mean and
darker grey boxes denoting an
expression ratio below the
mean. White boxes denote an
intermediate level. The tree
clusters and their shorter
Euclidean distance indicate
higher similarities. Similarity
between two metabolites is
represented by branch height,
therefore the lower a node is
vertically, the more similar its
sub-tree. Minimum similarity is
63%

PAG, xanthine, NDMA, acetate, and leucine and greater
3-PP. The potential health implications of altered metab-
olite levels in rumen fluid, due to different proportions of
barley grain in the diet, are discussed further below.
One of the most interesting observations from this study
was a multi-fold increase in the concentrations of methyl-
amine (MA) in the rumen of dairy cows fed with the
highest proportions of grain (i.e., 30 and 45%). To our
knowledge, this is the first report of such an association
between grain consumption and MA levels. Several
investigators have shown that bacterial decarboxylation of
amino acids such as glycine, tyrosine, and phenylalanine
(Davis and De Ropp 1961; Hill and Mangan 1964) and the
degradation of plant lipids, rich in choline (Hill and
Mangan 1964; Neill et al. 1978), contribute to the pro-
duction of methylated amines in ruminant animals. In
addition, research conducted by Hill and Mangan (1964)
indicated that an additional contributing factor in
production of methylated amines is the presence of an
acidic media. Indeed, the data from our study showed that
cows fed 30 and 45% barley grain had a rumen pH below
5.8 during 6–12 h post-feeding (data not shown). This pH
is at levels typically seen during SARA. The importance of
methylated amines to the health of dairy cows is related to
the fact that if they are absorbed into the blood circulation
they might be degraded by semicarbazide-sensitive amine
oxidases. This degradation process leads to the production
of harmful metabolites such as hydrogen peroxide and
formaldehyde, both of which are toxic and both of which
are associated with diseases such as diabetes, vascular
disorders, heart failure, and Alzheimer’s disease in humans
(Yu et al. 2006). To our knowledge, there is no information
about the role of methylated amines in the etiopathology of
metabolic diseases in dairy cows. It would be of interest to
explore their role on health issues of dairy cows. Intrigu-
ingly, Hill and Mangan (1964) and Neill et al. (1978)

123
Rumen metabolomics
demonstrated that methylated amines are converted into
methane by rumen microorganisms. Methane is a green-
house gas that contributes to atmospheric pollution. It is
well-known that a variety of factors, including the level
and the nature of dietary carbohydrate fermented in the
reticulorumen and the ratio between acetate and propio-
nate, affect the amount of methane emitted from cattle
(Johnson and Johnson 1995). Our data suggest that meth-
ylated amines might be an additional source of methane
that arises from the feeding of high-grain diets.
Although the concentration of NDMA was not signifi-
cantly different, explained by low statistical power, data
analysis indicated that cows fed the higher proportions of
barley grain (i.e., 30 and 45%) potentially had greater
concentrations of NDMA (N-nitrosodimethylamine).
NDMA is a semi-volatile compound released in the rumen
fluid due to the interaction of ingested nitrites or nitrates
with amines such DMA and TMA (Lijinsky 1999). Gas-
trointestinal NDMA has been reported to be a strong
hepatotoxic compound in sheep (Koppang 1964) and is
known as a powerful animal carcinogen capable of
inducing benign and malignant tumors in a variety of tis-
sues including liver, kidney, lung, and the nasal cavity
(Souliotis et al. 2002). The reason for elevated levels of
NDMA in the rumen fluid of cows fed higher proportions
of grain is not well understood presently; however, our
HCA revealed presence of a metabolite cluster that inclu-
ded NDMA and the amino acids glycine, valine, leucine,
glutamate, isoleucine as well as lactate. This association
suggests that higher concentrations of NDMA might be
related to microbial degradation of the aforementioned
amino acids and lactate. Indeed, Davis and De Ropp (1961)
reported that feeding rats with levels of glycine doubled the
amount of MA (a precursor to both DMA and NDMA)
excreted in the urine. Interestingly, Hashimoto et al. (1975)
reported that only pathogenic bacteria from human, rat, and
sheep intestines were able to catalyze the formation of
NDMA from DMA and nitrate in vitro. This suggests that
the feeding of large amounts of cereal grains to dairy cattle
might create favourable conditions for the growth and
activity of pathogenic bacteria in the rumen.
Our results also indicated a more than 2-fold increase,
although not statistically significant, in the amount of EtOH
in the rumen fluid when cows were fed 45% barley grain in
the diet. This is in agreement with previously reported data
on ruminants (sheep) that indicate that overfeeding with
wheat grain is associated with EtOH concentrations as high
as 8 mM (Allison et al. 1964). Kristensen et al. (2007) also
reported rumen EtOH concentrations above 2 mM in dairy
cows fed a TMR containing *54% corn silage and
*13.5% rolled barley grain. Moreover, Kristensen et al.
(2007) also reported that the EtOH concentration in rumen
fluid increased 5-fold within 1–3 h after the morning
591
feeding. In our study the amount of EtOH in the rumen
fluid of cows reached the greatest concentration (i.e.,
341 lM) on a 45% barley grain diet. The lower concen-
tration of rumen EtOH in our cows, compared to previous
reports, might be due to collection of rumen samples prior
to the morning feeding. Note that the cows in our experi-
ment were fed only once per day, at 0800. Although there
are no indications for the involvement of EtOH in the
etiopathology of metabolic disease in dairy cows, human
research implicates EtOH in multiple pathological states
(Estruch et al. 1993). Intriguingly, Enomoto et al. (2001)
showed that chronic exposure to EtOH increases gut per-
meability and portal vein translocation of endotoxin.
Additionally, research conducted by Pruett and Pruett
(2006) demonstrated that EtOH was not able to initiate an
acute phase response in C3H/HeJ (TLR-4 mutant) mice,
indicating that the response is mediated through the
endotoxin receptor, TLR-4. We also reported that cows fed
with larger amounts of barley grain (30 and 45% diet DM)
developed an inflammatory state with greater plasma con-
centrations of serum amyloid A, lipopolysaccharide-bind-
ing protein, and C-reactive protein (Emmanuel et al. 2008).
Recently, Khafipour et al. (2009) suggested an unknown
physiological factor that might affect gastrointestinal bar-
rier functions and facilitate translocation of endotoxin into
blood circulation and initiate an inflammatory response
during feeding of high-grain diets. It is postulated that
rumen EtOH might be the agent that facilitates transloca-
tion of endotoxin through rumen and colon walls
contributing to endotoxin-related disorders during feeding
of high-grain diets to dairy cows.
Another interesting cluster of metabolites that was
associated with high-grain diets and deserves further dis-
cussion was that of xanthine, uracil, alanine, and endo-
toxin. All four of these metabolites were elevated with
increasing amounts of dietary grain. Interestingly, all four
metabolites are degradation products of rumen bacteria.
McAllan and Smith (1973) showed that bacterial nucleic
acids (i.e., RNA or DNA) incubated with rumen fluid were
rapidly converted into xanthine, hypoxanthine, and uracil.
It is also known that the death of both Gram-negative and
Gram positive-bacteria is associated with the release of
endotoxin and alanine (Trent et al. 2006). It is well
established that the decrease in rumen pH in dairy cows fed
high-grain diets is associated with significant changes in
microbiota composition. Many microbial species are not
able to survive the stress of a low pH and lack of nutrients
(Slyter 1976). For example, Khafipour et al. (2009)
reported a decrease in the number of Gram-negative Bac-
teroidetes in rumen as a consequence of a high-grain diet.
Therefore, the increased concentrations of xanthine, uracil,
alanine, and endotoxin in the rumen fluid of cows fed high-
grain diets appear to be biomarkers of widespread bacterial
123
592
cell lysis and a fundamental change in the rumen
microbiota.
There are also two other mechanisms to explain the
higher concentration of alanine in rumen fluid. The first
one might be related to greater availability of glucose and
pyruvate due to the degradation of larger amounts of
starch, coupled with higher levels of ammonia. This
assumption is supported by data of Örlygsson et al. (1995),
who determined that alanine is produced via an amination
of pyruvate via a transamination reaction coupled to ala-
nine aminotransferase. This suggests that the presence of
high levels of pyruvate and ammonia favour the production
of alanine by rumen bacteria (Örlygsson et al. 1995). The
second reason for higher alanine content might be related
to the fact that feeding high amounts of readily available
carbohydrates to cattle is associated with lowering of
rumen pH and the sporulation of certain rumen bacteria to
survive the adverse conditions (Rieu-Lesme et al. 1996).
Sporulated bacteria use L-alanine to germinate; however
when rumen conditions are not favourable for growth they
convert L-alanine to D-alanine to postpone germination.
This process is known as autoinhibition (Gould 1970). D-
alanine is used by both Gram-negative and Gram-positive
bacteria in the biosynthesis of their cell wall components
(peptidoglycan and lipoteichoic acid) (Hoogenraad and
Hird 1970). It would be of interest to further explore the
latter hypothesis. To our knowledge, there are no known
health implications for high levels of alanine in the rumen
or gastrointestinal tract of dairy cows or other livestock
animals.
Our study showed opposing responses of 3-PP and
phenylacetate to the amount of dietary grain in the diet.
Thus, the concentration of 3-PP linearly decreased with
increasing proportions of grain in the diet, whereas an
opposite effect was observed with regards to rumen
phenylacetate. Research conducted in forage-fed sheep has
shown that the latter two compounds are the predominant
aromatic acids, with 3-PP and phenylacetate accounting for
about 50.8 and 13.5% of total aromatic acids in the rumen
fluid, respectively (Pagella 1998). Our results demonstrate
that the ratio between these aromatic compounds can be
changed by increasing the amount of grain in the diet.
In the rumen, 3-PP and phenylacetate are generated by
the activity of ruminal microbiota on plant constituents,
namely via the hydrogenation of phenolic compounds such
as p-coumaric, ferulic, and caffeic acids, followed by a
dehydroxylation process (Chesson et al. 1999). The con-
centration of ferulate in the rumen fluid did not change in
this study, and was generally low. This could be attributed
to the time of sampling, which was always done before the
morning feeding. An alternative origin of 3-PP and phen-
ylacetate is also possible via the deamination of aromatic
amino acids such as tyrosine and phenylalanine (Martin
123
B. N. Ametaj et al.
1982; Burlingame and Chapman 1983). Interestingly, our
HCA revealed the formation of a sub-cluster among
phenylacetate, tyrosine, and the branched chain fatty acids
isobutyrate and isovalerate. These branched chain fatty
acids are also synthesized from deamination of the amino
acids valine, isoleucine, leucine, and proline (Andries
et al. 1987). On the other hand, 3-PP formed a cluster
with benzoate and 3-HP, suggesting the presence of dif-
ferent origins of 3-PP and phenylacetate in the rumen of
cows fed with increasing amounts of barley grain. Over-
all, our data indicated that deamination processes for
various amino acids were favoured in the rumen for cows
on high grain diets, and this is reflected by a strong
positive association between high-grain diets and rumen
phenylacetate. The low concentration of 3-PP in a grain-
enriched diet might also be related to the changes in the
rumen microbiota and the availability of other compounds
in the rumen fluid. For example, Turlin et al. (2001)
demonstrated that the expression of genes coding for key
enzymes involved in 3-PP production such as 3-phenyl-
propionate dioxygenase and 3–phenylpropionate-20 ,30 -di-
hydrodiol dehydrogenase were strongly repressed in the
presence of glucose. Note that we found that glucose was
significantly increased in the rumen of cows fed higher
amounts of barley grain. Other than a role of 3-PP in the
growth of rumen bacteria and their protection from oxi-
dative stress, there are no data to relate changes in the
concentrations of 3-PP and phenylacetate with the health
of dairy cattle (Turlin et al. 2005).
Our study confirms previous findings with regard to
higher concentrations of VFA with increasing the propor-
tion of cereal grains in the diet. These VFA observations
were made using traditional analytical methods such as GC
and HPLC (Iqbal et al. 2009). Previous research has indi-
cated that feeding cattle with diets rich in readily degrad-
able carbohydrates, such as barley grain, is associated with
increased concentration of VFA in the rumen fluid. These
changes in the VFA profile arise from changes in the
propionate and valerate anabolic pathways (Bugaut 1987).
Accumulation of VFA in the rumen fluid is known to be
associated with the development of ARA and SARA and a
chain of other related metabolic disorders in dairy cattle.
Interestingly, the rumen concentration of lactate in our
study did not differ among diets despite large variations in
the amount of readily fermentable carbohydrates. This
result also agrees with previous reports (Nocek 1997; Iqbal
et al. 2009). Evidently lactate is rapidly converted to pro-
pionate via the acrylate pathway, found in several lactate-
utilizing rumen bacteria (Satter and Esdale 1968). Overall,
the consistency of our data with those reported by con-
ventional analytical techniques shows that NMR-based
metabolomics is a reliable approach for the evaluation of
rumen metabolic events.
Rumen metabolomics
The appropriate amount of cereal grains to feed dairy
cattle is a hotly debated issue among ruminant nutritionists
and health scientists. On one hand, cereal grains are nec-
essary dietary ingredients to support high milk production.
On the other hand, large amounts of grain may adversely
affect rumen metabolism as well as the health and pro-
ductivity of dairy cows (Nocek 1997; Iqbal et al. 2009). In
this regard, rumen data from this study clearly suggests that
diets containing 30 and 45% cereal grain generate a whole
variety of metabolites that have been or might be impli-
cated in the etiology and pathogenesis of different meta-
bolic diseases in dairy cows. Interestingly, the diet
containing 15% barley grain was shown to supply enough
energy and nutrients to support high milk production
(28.0 kg fat-corrected milk/d, Zebeli and Ametaj 2009)
without adversely affecting rumen metabolism, and the
overall metabolic health of dairy cows (Emmanuel et al.
2008).
5 Concluding remarks
In summary, 1H-NMR-based metabolomics and multivar-
iate analysis were used to provide concentration data on 46
rumen metabolites for cattle fed various grain-enriched
diets. With increasing amounts of dietary cereal grain we
found dramatic increases of rumen MA and endotoxin,
enhanced concentrations of glucose, alanine, maltose,
propionate, uracil, valerate, xanthine, and phenylacetate as
well as potential increases in the concentration of NDMA,
EtOH, and DMA. The only metabolite that dropped with
increasing amounts of dietary barley grain was 3-PP. The
PCA of the rumen data showed 4 distinct clusters of
metabolites corresponding to the 4 distinct cereal grain
diets. Metabolite levels corresponding to diets with 0 and
15% barley grain were highly similar and mapped closely
together, while the two other clusters (from 30 and 45%
grain diets) were located far apart. Avoiding digestive and
systemic health disturbances of ruminants through a heal-
thy nutrition is critical to ensure that milk and meat are
produced from healthy animals. Given that the feeding of
large amounts of barley grain to dairy cattle resulted in
major increases in a number of potentially harmful rumen
metabolites, our results suggest that a systematic evaluation
of milk and meat products of cows fed high grain diets
should also be performed to detect the presence of these
metabolites. Overall, our study suggests that 1H-NMR-
based metabolomics could be used as a reliable tool to
study the interactions between nutrition and health in dairy
cows.
Acknowledgements We acknowledge co-leading of the project
entitled ‘Profiling of Dairy Cattle Metabolome’ by Drs. Ametaj and
593
Wishart, which was supported financially by the Alberta Agricultural
Research Institute (AARI; Edmonton, Alberta, Canada), the Alberta
Livestock Industry Development Fund (ALIDF; Edmonton, Alberta,
Canada), and the Natural Sciences and Engineering Research Council
of Canada (NSERC; Ottawa, Ontario, Canada). The technical assis-
tance of D. G. V. Emmanuel, R. P. Pandian, and S. Sivaraman
(University of Alberta, Edmonton, Alberta, Canada) is highly
appreciated. We also are grateful to the technical staff of Dairy
Research and Technology Centre at the University of Alberta for their
help and care to the cows used in this study.
References
Allison, M. J., Dougherty, R. W., Bucklin, J. A., & Snyder, E. E.
(1964). Ethanol accumulation in the rumen after overfeeding
with readily fermentable carbohydrate. Science, 144, 54–55.
Ametaj, B. N., Bradford, B. J., Bobe, G., Nafikov, R. A., Lu, Y.,
Young, J. W., et al. (2005). Strong relationships between
mediators of the acute phase response and fatty liver in dairy
cows. Canadian Journal of Animal Science, 85, 165–175.
Andries, J. I., Buysse, F. X., Debrabander, D. L., & Cottyn, B. G.
(1987). Isoacids in ruminant nutrition: their role in ruminal and
intermediary metabolism and possible influences on perfor-
mances—a review. Animal Feed Science and Technology, 18,
169–180.
Bertram, H. C., Kristensen, N. B., Malmendal, A., Nielsen, N. C.,
Brod, R., Andersen, H. J., et al. (2005). A metabolomic
investigation of splanchnic metabolism using 1H NMR spec-
troscopy of bovine blood plasma. Analytica Chimica Acta,
536, 1–6.
Bertram, H. C., Kristensen, N. B., Vestergaard, M., Jensen, S. K.,
Sehested, J., Nielsen, N. C., et al. (2009). Metabolic character-
ization of rumen epithelial tissue from dairy calves fed different
starter diets using 1H NMR spectroscopy. Livestock Science,
120, 127–134.
Boudonck, K. J., Mitchell, M. W., Wulff, J., & Ryals, J. A. (2010).
Characterization of the biochemical variability of bovine milk
using metabolomics. Metabolomics. doi:10.1007/s11306-009-
0160-8.
Broadhurst, D. I., & Kell, D. B. (2006). Statistical strategies for
avoiding false discoveries in metabolomics and related exper-
iments. Metabolomics, 2, 171–196.
Bugaut, M. (1987). Occurrence, absorption and metabolism of short
chain fatty acids in the digestive tract of mammals. Comparative
Biochemistry and Physiology, 86B, 439–472.
Burlingame, R., & Chapman, P. J. (1983). Catabolism of phenylpro-
pionic acid and its 3-hydroxy derivative by Escherichia coli.
Journal of Bacteriology, 155, 113–121.
Canadian Council on Animal Care. (1993). Guide to the care and use
of experimental animals (2nd ed., Vol. 1). Ottawa: CCAC.
Chesson, A., Provan, G. J., Russell, W. R., Scobbie, L., Richardson,
A. J., & Stewart, C. (1999). Hydroxycinnamic acids in the
digestive tract of livestock and humans. Journal of the Science of
Food and Agriculture, 79, 373–378.
Davis, E. J., & De Ropp, R. S. (1961). Metabolic origin of urinary
methylamine in the rat. Nature, 190, 636–637.
Dieterle, F., Ross, A., Schlotterbeck, G., & Senn, H. (2006).
Probabilistic quotient normalization as robust method to account
for dilution of complex biological mixtures. Application in 1H
NMR metabonomics. Analytical Chemistry, 78, 4281–4290.
Dumas, M.-E., Barton, R. H., Toye, A., Cloarec, O., Blancher, Ch.,
Rothwell, A., et al. (2006). Metabolic profiling reveals a
contribution of gut microbiota to fatty liver phenotype in
123
594
insulin-resistant mice. Proceedings of National Academy of
Sciences USA, 103, 12511–12516.
Emmanuel, D. G. V., Dunn, S. M., & Ametaj, B. N. (2008). Feeding
high proportions of barley grain stimulates an inflammatory
response in dairy cows. Journal of Dairy Science, 91, 606–614.
Enomoto, N., Ikejima, K., Yamashina, S., Hirose, M., Shimizu, H.,
Kitamura, T., et al. (2001). Kupffer cell sensitization by alcohol
involves increased permeability to gut-derived endotoxin. Alco-
holism, Clinical and Experimental Research, 25, 51S–54S.
Estruch, R., Nicolás, J. M., Villegas, E., Jonqué, A., & Urbano-
Márquez, A. (1993). Relationship between ethanol-related
diseases and nutritional status in chronically alcoholic men.
Alcohol and Alcoholism, 28, 543–550.
Gould, G. W. (1970). Germination and the problem of dormancy.
Journal of Applied Bacteriology, 33, 34–49.
Hashimoto, S., Kawai, Y., & Mutai, M. (1975). In vitro N-
nitrosodimethylamine formation by some bacteria. Infection
and Immunity, 11, 1405–1406.
Hill, K. J., & Mangan, J. L. (1964). The formation and distribution of
methylamine in the ruminant digestive tract. Biochemistry
Journal, 93, 39–45.
Hoogenraad, N. J., & Hird, F. J. R. (1970). The chemical composition
of rumen bacteria and cell walls from rumen bacteria. British
Journal of Nutrition, 24, 119–127.
Iqbal, S., Zebeli, Q., Mazzolari, A., Bertoni, G., Dunn, S. M., et al.
(2009). Feeding barley grain steeped in lactic acid modulates
rumen fermentation patterns and increases milk fat content in
dairy cows. Journal of Dairy Science, 92, 6023–6032.
Jenkins, T. C., & McGuire, M. A. (2006). Major advances in
nutrition: impact on milk composition. Journal of Dairy Science,
89, 1302–1310.
Johnson, K. A., & Johnson, D. E. (1995). Methane emissions from
cattle. Journal of Animal Science, 73, 2483–2492.
Khafipour, E., Li, S., Plaizier, J. C., & Krause, D. O. (2009). Rumen
microbiome composition determined using two nutritional
models of subacute ruminal acidosis. Applied and Environmental
Microbiology, 75, 7115–7124.
Koppang, N. (1964). An outbreak of toxic liver injury in ruminants.
Nord Veterinaermed, 16, 305–322.
Kristensen, N. B., Storm, A., Raun, B. M., Røjen, B. A., & Harmon,
D. L. (2007). Metabolism of silage alcohols in lactating dairy
cows. Journal of Dairy Science, 90, 1364–1377.
Lijinsky, W. (1999). N-Nitroso compounds in the diet. Mutation
Research, 443, 129–138.
Littell, R. C., Henry, P. R., & Ammerman, C. B. (1998). Statistical
analysis of repeated measures data using SAS procedures.
Journal of Animal Science, 76, 1216–1231.
Martin, A. K. (1982). The origin of urinary aromatic compounds
excreted by ruminants 3. The metabolism of phenolic com-
pounds to simple phenols. British Journal of Nutrition, 48,
497–507.
McAllan, A. B., & Smith, R. H. (1973). Degradation of nucleic acids
in the rumen. British Journal of Nutrition, 29, 331–345.
Neill, A. R., Grime, D. W., & Dawson, R. M. (1978). Conversion of
choline methyl groups through trimethylamine into methane in
the rumen. Biochemistry Journal, 170, 529–535.
Nocek, J. E. (1997). Bovine acidosis: implications on laminitis.
Journal Dairy Science, 80, 1005–1028.
NRC. (2001). Nutrient requirements of dairy cattle (7th rev. edn).
National Academy of Sciences, Washington, DC.
Örlygsson, J., Anderson, R., & Svensson, B. H. (1995). Alanine as an
end product during fermentation of monosaccharides by Clos-
tridium strain P2. Antonie van Leeuwenhoek, 68, 273–280.
Pagella, J. H. (1998). Urinary benzylated compounds as potential
markers of forage intake and metabolism of their precursors in
ruminants. PhD Dissertation, Aberdeen University, UK.
123
B. N. Ametaj et al.
Pruett, B. S., & Pruett, S. B. (2006). An explanation for the
paradoxical induction and suppression of an acute phase
response by ethanol. Alcohol, 39, 105–110.
Rieu-Lesme, F., Dauga, C., Morvan, B., Bouvet, O. M. M., Grimont,
P. A. D., & Doré, J. (1996). Acetogenic sporulating cocci
isolated from the rumen. Research in Microbiology, 147,
753–764.
Satter, L. D., & Esdale, W. J. (1968). In vitro lactate metabolism by
ruminal ingesta. Applied Microbiology, 16, 680–688.
Saude, E. J., Slupsky, C. M., & Sykes, B. D. (2006). Optimization of
NMR analysis of biological fluids for quantitative accuracy.
Metabolomics, 2, 113–123.
Seo, J. (2005). Information visualization design for multidimensional
data: integrating the rank-by-feature framework with hierarchi-
cal clustering. Ph.D. Dissertation, University of Maryland.
Slyter, L. L. (1976). Influence of acidosis on rumen function. Journal
of Animal Science, 43, 910–929.
Souliotis, V. L., Henneman, J. R., Reed, C. D., Chhabra, S. K.,
Diwan, B. A., Anderson, L. M., et al. (2002). DNA adducts and
liver DNA replication in rats during chronic exposure to N-
nitrosodimethylamine (NDMA) and their relationships to the
dose-dependence of NDMA hepatocarcinogenesis. Mutation
Research, 500, 75–87.
Tajima, K., Arai, S., Ogata, K., Nagamine, T., Matsui, H., Nakamura,
M., et al. (2000). Rumen bacterial community transition during
adaptation to high-grain diet. Anaerobe, 6, 273–284.
Trent, M. S., Stead, C. M., Tran, A. X., & Hankins, J. V. (2006).
Diversity of endotoxin and its impact on pathogenesis. Journal
of Endotoxin Research, 12, 205–223.
Turlin, E., Perrotte, M., Danchin, A., & Biville, F. (2001). Regulation
of the early steps of 3-phenylpropionate catabolism in Esche-
richia coli. Journal of Molecular Microbiology and Biotechnol-
ogy, 3, 127–133.
Turlin, E., Sismeiro, O., Le Caer, J. P., Labas, V., Danchin, A., &
Biville, F. (2005). 3-phenylpropionate catabolism and the
Escherichia coli oxidative stress response. Research in Micro-
biology, 156, 312–321.
Vinayavekhin, N., Homan, E. A., & Saghatelian, A. (2010). Exploring
disease through metabolomics. American Chemical Society
Chemical Biology, 5, 91–103.
Weljie, A. M., Newton, J., Mercier, P., Carlson, E., & Slupsky, C. M.
(2006). Targeted profiling: Quantitative analysis of 1H NMR
metabolomics data. Analytical Chemistry, 78, 4430–4442.
Wishart, D. S. (2008a). Metabolomics: Applications to food science
and nutrition research. Trends in Food Science and Technology,
19, 482–493.
Wishart, D. S. (2008b). Quantitative metabolomics using NMR.
Trends in Analytical Chemistry, 27, 228–237.
Wishart, D. S., Lewis, M. J., Morrissey, J. A., Flegel, M. D., Jeroncic,
K., Xiong, Y., et al. (2008). The human cerebrospinal fluid
metabolome. Journal of Chromatography B, Analytical Tech-
nologies in the Biomedical and Life Sciences, 871, 164–173.
Xia, J., Psychogios, N., Young, N. & Wishart, D. S. (2009).
MetaboAnalyst: a web server for metabolomic data analysis
and interpretation. Nucleic Acids Research, 37(Web Server
issue), W652–W660.
Yu, P., Xin, H., Lu, L., Fan, H., Kazachkov, M., Jiang, Z. J., et al.
(2006). Involvement of semicarbazide-sensitive amine oxidase-
mediated deamination in lipopolysaccharide-induced pulmonary
inflammation. American Journal of Pathology, 168, 718–726.
Zebeli, Q., & Ametaj, B. N. (2009). Relationships between rumen
lipopolysaccharide and mediators of inflammatory response with
milk fat production and efficiency in dairy cows. Journal of
Dairy Science, 92, 3800–3809.