Kanglexin, a new anthraquinone compound, attenuates lipid accumulation by activating the AMPK/SREBP-2/PCSK9/LDLR signalling pathway

Article abstract of DOI:10.1016/j.biopha.2020.110802

Hyperlipidaemia is one of the major risk factors for atherosclerosis, coronary heart disease, stroke and diabetes. In the present study, we synthesized a new anthraquinone compound, 1,8-dihydroxy-3-succinic acid monoethyl ester-6-methylanthraquinone, and named it Kanglexin (KLX). The aim of this study was to evaluate whether KLX has a lipid-lowering effect and to explore the potential molecular mechanism. In this study, Sprague-Dawley rats were fed a high fat diet (HFD) for 5 weeks to establish a hyperlipidaemia model; then, the rats were orally administered KLX (20, 40, and 80 mg kg^?1·d^?1) or atorvastatin calcium (AT, 10 mg kg^?1·d^?1) once a day for 2 weeks. KLX had prominent effects on reducing blood lipids, hepatic lipid accumulation, body weight and the ratio of liver weight/body weight. Furthermore, KLXdramatically reduced the total cholesterol (TC) and triglyceride (TG) levels and lipid accumulation in a HepG2 cell model of dyslipidaemia induced by 1 mmol/L oleic acid (OA). KLX may decrease lipid levels by phosphorylating adenosine monophosphate-activated protein kinase (AMPK) and the downstream sterol regulatory element binding protein 2 (SREBP-2)/proprotein convertase subtilisin/kexin type 9 (PCSK9)/low-density lipoprotein receptor (LDLR) signalling pathway in the HFD rats and OA-treated HepG2 cells. The effects of KLX on the AMPK/SREBP-2/PCSK9/LDLR signalling pathway were abolished when AMPK was inhibited by compound C (a specific AMPK inhibitor) in HepG2 cells. In summary, KLX has an efficient lipid-lowering effect mediated by activation of the AMPK/SREBP-2/PCSK9/LDLR signalling pathway. Our findings may provide new insight into and evidence for the discovery of a new lipid-lowering drug for the prevention and treatment of hyperlipidaemia, fatty liver, and cardiovascular disease in the clinic.

Full text of DOI:10.1016/j.biopha.2020.110802

J. Dairy Sci. 97:3761–3776
http://dx.doi.org/10.3168/jds.2013-7358
© American Dairy Science Association®, 2014.
Effect of replacing grass silage with red clover silage on nutrient
digestion, nitrogen metabolism, and milk fat composition in lactating
cows fed diets containing a 60:40 forage-to-concentrate ratio
A. Halmemies-Beauchet-Filleau,*† A. Vanhatalo,† V. Toivonen,* T. Heikkilä,* M. R. F. Lee,‡1
and K. J. Shingfield*2
*MTT Agrifood Research Finland, Animal Production Research, FI-31600 Jokioinen, Finland
†University of Helsinki, Department of Agricultural Sciences, PO Box 28, FI-00014 University of Helsinki, Finland
‡Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Gogerddan, Aberystwyth, Ceredigion,
SY23 3EB, United Kingdom
ABSTRACT
RCS decreased linearly milk fat 4:0 to 8:0, 14:0, and
16:0 concentrations and increased linearly 18:2n-6 and
18:3n-3 concentrations, in the absence of changes in
cis-9 18:1, cis-9,trans-11 18:2, or total trans fatty acid
concentration. Inclusion of RCS in the diet progres-
sively increased the apparent transfer of 18-carbon
PUFA from the diet into milk, but had no effect on
the amount of 18:2n-6 or 18:3n-3 at the omasum re-
covered in milk. In conclusion, forage species modified
ruminal N metabolism, the flow of AA at the omasum,
and whole-body N partitioning. A lower efficiency of
N utilization for milk protein synthesis with RCS rela-
tive to GS was associated with decreased availability of
AA for absorption, with some evidence of an imbalance
in the supply of AA relative to requirements. Higher
enrichment of PUFA in milk for diets based on RCS
was related to an increased supply for absorption, with
no indication that forage species substantially altered
PUFA bioavailability.
Diets based on red clover silage (RCS) typically in-
crease the concentration of polyunsaturated fatty acids
(PUFA) in ruminant meat and milk and lower the ef-
ficiency of N utilization compared with grass silages
(GS). Four multiparous Finnish Ayrshire cows (108 d
postpartum) fitted with rumen cannulas were used in a
4 × 4 Latin square design with 21-d periods to evaluate
the effect of incremental replacement of GS with RCS
on milk production, nutrient digestion, whole-body N
metabolism, and milk fatty acid composition. Treat-
ments comprised total mixed rations offered ad libitum,
containing 600 g of forage/kg of diet dry matter (DM),
with RCS replacing GS in ratios of 0:100, 33:67, 67:33,
and 100:0 on a DM basis. Intake of DM and milk yield
tended to be higher when RCS and GS were offered as
a mixture than when fed alone. Forage species had no
influence on the concentration or secretion of total milk
fat, whereas replacing GS with RCS tended to decrease
milk protein concentration and yield. Substitution of
GS with RCS decreased linearly whole-tract apparent
organic matter, fiber, and N digestion. Forage species
had no effect on total nonammonia N at the omasum,
whereas the flow of most AA at the omasum was higher
for diets based on a mixture of forages. Replacing GS
with RCS progressively lowered protein degradation in
the rumen, increased linearly ruminal escape of dietary
protein, and decreased linearly microbial protein syn-
thesis. Incremental inclusion of RCS in the diet tended
to lower whole-body N balance, increased linearly the
proportion of dietary N excreted in feces and urine,
and decreased linearly the utilization of dietary N for
milk protein synthesis. Furthermore, replacing GS with
Key words: red clover, grass, milk fatty acid, nitro-
gen metabolism
INTRODUCTION
Red clover (Trifolium pratense) is a common forage
legume grown in northern Europe, the United States,
and Canada. Forage legumes form an integral part of
organic and low-input milk-production systems, owing
to the ability to capture atmospheric N2, lowering the
necessity for additional N. Given the rising costs of
inorganic N fertilizers and attempts to decrease the en-
vironmental footprint of ruminant livestock production,
red clover may also be advantageous in conventional
milk-production systems. Compared with grasses, red
clover has a high intake and milk-production potential
(Dewhurst et al., 2003b; Moorby et al., 2009; Stein-
shamn, 2010), but the efficiency of dietary N utilization
for milk protein synthesis is often lower (Dewhurst et
al., 2003b; Moorby et al., 2009; Vanhatalo et al., 2009).
It has been postulated that either an excessive or an
Received August 9, 2013.
Accepted February 10, 2014.
1Current address: School of Veterinary Science, University of
Bristol, Langford, Somerset, BS40 5DU, United Kingdom.
2Corresponding author: kevin.shingfield@mtt.fi
3761

3762
HALMEMIES-BEAUCHET-FILLEAU ET AL.
imbalanced supply of AA available to the mammary Finland) in accordance with guidelines established by
glands, or possibly both, may limit the conversion ef- the European Community Council Directives 86/609/
ficiency of dietary N into milk with red clover silage EEC. Four multiparous Finnish Ayrshire cows (599
(RCS) diets, with a potential deficiency of Met being 26.0 kg of BW, 108 5.8 DIM, and producing 30.0
implicated (Lee et al., 2009; Vanhatalo et al., 2009). 1.84 kg of milk/d) fitted with rumen cannulas (100
Meat and milk from ruminants are characterized by mm i.d.; Bar Diamond Inc., Parma, ID) were allocated
high concentrations of SFA and a low abundance of at random to experimental diets according to a 4 × 4
PUFA, due at least in part, to extensive biohydrogena- Latin square with 21-d periods. A detailed description
tion of dietary unsaturated FA in the rumen (Dewhurst of experimental treatments, management of grass and
et al., 2003a; Shingfield et al., 2013). Ruminant-derived red clover leys, and preparation of experimental si-
foods are an important source of lipid in the human lages are provided in a companion paper (Halmemies-
diet (Shingfield et al., 2013) and, therefore, consider- Beauchet-Filleau et al., 2013c). In brief, experimental
able interest exists in developing nutritional strategies treatments comprised TMR containing 600 g of forage/
to lower 12:0, 14:0, and 16:0 and increase 18:3n-3 in kg of diet DM with RCS replacing GS in the diet in the
ruminant milk fat with potential to improve long-term DM ratio of 0:100, 33:67, 67:33, and 100:0 (treatments
human health (Gebauer et al., 2006; Shingfield et al., GS, GRC, RCG, and RCS, respectively). Silages
2008). It is well established that milk from diets based were prepared from mixed timothy (Phleum pratense
on RCS contains much higher 18:2n-6 and 18:3n-3 con- L. ‘Tuukka’) and meadow fescue (Festuca pratensis
centrations compared with ensiled grass (Dewhurst et Huds. ‘Antti’ and ‘Ilmari’) and red clover (Trifolium
al., 2006; Vanhatalo et al., 2007; Moorby et al., 2009). pratense L. ‘Jokioinen’) swards treated with 5 L/t of
At least part of the enrichment in milk can be attrib- formic acid-based additive (760 g of formic acid and
uted to higher amounts of 18:2n-6 and 18:3n-3 escaping 55 g of ammonium formate, AIV 2 Plus; Kemira Ltd.,
the rumen (Dewhurst et al., 2003a,b), but the influence Helsinki, Finland). Forages were supplemented with
of forage species on the digestion, absorption, and utili- a standard concentrate (Table 1). Diets were offered
zation of FA for milk fat synthesis in lactating cows has ad libitum as 4 equal meals at 0600, 0800, 1800, and
not been extensively investigated.
2000 h. The chemical composition of individual feed
Red clover has a higher polyphenol oxidase (PPO) ingredients and experimental diets is shown in Table
activity compared with grasses, differences that have 1. Cows were housed in a dedicated metabolism unit
been suggested to contribute to the effects of forage fitted with individual tie-stalls with continuous access
species on proteolysis and lipolysis in silo and in vivo to water. Cows were milked twice daily at 0700 and
(Lee et al., 2004; Merry et al., 2006; Van Ranst et al., 1645 h. At the end of the experiment, cows weighed
2011). Previous investigations comparing RCS and 603 29.3 kg.
grass silage (GS) diets have characterized the effects of
forage species on ruminal N and lipid metabolism and
milk FA composition in lactating cows under condi-
tions typical for organic or low-input milk production
(Dewhurst et al., 2003a,b; Moorby et al., 2009).
Measurements and Sampling
Individual cow intakes were recorded throughout the
experiment, but only measurements for the last 7 d of
The aim of the present study was to provide further each period were used for statistical analysis. During
insight into the influence of forage species on the trans- this time, representative samples of silage and concen-
formations of dietary protein in the rumen, efficiency of trates were collected, composited, and stored at −20°C
N utilization, and bioavailability of FA in cows fed diets before chemical analysis. The chemical composition of
relevant to more intensive milk-production systems. feeds, including the correction of silage DM content for
Four cows in a 4 × 4 Latin square design were used volatile losses was determined using standard methods
to test the hypotheses that the effects of RCS on the (Halmemies-Beauchet-Filleau et al., 2013a). Starch in
efficiency of N utilization and milk FA composition are feeds was analyzed according to Salo and Salmi (1968).
related to changes in the amount and bioavailability of The concentration of indigestible NDF (iNDF) in si-
AA and FA available for absorption.
lages and concentrates was determined in duplicate by
incubating 0.5- to 1-g samples within polyester bags
(60 × 120 mm, 17-μm pore size) in the rumen for 12
d (Ahvenjärvi et al., 2000). The OM content of the
indigestible residue was determined by ashing at 600°C
for 18 h. Body condition scores were recorded on a scale
MATERIALS AND METHODS
Animals, Experimental Design,
and Experimental Diets
All experimental procedures were approved by the of 1 (thin) to 5 (fat) at the start of the experiment and
National Animal Ethics Committee (Hämeenlinna, at the end of each experimental period.
Journal of Dairy Science Vol. 97 No. 6, 2014

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EFFECT OF FORAGE SPECIES ON NITROGEN USE AND MILK FAT
Table 1. Chemical composition of dietary ingredients and experimental diets
Dietary ingredient
Red clover
Experimental diet1
Grass
Item
silage2
silage3
23.3
Concentrate4
89.8
5.27
GS
GRC
50.0
RCG
50.0
RCS
49.9
DM, %
23.6
50.1
% of DM
Ash
8.72
15.6
52.9
9.82
3.29
1.03
10.3
20.0
33.9
9.16
2.08
1.63
7.34
17.4
40.6
7.61
4.71
12.1
7.66
18.3
36.8
7.48
4.47
12.2
7.97
19.2
33.0
7.35
4.23
12.3
8.29
20.0
29.2
7.22
3.98
12.4
CP
20.1
22.1
4.30
6.84
28.6
NDF
Indigestible NDF
Water-soluble carbohydrates
Starch
AA, g/100 g of CP
Arg
4.25
1.50
4.16
7.51
5.37
2.41
5.02
4.45
5.49
6.63
9.93
1.08
10.7
4.75
1.14
5.35
4.22
2.79
17.2
40.2
46.6
86.8
4.72
2.00
4.84
8.25
4.91
2.15
5.31
4.73
7.08
6.65
11.2
1.03
10.1
5.12
0.14
4.80
4.86
3.41
20.2
44.0
47.3
91.3
5.66
2.38
3.67
6.69
4.81
2.30
4.26
4.01
4.98
4.17
6.85
2.32
4.90
1.91
3.93
7.13
5.11
2.36
4.67
4.25
5.25
5.49
8.51
1.65
4.97
2.00
4.09
7.31
5.02
2.30
4.75
4.32
5.61
5.55
8.85
1.61
14.0
4.73
0.45
5.86
4.42
3.02
17.0
40.4
48.5
88.8
5.04
2.08
4.24
7.47
4.94
2.26
4.82
4.38
5.94
5.61
9.17
1.58
13.7
4.80
0.28
5.72
4.54
3.14
17.7
41.2
48.5
89.7
5.10
2.15
4.37
7.62
4.87
2.21
4.89
4.44
6.24
5.66
9.45
1.55
13.4
4.88
0.12
5.59
4.66
3.25
18.2
41.9
48.6
90.5
His
Ile
Leu
Lys
Met
Phe
Thr
Val
Ala
Asp
Cys
Glu
18.4
14.3
Gly
4.51
0.08
6.78
4.35
3.01
4.64
0.65
6.01
4.28
2.89
Orn
Pro
Ser
Tyr
∑ Branched-chain AA5
∑ EAA6
15.3
16.3
38.8
50.5
89.2
39.6
48.4
87.9
∑ NEAA7
∑ Total AA
1Refers to diets containing a 60:40 forage:concentrate ratio based on grass silage (GS), red clover silage (RCS),
or a 2:1 (GRC) or 1:2 (RCG) mixture (DM basis) of GS and RCS, respectively.
2Mean fermentation characteristics of grass silage: pH 3.97; % of DM: lactic acid: 6.32, acetic acid: 1.97, propi-
onic acid: 0.02, and butyric acid: 0.04; % of total N: ammonium-N: 4.46 and soluble N: 61.5.
3Mean fermentation characteristics of red clover silage: pH 4.00; % of DM: lactic acid: 10.1, acetic acid: 2.49,
propionic acid: 0.02, and butyric acid: 0.01; % of total N: ammonium-N: 5.28 and soluble N: 38.4.
4Concentrate comprised (% of DM in TMR) rolled barley (18), molassed sugar beet pulp (9), solvent-extracted
rapeseed meal (11.5; Raisio Feed Ltd., Raisio, Finland), and a proprietary vitamin and mineral supplement
(1.5; Onni; Melica Finland Ltd., Vaasa, Finland) declared as containing (g/kg) calcium (205), magnesium
(72), sodium (85), phosphorus (27), zinc (1.46), manganese (0.35), copper (0.27); (mg/kg) iodine (39), cobalt
(27), selenium (20); and (IU/g) retinyl acetate (120), cholecalciferol (25), and dl-α-tocopheryl acetate (0.34).
5Sum of Ile, Leu, and Val.
6Sum of Arg, His, Ile, Leu, Lys, Met, Phe, Thr, and Val.
7Sum of Ala, Asp, Cys, Glu, Gly, Orn, Pro, Ser, and Tyr.
Omasal digesta flow was determined using the oma- feeding cycle. At the end of the experiment, digesta
sal sampling technique (Ahvenjärvi et al., 2000) using samples were separated into large-particle, small-parti-
Cr-EDTA, Yb-acetate, and iNDF as markers for liquid, cle, and liquid phases by filtration and centrifugation,
small particles, and large particles, respectively (Hal- freeze-dried, and analyzed for DM, OM, VFA, Cr, Yb,
memies-Beauchet-Filleau et al., 2013c). Spot samples and iNDF (Halmemies-Beauchet-Filleau et al., 2013c).
(500 mL) of digesta entering the omasal canal were col- Based on marker concentrations, the relative propor-
lected 3 times daily at 4-h intervals on d 17 to 20 using tions of the liquid, small-particle, and large-particle
the omasal sampling device. Sampling started at 0600 fractions in true digesta were calculated (Ahvenjärvi et
h and was advanced by 1 h each day to cover a 12-h pe- al., 2000). Thereafter, appropriate amounts of freeze-
riod. Digesta collected over the 12-h interval from 0600 dried digesta were weighed to yield a composite sample
to 1800 h was considered representative for the entire for chemical analysis.
Journal of Dairy Science Vol. 97 No. 6, 2014

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HALMEMIES-BEAUCHET-FILLEAU ET AL.
The flow of microbial N at the omasum was esti- and hexane (0.2:1:2.5:2.5, vol/vol, respectively) and
mated using 15.0 g/d of ammonium sulfate (Isotec Inc., converted to FAME using methanolic sodium methoxide
Miamisburg, OH) enriched (10%) with 15N (317 mg of as a catalyst (Halmemies-Beauchet-Filleau et al., 2011).
15N/d) as a marker. Ammonium sulfate was dissolved
Samples of FAME were quantified using a gas chro-
in the same solution containing Yb-acetate and infused matograph (model 6890; Hewlett-Packard, Wilming-
into the rumen from d 15 at 0600 to 1800 h on d 21 of ton, DE) equipped with a flame-ionization detector,
each period. Immediately before the start of the marker automatic injector, split injection port, and a 100-m
infusion, samples of rumen contents for each cow were fused silica capillary column (0.25-mm i.d.) coated
collected for the determination of background 15N abun- with a 0.2-μm film of cyanopropyl polysiloxane (CP-
dance. Samples of digesta (500 mL) for the analysis of Sil 88, Chromopack 7489; Chromopack, Middelburg,
microbial 15N enrichment were collected (n = 4) from the Netherlands). The total FAME profile in a 2-μL
the reticulorumen at 1500, 1200, 0900, and 0600 h on d sample at a split ratio of 1:50 was determined using a
17, 18, 19, and 20, respectively. Microbes were isolated temperature gradient program and hydrogen as a car-
from ruminal digesta by differential centrifugation and rier gas operated at constant pressure (137.9 kPa) at a
filtration (Ahvenjärvi et al., 2000). Enrichment of 15N flow rate of 0.5 mL/min. Isomers of 18:1 were further
in reticulorumen and omasal digesta was analyzed in resolved in a separate analysis under isothermal condi-
triplicate using a Roboprep-CN analyzer (Europa Sci- tions at 170°C. Peaks were identified by comparison
entific Ltd., Crewe, UK) and VG Micromass 622 mass of retention times with authentic FAME standards.
spectrometer (VG Micromass Ltd., Winsford, UK) Fatty acid methyl esters not available as commercial
according to Esala (1991). Concentrations of AA in standards were identified based on GC-MS analysis of
feeds and reconstituted omasal digesta were analyzed 4,4-dimethyloxoline derivatives prepared from FAME.
following acid hydrolysis (6 M HCl at 110°C for 23 h) Preparation of 4,4-dimethyloxoline derivatives, param-
according to the European Commission (1998) direc- eters used for GC-MS analysis, and interpretation mass
tive using a Waters MassTrak ultra performance liquid spectra were in accordance with earlier reports (Hal-
chromatography (UPLC) kit (Waters Corp., Milford, memies-Beauchet-Filleau et al., 2011). Relative reten-
MA) and the UPLC AA analysis solution provided by tion time and order of elution was used to differentiate
the manufacturer.
Whole-tract apparent digestibility coefficients were (Halmemies-Beauchet-Filleau et al., 2013b).
determined by total fecal collection over 96 h, start-
The distribution of CLA isomers in milk samples
between diastereomers of 3,7,11,15-tetramethyl-16:0
ing at 1800 h on d 17 of each experimental period. was determined using an HPLC system (model 1090;
Subsamples (5%, wt/wt) were stored at −20°C until Hewlett-Packard, Wilmington, DE) equipped with 4
analyzed for DM, OM, NDF, iNDF, N, Cr, and Yb silver-impregnated silica columns (ChromSpher 5 Lip-
using the same methods applied to feeds and omasal ids, 250 × 4.6 mm, 5-μm particle size; Varian Ltd.,
digesta.
Walton-on-Thames, UK) coupled in series. Methyl es-
Milk yield was recorded daily throughout the experi- ters of CLA were separated under isothermal conditions
ment, but only measurements made from d 15 to 21 of at 22°C using 0.1% (vol/vol) acetonitrile in heptane at
each period were used for statistical analysis. Samples a flow rate of 1 mL/min and monitoring column efflu-
of milk were collected from each cow over 4 consecu- ent at 233 and 210 nm (Halmemies-Beauchet-Filleau et
tive milkings starting at 1645 h on d 19. Milk samples al., 2011).
treated with preservative (bronopol; Valio Ltd., Hel-
Milk FA composition was expressed as a weight per-
sinki, Finland) were analyzed for milk fat, CP, and centage of total FA using theoretical relative response
lactose (MilkoScan 605 analyzer; Foss Electric A/S, factors to account for the carbonyl deficiency in the
Hillerød, Denmark). Samples of unpreserved milk were flame-ionization detector response for 4- to 10-carbon-
also collected, stored at 4°C, composited according to containing FAME (Halmemies-Beauchet-Filleau et al.,
yield, and analyzed for urea concentration, measured 2011). Concentrations of specific conjugated isomers
as ammonia following urease hydrolysis (Shingfield et were calculated based on proportionate peak area re-
al., 2002). Additional samples of unpreserved milk com- sponses determined by HPLC and the sum of trans-
posited according to yield were stored at −20°C until 7,cis-9 CLA, trans-8,cis-10 CLA, and cis-9,trans-11
analyzed for FA composition.
CLA weight percentage determined by GC.
Lipid Analysis
Calculations
Lipid in 1-mL milk samples was extracted 3 times
Metabolizable energy content of experimental diets
with a mixture of ammonia, methanol, diethyl ether, (MJ) was calculated as 0.016 × digestible OM in DM
Journal of Dairy Science Vol. 97 No. 6, 2014

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EFFECT OF FORAGE SPECIES ON NITROGEN USE AND MILK FAT
RESULTS
(g/kg; Ministry of Agriculture, Fisheries and Food,
1975) determined by total fecal collection.
Nutrient Intake
Ruminal AA balance (g/d) was calculated as flow of
AA at the omasum (g/d) − intake of AA (g/d).
Ruminal protein degradability (%) was calculated as
follows: ({[N intake (g/d) – ammonia-N intake (g/d)] –
flow of dietary NAN at the omasum (g/d)}/[N intake
(g/d) − ammonia-N intake (g/d)]) × 100.
Ruminal AA degradability (%) was calculated as fol-
lows: {[intake of AA (g/d) − flow of dietary AA at the
omasum (g/d)]/intake of AA (g/d)} × 100.
Flow of dietary AA at the omasum (g/d) was calcu-
lated as total flow of AA at the omasum (g/d) − flow
of microbial AA at the omasum (g/d), assuming that
AA represented, on average, 73.4% of microbial NAN
entering the omasal canal (Korhonen et al., 2002), and
the AA profile of microbial NAN measured across 18
dietary treatments, based on the mean of 43 determina-
tions for cows fed diets based on GS, RCS, or mixtures
of both forage species [A. Vanhatalo (University of
Helsinki, Helsinki, Finland) and P. Huhtanen (Swedish
University of Agricultural Sciences, Umeå, Sweden),
unpublished data]. The assumption underlying these
calculations was considered valid, given that differences
in diet composition have minimal influence on the AA
composition of microbial NAN fractions at the omasum
(Korhonen et al., 2002).
Intakes of DM, OM, starch, and N tended (P ≤ 0.08)
to be higher when RCS and GS were offered together
than when fed as the sole forage in the diet (Table 2).
Increases in the proportion of RCS in the diet decreased
linearly (P < 0.01) NDF intake (Table 2). Replacing GS
with RCS in the diet increased linearly (P < 0.01) AA
intake from 3,175 to 3,600 g/d (Supplemental Table S1;
http://dx.doi.org/10.3168/jds.2013-7358). The intake
of most AA increased linearly (P < 0.05) in response to
RCS in the diet, except that of Cys, Glu, Lys, Met, and
Pro, which were unaffected (P > 0.10) by differences
in forage species, whereas Orn decreased linearly (P <
0.01) to RCS in the diet (Supplemental Table S1).
Omasal Flow of Nutrients
Increases in the proportion of RCS in the diet de-
creased linearly (P < 0.05) the flow of OM and NDF
at the omasum (Table 2). Treatments had no effect
(P > 0.10) on total NAN at the omasum. However,
substituting GS for RCS in the diet decreased linearly
(P < 0.01) microbial NAN, but increased linearly (P <
0.01) dietary NAN at the omasum (Table 2). Feeding
mixtures of GS and RCS increased (P < 0.05; Met,
Phe, Val, Pro, and Ser), or tended to increase (P ≤
0.10; Arg, His, Leu, Thr, Ala, Glu, and Gly) the flow of
most AA at the omasum compared with either forage
species alone (Table 2).
Whole-body N balance (g/d) was calculated as N
intake (g/d) − [N in milk (g/d) + N excreted in feces
(g/d) + N excreted in urine (g/d)].
The transfer efficiency (%) of FA from the diet into
milk was calculated as FA secretion in milk (g/d per FA
intake; g/d) × 100. Efficiency of transfer (%) of FA at
the omasum into milk was calculated as FA secretion
in milk (g/d per FA flow at the omasum; g/d) × 100.
Nutrient Digestibility
Treatments had no effect (P > 0.10) on the extent
of nutrient digestion in the rumen, whereas replacing
GS with RCS in the diet decreased linearly (P < 0.04)
whole-tract DM, OM, NDF, and N digestibility (Table
3).
Statistical Analysis
Data were analyzed by ANOVA with a model that
included the fixed effects of treatment and period
and random effects cow using PROC MIXED of SAS
(version 9.2; SAS Institute Inc., Cary, NC). Sums of
squares for treatment effects were further separated us-
ing orthogonal contrasts into single-degree-of-freedom
comparisons to test for the significance of linear and
quadratic components of response to incremental re-
placement of GS with RCS in the diet. Least squares
means are reported, with treatment effects declared
significant at P ≤ 0.05, with P-values between 0.05 and
0.10 considered a trend toward significance. Relation-
ships between flows of FA at the omasum and the secre-
tion of FA in milk were evaluated by linear regression
analysis using PROC REG of SAS.
Ruminal and Whole-Body N Metabolism
Ruminal balance of total AA was negative for all diets
(Supplemental Table S2; http://dx.doi.org/10.3168/
jds.2013-7358). Replacing GS with RCS in the diet
progressively lowered (P < 0.05) ruminal balance of
branched-chain AA (BCAA) and EAA, except for Lys
and Met, which were unaffected (P > 0.10). Net disap-
pearance of NEAA, Glu, and Pro, was relatively high
on all diets, whereas comparisons of intake and flow at
the omasum indicated a net synthesis of Ile, Lys, Asp,
and Tyr in the rumen (Supplemental Table S2).
Journal of Dairy Science Vol. 97 No. 6, 2014

3766
HALMEMIES-BEAUCHET-FILLEAU ET AL.
Table 2. Effect of replacing grass silage with red clover silage in the diet on the intake and the flow of nutrients at the omasum, and BCS in
lactating cows
Treatment1
GRC
P-value2
Item
GS
RCG
RCS
SEM
L
Q
Intake
DM, kg/d
OM, kg/d
NDF, kg/d
pdNDF,3 kg/d
Starch, kg/d
N, g/d
19.9
18.4
20.1
18.6
20.1
18.5
18.4
16.9
1.06
0.98
0.360
0.278
0.135
31.8
0.06
0.031
<0.001
<0.001
0.28
0.06
0.06
0.06
0.09
0.08
0.08
0.016
0.17
8.05
7.38
6.63
5.36
6.50
2.39
552
206
0.03
5.83
2.45
587
209
0.03
5.11
2.48
617
207
0.03
4.00
2.28
589
186
−0.06
0.08
ME, MJ/d
Change in BCS
Omasal flow
DM, kg/d
OM, kg/d
NDF, kg/d
pdNDF, kg/d
N, g/d
9.8
0.039
0.008
0.09
14.2
11.0
3.92
2.41
563
555
380
174
13.9
10.8
3.62
2.12
580
571
349
222
14.2
11.0
3.35
1.87
624
614
347
267
12.8
9.84
2.79
1.47
589
1.04
0.81
0.08
0.044
<0.001
0.003
0.25
0.25
0.002
0.002
0.25
0.18
0.37
0.71
0.28
0.29
0.28
0.53
0.272
0.212
46.7
NAN, g/d
Microbial NAN, g/d
Dietary NAN, g/d
AA, g/d
Arg
581
45.9
288
24.9
293
26.1
151
58.3
162
63.9
174
69.8
164
66.6
168
259
208
78.4
164
161
188
196
344
50.1
429
178
4.52
143
145
161
614
12.0
4.90
12.7
19.9
16.8
6.23
11.9
12.4
14.3
16.1
28.2
4.06
34.1
13.6
0.422
10.7
10.8
13.0
46.9
110.4
128.3
238.6
0.08
0.018
0.36
0.11
0.89
0.90
0.06
0.32
0.16
0.67
0.69
0.13
0.47
0.06
0.96
0.06
0.08
0.20
0.17
0.20
0.25
0.23
0.10
0.09
0.11
0.09
0.16
0.042
0.049
0.07
0.049
0.06
0.15
0.39
0.10
0.09
0.25
0.045
0.047
0.13
0.08
0.08
0.08
0.08
His
Ile
162
239
209
172
259
217
181
275
227
Leu
Lys
Met
78.9
84.1
87.2
Phe
149
154
176
194
338
165
166
192
207
361
174
172
203
215
369
Thr
Val
Ala
Asp
Cys
44.5
417
159
47.7
445
175
51.6
462
190
Glu
Gly
Orn
4.49
130
132
142
577
1,377
1,561
2,938
4.84
143
146
169
622
1,480
1,699
3,179
4.84
153
156
164
659
1,564
1,765
3,329
Pro
Ser
Tyr
∑ Branched-chain AA4
∑ EAA5
∑ NEAA6
∑ Total AA
1,456
1,651
3,108
1Refers to diets containing a 60:40 forage:concentrate ratio based on grass silage (GS), red clover silage (RCS), or a 2:1 (GRC) or 1:2 (RCG)
mixture (DM basis) of GS and RCS, respectively.
2Significance of linear (L) and quadratic (Q) components of response to incremental replacement of GS with RCS in the diet.
3pdNDF = potentially digestible NDF.
4Sum of Ile, Leu, and Val.
5Sum of Arg, His, Ile, Leu, Lys, Met, Phe, Thr, and Val.
6Sum of Ala, Asp, Cys, Glu, Gly, Orn, Pro, Ser, and Tyr.
Increases in the proportion of RCS in the diet de- including Met and Cys. Substitution of GS with RCS
creased linearly (P < 0.01) protein degradability in the also tended to lower (P = 0.08) the energy efficiency of
rumen (Table 3). However, the extent of degradation ruminal microbial protein synthesis (Table 3).
for individual AA also varied according to AA struc-
Replacing GS with RCS in the diet decreased linearly
ture (Table 3). Ruminal degradability of His and Pro (P < 0.05) N secretion in milk, increased linearly (P <
was particularly high on all diets, whereas the opposite 0.01) fecal N excretion, and tended to increase (P =
was true for Ile, Lys, and Tyr. Replacing GS with RCS 0.07) urinary N excretion (Table 4). Overall, increases
in the diet decreased in a linear or quadratic manner in the proportion of RCS in the diet tended to lower (P
(P < 0.05) the degradability of most AA in the rumen, = 0.08) whole-body N retention (Table 4).
Journal of Dairy Science Vol. 97 No. 6, 2014

3767
EFFECT OF FORAGE SPECIES ON NITROGEN USE AND MILK FAT
Table 3. Effect of replacing grass silage with red clover silage in the diet on energy efficiency of microbial protein synthesis in the rumen and
ruminal and whole-tract nutrient digestibility coefficients in lactating cows
Treatment1
P-value2
Item
GS
GRC
RCG
RCS
SEM
L
Q
Ruminal
Energy efficiency of microbial protein synthesis,
31.6
67.5
29.5
61.2
29.8
55.5
27.5
49.2
1.43
3.10
0.08
0.94
1.00
g of N/kg of OMTDR3
Protein degradability,4 %
0.001
AA degradability,4 %
Arg
62.9
69.2
55.1
61.2
56.5
62.9
64.9
63.7
61.6
64.4
58.0
64.4
63.1
63.0
70.2
64.0
52.0
60.0
61.5
63.0
62.3
55.7
61.0
47.7
53.0
48.7
53.5
54.9
53.7
55.0
56.4
50.1
57.0
55.5
52.6
62.6
55.5
27.0
52.4
53.3
53.6
53.5
52.6
57.2
46.7
50.9
45.3
51.9
53.0
53.4
54.9
55.3
51.8
52.2
53.8
47.6
59.7
53.4
37.4
51.3
51.5
52.5
52.1
51.4
55.4
46.7
49.9
43.9
52.4
51.3
51.7
56.3
55.5
51.3
47.6
52.4
46.4
58.3
53.8
30.1
51.3
50.8
51.0
50.9
2.18
2.00
2.72
2.53
3.68
3.77
2.01
2.59
2.40
2.95
3.22
3.36
2.59
2.56
1.76
2.26
5.31
2.50
2.53
2.42
2.46
<0.001
<0.001
0.036
0.003
0.019
0.025
<0.001
0.004
0.07
0.08
0.022
0.15
0.08
0.32
0.10
0.025
0.06
0.042
0.10
0.17
0.67
0.13
0.07
0.029
0.015
0.15
0.08
0.07
0.06
0.07
His
Ile
Leu
Lys
Met
Phe
Thr
Val
Ala
0.027
0.14
Asp
Cys
0.008
0.005
<0.001
<0.001
0.002
0.06
0.014
0.005
0.003
0.004
Glu
Gly
Pro
Ser
Tyr
∑ Branched-chain AA5
∑ EAA6
∑ NEAA7
∑ Total AA
True digestibility, %
OM
65.0
64.0
62.8
62.2
0.02
0.21
0.91
Apparent digestibility, %
DM
28.8
40.3
30.7
41.7
51.0
63.9
1.12
29.7
40.8
31.1
42.3
48.2
63.7
0.82
2.61
2.23
2.30
2.82
3.91
0.59
0.58
0.28
0.91
0.71
0.91
0.99
0.85
0.93
0.89
OM
NDF
51.5
49.5
pdNDF8
63.1
63.4
N
−1.70
−1.13
Whole-tract apparent digestibility, %
DM
OM
NDF
pdNDF
N
67.8
69.9
59.9
69.4
66.5
68.4
70.4
59.1
70.6
65.5
67.6
69.7
56.5
65.6
62.9
66.4
68.8
53.4
65.3
61.1
0.52
0.44
0.81
1.20
0.82
0.007
0.032
<0.001
0.011
0.002
0.020
0.06
0.07
0.50
0.61
1Refers to diets containing a 60:40 forage:concentrate ratio based on grass silage (GS), red clover silage (RCS), or a 2:1 (GRC) or 1:2 (RCG)
mixture (DM basis) of GS and RCS, respectively.
2Significance of linear (L) and quadratic (Q) components of response to incremental replacement of GS with RCS in the diet.
3OMTDR = OM truly digested in the rumen.
4Calculated taking into account the contribution of microbial N at the omasum.
5Sum of Ile, Leu, and Val.
6Sum of Arg, His, Ile, Leu, Lys, Met, Phe, Thr, and Val.
7Sum of Ala, Asp, Cys, Glu, Gly, Orn, Pro, Ser, and Tyr.
8pdNDF = potentially digestible NDF.
Milk Production
in the diet increased linearly (P < 0.02) milk urea and
lactose concentrations (Table 5).
Diets containing both forage species resulted in
higher (P < 0.05) milk yields compared with either GS
or RCS alone (Table 5). Treatments had no influence
(P > 0.10) on the concentration or secretion of milk fat,
whereas milk protein concentration and yield tended to
decrease linearly (P = 0.07) with increasing RCS in the
diet (Table 5). Furthermore, substituting GS with RCS
Milk FA Composition
Treatment effects on the composition of major FA in
milk are presented in Table 6. A detailed description of
treatment effects on monoenoic, octadecadienoic, and
odd- and branched-chain FA (OBCFA) are reported
Journal of Dairy Science Vol. 97 No. 6, 2014

3768
HALMEMIES-BEAUCHET-FILLEAU ET AL.
Table 4. Effect of replacing grass silage with red clover silage in the diet on N partitioning and whole-animal
N balance in lactating cows
Treatment1
P-value2
Item
GS
GRC
1.67
RCG
RCS
SEM
L
Q
N balance, g/d
N in milk, g/d
N in feces, g/d
N in urine, g/d
Partitioning, %
N in milk
9.62
145
185
212
0.29
144
228
245
−25.4
137
230
247
15.61
2.2
12.9
12.6
0.08
0.45
0.11
0.34
0.22
147
0.039
0.003
0.019
202
236
26.4
33.5
38.4
25.3
34.5
40.4
23.6
37.1
40.0
23.5
38.9
42.0
1.53
0.80
1.60
0.042
0.002
0.07
0.62
0.62
0.98
N in feces
N in urine
1Refers to diets containing a 60:40 forage:concentrate ratio based on grass silage (GS), red clover silage (RCS),
or a 2:1 (GRC) or 1:2 (RCG) mixture (DM basis) of GS and RCS, respectively.
2Significance of linear (L) and quadratic (Q) components of response to incremental replacement of GS with
RCS in the diet.
in Supplemental Tables S3, S4, and S5, respectively
Replacing GS with RCS had no effect (P > 0.10) on
(http://dx.doi.org/10.3168/jds.2013-7358). Replacing milk fat 18:0 or cis-9 18:1 concentration, but increased
GS with RCS in the diet decreased linearly (P ≤ 0.05) linearly (P ≤ 0.05) the abundance of several cis 18:1
the proportions of several short- and medium-chain (Δ11–16) and trans 18:1 (Δ6–10, 12–16) isomers (Table
SFA in milk fat (4:0–8:0, 14:0, and 16:0) and increased 6; Supplemental Table S3; http://dx.doi.org/10.3168/
linearly (P < 0.01) 18:2n-6 and 18:3n-3 concentrations jds.2013-7358).
(Table 6). However, forage species had no effect (P >
Forage species also resulted in relatively small, albeit
0.05) on the total secretion of short- and medium-chain often significant, differences in the concentration of
SFA in milk, whereas the output of 18:2n-6 and 18:3n- 18:2 isomers in milk (Supplemental Table S4; http://
3 was increased (P < 0.05) in direct relation to RCS dx.doi.org/10.3168/jds.2013-7358). Replacing GS with
in the diet (Supplemental Figure S1; http://dx.doi. RCS increased linearly (P < 0.04) cis-9,trans-12 18:2
org/10.3168/jds.2013-7358). Substituting GS with RCS and Δ9,13 18:2 abundance and decreased (P < 0.02)
in the diet increased linearly (P < 0.02) the efficiency of trans-9,cis-12 18:2, trans-10,trans-14 18:2, and trans-
18:2n-6 [from 11 to 15% (SE = 1.5)] and 18:3n-3 [from 11,trans-15 18:2 concentrations. Treatments had no ef-
3 to 9% (SE = 0.7)] transfer from the diet into milk, fect (P > 0.05) on milk fat cis-9,trans-11 CLA concen-
whereas the apparent transfer of both PUFA from the tration or secretion, whereas substituting GS for RCS
omasum into milk was similar (P > 0.10) among treat- increased linearly (P ≤ 0.05) the relative abundance
ments, averaging 55 and 54% for 18:2n-6 and 18:3n-3, of several minor CLA isomers in milk fat, including
respectively.
trans-7,cis-9 CLA, trans-8,cis-10 CLA, trans-10,cis-12
Table 5. Effect of replacing grass silage with red clover silage in the diet on milk production in lactating cows
Treatment1
P-value2
Item
GS
GRC
RCG
RCS
SEM
L
Q
Yield
Milk, kg/d
ECM, kg/d
Fat, g/d
27.2
26.9
28.3
28.9
28.7
28.2
994
27.8
27.5
0.38
0.64
73.4
16.9
5.0
0.23
0.74
0.95
0.07
0.019
0.041
0.08
0.92
0.16
0.45
1,003
1,054
1,031
Protein, g/d
Lactose, g/d
Concentration
Fat, g/kg
Protein, g/kg
Lactose, g/kg
Urea, mmol/L
927
1,240
942
1,248
918
879
1,263
1,248
3.83
4.02
3.79
3.94
0.280
0.065
0.042
0.227
0.95
0.07
0.019
0.010
0.92
0.16
0.47
0.70
3.54
4.73
4.32
3.60
4.76
4.65
3.51
4.76
5.26
3.36
4.82
5.41
1Refers to diets containing a 60:40 forage:concentrate ratio based on grass silage (GS), red clover silage (RCS),
or a 2:1 (GRC) or 1:2 (RCG) mixture (DM basis) of GS and RCS, respectively.
2Significance of linear (L) and quadratic (Q) components of response to incremental replacement of GS with
RCS in the diet.
Journal of Dairy Science Vol. 97 No. 6, 2014

3769
EFFECT OF FORAGE SPECIES ON NITROGEN USE AND MILK FAT
Table 6. Effect of replacing grass silage with red clover silage in the diet on milk fatty acid composition in
lactating cows
Treatment1
P-value2
FA,
g/100 g of FA
GS
GRC
RCG
RCS
SEM
L
Q
4:0
3.30
2.00
1.22
2.92
0.32
3.59
0.10
0.09
3.27
1.99
1.22
2.90
0.33
2.56
0.10
0.09
3.14
1.94
1.20
2.93
0.33
3.61
0.10
0.09
12.4
1.08
0.02
33.1
1.74
0.30
7.88
15.2
16.2
1.06
2.92
1.61
2.64
0.56
0.72
0.94
0.022
0.073
0.18
0.25
0.03
0.01
0.08
0.06
0.09
0.13
0.08
0.005
0.014
0.08
0.003
0.01
0.02
0.004
0.45
3.57
70.9
23.7
4.88
94.0
3.05
1.88
1.18
2.93
0.30
3.62
0.09
0.08
12.0
0.95
0.01
32.5
1.70
0.34
8.19
15.3
16.4
1.15
3.10
1.83
2.86
0.57
0.74
1.19
0.023
0.072
0.20
0.27
0.03
0.02
0.08
0.06
0.10
0.15
0.09
0.005
0.014
0.09
0.003
0.01
0.01
0.004
0.46
3.70
70.1
24.0
5.43
94.0
0.148
0.079
0.041
0.123
0.024
0.139
0.006
0.006
0.28
0.075
0.001
0.88
0.039
0.012
0.348
0.451
0.49
0.047
0.120
0.084
0.115
0.037
0.044
0.052
0.0013
0.0048
0.006
0.009
0.002
0.002
0.012
0.007
0.008
0.006
0.003
0.0004
0.0018
0.008
0.0003
0.001
0.001
0.0003
0.018
0.431
0.800
0.586
0.235
0.034
0.006
0.010
0.019
0.76
0.59
0.35
0.47
0.80
0.207
0.72
0.47
0.33
0.57
0.30
0.041
0.39
0.71
0.16
0.22
0.91
0.84
0.52
0.81
0.54
0.94
0.68
0.70
0.33
0.86
0.22
0.21
0.73
0.07
0.21
0.36
0.90
0.79
0.91
0.20
0.64
0.68
0.78
0.09
0.73
0.49
0.41
0.77
0.28
0.96
0.96
0.98
0.57
6:0
8:0
10:0
cis-9 10:1
12:0
cis-9 12:1
trans-11 12:1
14:0
cis-9 14:1
trans-9 14:1
16:0
∑ cis 16:1
∑ trans 16:1
18:0
0.26
0.67
0.47
0.17
12.7
12.5
0.009
0.06
1.12
0.02
33.6
1.76
0.30
8.85
14.9
15.8
1.05
1.10
0.01
33.5
1.75
0.31
8.33
15.0
15.9
1.12
0.06
0.022
0.31
0.027
0.13
cis-9 18:1
∑ cis 18:1
trans-11 18:1
∑ trans 18:1
18:2n-6
0.24
0.13
0.10
2.74
1.23
2.18
0.51
0.65
0.51
0.018
0.055
0.16
0.22
0.02
0.01
0.06
0.05
0.07
0.10
0.07
0.004
0.011
0.07
0.005
0.02
0.03
0.005
0.41
3.67
2.90
1.40
2.42
0.56
0.72
0.71
0.019
0.067
0.17
0.24
0.02
0.01
0.07
0.05
0.08
0.11
0.08
0.004
0.012
0.07
0.004
0.02
0.03
0.005
0.43
4.82
0.005
<0.001
<0.001
0.30
∑ 18:23
cis-9,trans-11 CLA
∑ CLA
0.24
18:3n-3
18:3n-6
<0.001
0.011
0.035
<0.001
0.003
<0.001
<0.001
<0.001
0.002
<0.001
<0.001
<0.001
0.17
cis-9,trans-11,cis-15 18:3
20:0
∑ 20:1
20:2n-6
20:3n-3
20:3n-6
20:4n-3
20:4n-6
20:5n-34
22:0
22:2n-6
22:4n-6
0.003
0.005
0.012
<0.001
<0.001
0.027
0.048
0.58
0.009
0.16
<0.001
0.85
22:5n-3
22:6n-3
cis-15 24:1
26:0
28:0
∑ unidentified FA
∑ trans FA
∑ SFA
72.6
23.2
3.83
94.0
71.7
23.5
4.37
94.0
∑ MUFA
∑ PUFA
FA, g/100 g of fat
1Refers to diets containing a 60:40 forage:concentrate ratio based on grass silage (GS), red clover silage (RCS),
or a 2:1 (GRC) or 1:2 (RCG) mixture (DM basis) of GS and RCS, respectively.
2Significance of linear (L) and quadratic (Q) components of response to incremental replacement of GS with
RCS in the diet.
3Excludes isomers of CLA.
4Coelutes with 24:0.
CLA, trans-9,trans-11 CLA, and trans-11,trans-13 CLA increased linearly (P < 0.01) 15:0, 17:0, trans 15:1
(Table 6; Supplemental Table S4 and Supplemental (Δ5 and 7), cis-9 17:1, and 18-carbon oxygenated FA
Figure S1; http://dx.doi.org/10.3168/jds.2013-7358).
abundance and decreased linearly (P ≤ 0.05) concen-
Treatments also altered the abundance of OBCFA trations of several 12- to 17-carbon iso and anteiso FA.
in milk fat (Supplemental Table S5; http://dx.doi. However, increases in the proportion of RCS in the diet
org/10.3168/jds.2013-7358). Substituting GS for RCS had no effect (P > 0.10) on the concentration (Supple-
Journal of Dairy Science Vol. 97 No. 6, 2014

3770
mental Table S5) or secretion (data not presented) of al., 2009). In contrast, other studies indicate the intake
HALMEMIES-BEAUCHET-FILLEAU ET AL.
S3,R7,R11,15-tetramethyl-16:0 in milk. potential of RCS to be higher than GS or a mixture
Associations between the flow at the omasum and of both forages (Dewhurst et al., 2003b; Moorby et
secretion in milk for specific FA are documented in al., 2009). The reason for the lower intake of the RCS
Figure 1 and Supplemental Table S6 (http://dx.doi. than GS treatment in the current experiment is not
org/10.3168/jds.2013-7358). Across all treatments a obvious. Concentrations of water-soluble carbohydrates
close linear relationship existed between the flow at the and starch did not differ between ensiled forages, sug-
omasum and secretion in milk for 18:2n-6 and 18:3n-3 gesting that other factors were responsible. Differences
(R2 = 0.95 and 1.00, respectively; P < 0.02; Figure 1). in herbage composition, extent of fermentation in silo,
In contrast, associations for the most-abundant 15- and and digestibility are known to influence the intake
17-carbon OBCFA were relatively weak (P > 0.10), potential of forages in lactating cows (Huhtanen et
with the exception of iso 15:0 (R2 = 0.95; P < 0.02) al., 2007; Kuoppala et al., 2009). However, the more
and iso 17:0 (R2 = 0.73; P = 0.08; Supplemental Table extensive fermentation in silo in terms of higher lactic
S6; http://dx.doi.org/10.3168/jds.2013-7358). Further- acid concentration for RCS than GS may only partly
more, a close linear relationship also existed between explain the lower intake of RCS in the present study. In
the flow of NAN and the sum of 13- to 18-carbon OB- earlier investigations, the intake of RCS was found to
CFA (collectively 15:0; 17:0; iso 13:0, 14:0, 15:0, 16:0, be lower than GS, even when both silages were restric-
17:0, and 18:0; anteiso 13:0, 15:0, and 17:0; ∑15:1; and tively fermented in the silo and no differences existed
∑17:1; R2 = 0.83; P = 0.06) at the omasum, but not in lactic acid concentration between the ensiled forages
between the flow of NAN and secretion of OBCFA in (Kuoppala et al., 2009; Vanhatalo et al., 2009).
milk (R2 = 0.30; P > 0.10; Supplemental Table S7;
http://dx.doi.org/10.3168/jds.2013-7358).
Ruminal pool sizes of DM and NDF have been deter-
mined as being lower or similar in cows fed diets based
on RCS compared with GS (Bertilsson and Murphy,
2003; Dewhurst et al., 2003a; Kuoppala et al., 2009).
In the current experiment, the NDF concentration of
DISCUSSION
Both experimental silages were well conserved and of RCS was lower than GS, but the extent of nutrient
high nutritive and fermentation quality as indicated by digestion in the rumen was similar among treatments.
the low pH, concentration of fermentation acids, and These observations would tend to suggest that meta-
proportions of ammonium N in total N. Even though bolic rather than physical factors limited the intake of
the DM content of red clover was lower than grass at the RCS treatment. Even though the regulation of in-
the time of ensiling (19 vs. 23% for red clover and grass take involves a complex interaction between numerous
swards, respectively), the DM contents of GS and RCS factors, evidence exists to suggest that an imbalance or
were similar, suggesting higher effluent losses during lower bioavailability of AA, or both, may restrict the
the ensilage of red clover compared with grass. A higher intake of ensiled red clover harvested at an early stage
ash and CP concentration and lower concentration of
NDF in RCS compared with GS reflect differences
that are typical between ensiled grasses and red clover
(Dewhurst et al., 2003b; Lee et al., 2003; Vanhatalo
et al., 2009). However, the lower NDF concentration
of RCS was not associated with an increase in iNDF
concentration compared with GS, which often occurs
for red clover grown in Northern Europe (Bertilsson
and Murphy, 2003; Kuoppala et al., 2009). It appears
that the harvesting of red clover (late-bud stage) at an
earlier stage of maturity compared with grass (head-
ing stage) also contributed to the higher CP and lower
NDF concentration of RCS relative to GS.
Nutrient Intake
Diets based on a mixture of forages resulted in higher
DMI compared with either GS or RCS alone, which
is in agreement with several earlier reports (Bertilsson
and Murphy, 2003; Dewhurst et al., 2003a; Kuoppala et
Figure 1. Relationship between the flow at the omasum and secre-
tion in milk for 18:2n-6 and 18:3n-3 in lactating cows fed diets contain-
ing a 60:40 forage-to-concentrate ratio based on grass silage, red clover
silage, or a mixture of both ensiled forages. Each point represents the
treatment mean for n = 4 cows.
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3771
EFFECT OF FORAGE SPECIES ON NITROGEN USE AND MILK FAT
of maturity (Vanhatalo et al., 2009). Consistent with ated with higher postruminal NAN flow, principally
the relative ratio of AA to energy being an important through greater ruminal escape of dietary protein, and
determinant of intake potential, the lower intake of to a lesser extent microbial NAN (Dewhurst et al.,
the RCS than GS treatment was accompanied by a 2003a; Vanhatalo et al., 2006, 2009). Forage species had
decrease in the proportion of dietary N recovered in no effect on NAN at the omasum, such that the higher
milk, and an increase in the partitioning of N intake in escape of dietary protein on RCS-containing diets com-
feces and urine.
pensated for a decrease in microbial protein synthesis
compared with GS. In the present experiment, red clo-
ver was harvested at a relatively early growth stage, al-
though microbial NAN at the omasum can be increased
Diet Digestibility
Despite differences in the NDF and CP concentra- when red clover is harvested at a later stage of growth,
tion of silages, substituting GS for RCS had no major due to a higher DMI potential (Vanhatalo et al., 2009).
influence on apparent nutrient digestion in the rumen, Often, ruminal microbial protein synthesis has been
consistent with the limited differences between these reported energetically more efficient in cows fed RCS
forages species reported previously (Dewhurst et al., compared with GS (Dewhurst et al., 2003a; Vanhatalo
2003a; Vanhatalo et al., 2006). Increases in forage NDF et al., 2006, 2009), whereas the reverse was true in the
concentration during plant growth are often associated present study. Overall, the increases in net ruminal AA
with a decrease in ruminal NDF digestibility (Kuop- degradation (Supplemental Table S2; http://dx.doi.
pala et al., 2010). Even though the NDF concentra- org/10.3168/jds.2013-7358), in addition to a lower DMI
tion of GS was higher than RCS, forage species had no and higher lactic acid concentration during ensiling,
influence on the extent of NDF digestion in the rumen, suggests that the supply of energy, rather than AA,
which may be related to the lower ratio of iNDF to constrained the synthesis of microbial protein in the
NDF in GS compared with RCS (0.19 vs. 0.27). Grasses rumen when RCS replaced GS in the diet.
and forage legumes are also known to exhibit different
Higher escape of dietary N from the rumen and de-
ruminal digestion, passage, and comminution kinetics creases in total-tract N digestibility when RCS replaced
as a consequence of variation in morphology and cel- GS in the diet may be related to the inherent differences
lular structures (Wilson and Kennedy, 1996; Rinne and in PPO activity between forages, but the underlying
Nykänen, 2000). The passage rate of potentially digest- mechanism and mode of action remains uncertain (Lee
ible NDF in the rumen has been reported to be higher et al., 2004; Lourenço et al., 2008; Van Ranst et al.,
in cows fed diets based on RCS than GS (Kuoppala 2011). Previous studies have also reported that com-
et al., 2009). It therefore appears plausible that the pared with grasses, the protein of RCS is inherently
lack of treatment effects on ruminal fiber digestion in less degradable in the rumen (Dewhurst et al., 2003a;
the present study was related to the decrease in NDF Vanhatalo et al., 2006, 2009). Recent reports suggest
intake when RCS replaced GS in the diet and a shorter that the higher fecal N output for diets containing RCS
retention time of red clover than grass forage particles is not related to greater excretion of particle-associated
in the rumen.
CP, but more likely to be explained by incomplete di-
Although forage species had limited effects on rumi- gestion (Huhtanen et al., 2014).
nal digestion, substituting GS for RCS lowered whole-
Red clover has an inherently higher activity of PPO
tract OM, NDF, and N digestibility, indicating lower than grasses, a stress-activated enzyme that catalyzes
availability of nutrients in the small intestine. Previous the oxidation of phenols and diphenols to quinones,
studies have reported that RCS in the diet has no effect which will readily react with nucleophilic binding sites
(Bertilsson and Murphy, 2003; Vanhatalo et al., 2006; (Igarashi and Yasui, 1985). Such binding sites can be
Kuoppala et al., 2009) or may decrease (Moorby et al., found on sulfur-containing AA, resulting in the for-
2009) whole-tract OM and total NDF digestion com- mation of protein-bound phenols that may serve as a
pared with GS. It is possible that the decrease in whole- physical barrier to digestive enzymes (Van Ranst et al.,
tract N digestibility with increased RCS in the diet is 2011). It might, therefore, be expected that the flow of
related to a higher proportion of AA at the omasum bound phenols at the omasum would be increased when
originating from forage protein that has lower bioavail- RCS replaced GS in the diet, but forage species had no
ability relative to microbial NAN (Steinshamn, 2010).
influence on the amounts of these compounds escaping
the rumen (Halmemies-Beauchet-Filleau et al., 2013c).
Although the net disappearance of sulfur-containing
AA Met and Cys in the rumen did not differ between
Nitrogen Metabolism
Omasal Flow of NAN. A higher CP concentration forage species (Supplemental Table S2; http://dx.doi.
of red clover relative to grasses has often been associ- org/10.3168/jds.2013-7358), RCS in the diet progres-
Journal of Dairy Science Vol. 97 No. 6, 2014

3772
sively lowered ruminal Met and Cys degradability
HALMEMIES-BEAUCHET-FILLEAU ET AL.
Nitrogen Partitioning. Whole-body N balance
from 63 to 52% and from 64 to 48%, respectively. was positive on all diets, other than when RCS was fed
Furthermore, the decrease in ruminal degradability of alone. Differences in BCS across treatments also suggest
Cys (−17 percentage units) to RCS in the diet was that body reserves were used to support milk produc-
higher compared with that of total AA (−11 percentage tion for the RCS diet. These findings may be related to
units), which is consistent with a possible role of PPO lower bioavailability of AA with the RCS treatment, as
in protecting sulfur-containing AA from metabolism in indicated by the lower whole-tract N digestibility and a
the rumen.
possible imbalance of AA available for absorption com-
It is also possible that differences in ruminal diges- pared with diets containing GS. The effects of forage
tion kinetics offer an explanation for the effects of for- species on N retention in lactating cows are, however,
age species on ruminal protein degradation. Diets based inconsistent. Replacing GS with RCS in the diet has
on RCS alone or as a mixture with GS have in some been shown to have no effect (Bertilsson and Murphy,
(Bertilsson and Murphy, 2003; Bayat et al., 2010), but 2003; Moorby et al., 2009) or marginally improve (Lee
not in all cases (Dewhurst et al., 2003a), been demon- et al., 2009; Vanhatalo et al., 2009) whole-body N bal-
strated to alter particle size distribution in the rumen, ance. This may be related to differences in the genetic
in particular by increasing the proportion of very small merit and production potential of the animal, quality of
particles (<0.038 mm; Bayat et al., 2010) capable of the silage fed, and the forage-to-concentrate ratio used
escaping the rumen.
Omasal Flow of AA. Replacing GS with RCS had
within the studies.
Replacing GS with RCS lowered the conversion of
no major effect on the postruminal supply of AA in the dietary N into milk N from 26.4 to 23.5%, which rep-
present work, whereas earlier studies reported higher resents a typical response for lactating cows (Dewhurst
flows of all AA with RCS relative to GS, other than for et al., 2003b; Moorby et al., 2009; Vanhatalo et al.,
Met and Cys, which were unaffected by forage species 2009). A lower recovery of N in milk was accompanied
(Vanhatalo et al., 2009). Diets based on a mixture of by increased partitioning of N into urine and feces, in
GS and RCS increased the flow of most AA at the agreement with earlier reports (Lee et al., 2009; Van-
omasum that paralleled the improvements in DMI. hatalo et al., 2009).
Positive production responses to rumen-protected AA
and intravenous or postruminal infusions of AA serve
to highlight the importance of the balance of absorbed
AA on lactational performance (Schwab et al., 1992;
Milk Production
Feeding GS and RCS as a mixture increased milk
Vanhatalo et al., 1999; Robinson, 2010). Some evidence yield compared with GS or RCS as the sole forage,
exists that Met is the first-limiting AA when RCS which reflected the higher DMI of diets containing both
replaces GS in diets supplemented with cereal-based forages. Diets based on a mixture of GS and RCS have,
concentrate containing rapeseed meal (Vanhatalo et al., in some cases, been shown to improve milk yield com-
2009). Even though diets based on both GS and RCS pared with either forage alone (Moorby et al., 2009),
increased Met at the omasum compared with either for- whereas in others, milk yield has been increased in
age alone, the relative proportions of Met progressively direct response to RCS in the diet (Dewhurst et al.,
decreased as the amount of RCS in the diet increased 2003b; Steinshamn, 2010). Despite a lower DM and ME
(from 2.7 to 2.5 g/100 g of total AA). Replacing GS intake for RCS (−1.5 kg/d and −20 MJ/d, respectively)
with RCS is also known to decrease the transfer of Met relative to GS, the ECM yield was higher (+0.6 kg/d),
from the small intestine into plasma (Vanhatalo et al., indicating an improved apparent conversion of dietary
2009) and the availability of Met for absorption (Lee et energy into milk when RCS was fed as the sole forage,
al., 2009). In maize silage-based diets, Lys is often rate which is in agreement with earlier reports (Vanhatalo et
limiting for milk protein synthesis (Schwab et al., 1992). al., 2009). However, improvements in energy utilization
In the present study, RCS in the diet progressively de- for milk production may be more apparent than true,
creased the Lys concentration of omasal digesta from considering the implicated higher use of body reserves
7.1 to 6.7 g/100 g of total AA, which can be attributed in cows fed RCS.
to the lower Lys concentration of RCS relative to GS
Replacing GS with RCS in the diet increased linearly
as well as less net synthesis of Lys in the rumen. For all the secretion of lactose in milk, which can be attributed
diets, the flow of Lys at the omasum exceeded intake, to the higher lactic acid concentration of RCS relative
on average, by 7 to 12%, providing further evidence to GS and associated with increases in ruminal molar
that rumen bacteria and protozoa are capable of EAA propionate proportions (Halmemies-Beauchet-Filleau
synthesis (Onodera, 1986; El-Waziry, 2012).
et al., 2013c).
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3773
EFFECT OF FORAGE SPECIES ON NITROGEN USE AND MILK FAT
Often, replacing GS with RCS lowers milk fat con- et al., 2007; Moorby et al., 2009), but not in all cases
centration, which has been attributed to decreases (Dewhurst et al., 2003b, experiment 1; Steinshamn,
in 2:0 and 4:0 availability (Steinshamn, 2010). Even 2010). Long-chain FA containing 16 or more carbon
though RCS in the diet progressively decreased the mo- atoms inhibit synthesis of short- and medium-chain
lar ratio of lipogenic to glucogenic VFA in the rumen SFA de novo in the mammary glands of ruminants,
and increased 18:2n-6 and 18:3n-3 at the omasum (Hal- with the effects being more potent for FA with a lon-
memies-Beauchet-Filleau et al., 2013c), these changes ger carbon chain or higher degree of unsaturation, or
were not associated with alterations in the secretion of both (Shingfield et al., 2010). Daily secretion of 4- to
4- to 16-carbon SFA in milk or milk fat output.
16-carbon SFA in milk was unaffected by treatment,
Despite a marginally higher N intake of cows fed despite progressive increases in PUFA at the omasum
a mixture of both forages, RCS tended to lower milk to RCS in the diet (from 46 to 65 g/d; Halmemies-
protein concentration and secretion, which appears to Beauchet-Filleau et al., 2013c). Decreases in milk fat
be related to decreases in microbial protein supply and concentration of short- and medium-chain SFA for
lower AA bioavailability in the small intestine. In some RCS diets relative to GS in the current study was due
studies, RCS has increased milk protein yield compared to increased incorporation of preformed FA into milk
with GS (Bertilsson and Murphy, 2003, yr 1; Dewhurst triacylglycerols, rather than an overall decrease in the
et al., 2003b; Vanhatalo et al., 2006) but not in all cases synthesis of SFA de novo.
(Bertilsson and Murphy, 2003, yr 2; Vanhatalo et al.,
2009).
Nonconjugated 18:2 and 18:3 Intermediates
of Biohydrogenation. Inclusion of RCS in the diet
enriched cis-9,trans-11,cis-15 18:3 and lowered trans-
11,trans-15 18:2 concentrations in milk, which re-
flected the influence of forage species on the flow of
Milk FA
cis-9 18:1, 18:2n-6, and 18:3n-3. Consistent with these biohydrogenation intermediates at the omasum
earlier reports, substituting GS for RCS in the diet (Halmemies-Beauchet-Filleau et al., 2013c). It is pos-
increased milk fat 18:2n-6 and 18:3n-3 concentration sible that a proportion of cis-9,trans-11,cis-15 18:3 in
(Dewhurst et al., 2006; Vanhatalo et al., 2007; Moorby milk could originate from the action of stearoyl-CoA
et al., 2009). However, the enrichment of PUFA in milk desaturase on trans-11,cis-15 18:2 in the mammary
fat was not solely explained by differences in 18:2n- gland. Across all treatments, the flow of trans-11,cis-15
6 and 18:3n-3 intake between forage species (108 and 18:2 at the omasum was only moderately associated
160 vs. 120 and 133 for GS and RCS, respectively; with cis-9,trans-11,cis-15 18:3 secretion in milk, with
Halmemies-Beauchet-Filleau et al., 2013c). Replacing the regression having a slope 3 times lower compared
GS with RCS in the diet progressively increased the with cis-9,trans-11,cis-15 18:3 at the omasum. Overall,
output of 18:2n-6 and 18:3n-3 in milk in direct relation evidence from this and earlier studies (Lerch et al.,
to increases in the flows of these PUFA at the omasum 2012) do not support endogenous synthesis as a major
(from 24 to 32 and from 11 to 21 g/d, respectively; source of cis-9,trans-11,cis-15 18:3 in bovine milk.
Halmemies-Beauchet-Filleau et al., 2013c). A similar
CLA. Replacing GS with RCS in the diet had no
efficiency of transfer of 18-carbon PUFA from the effect on milk fat cis-9,trans-11 CLA concentration,
omasum into milk among treatments also suggests that consistent with earlier findings (Dewhurst et al.,
forage species does not substantially alter the bioavail- 2003b;Vanhatalo et al., 2007; Lee et al., 2009), but
ability of PUFA in the small intestine of lactating cows. progressively increased the abundance of trans-7,cis-9
Forage species had limited influence on milk fat cis-9 CLA, trans-10,cis-12 CLA, and trans-11,trans-13 CLA,
18:1 concentrations consistent with the similar flow of as reported previously (Vanhatalo et al., 2007).
18:0 and cis-9 18:1 at the omasum across treatments
Secretion of trans-10,cis-12 CLA in milk increased
(Halmemies-Beauchet-Filleau et al., 2013c) and earlier in direct response to RCS in the diet (from 7.3 to 14
comparisons (Lee et al., 2009), whereas in other studies mg/d), despite decreases in trans-10,cis-12 CLA at the
RCS has been reported to increase milk fat cis-9 18:1 omasum from 160 to 110 mg/d (Halmemies-Beauchet-
concentration (Vanhatalo et al., 2007; Moorby et al., Filleau et al., 2013c). These findings may be related
2009).
to forage species influencing the absorption or the
Short- and Medium-Chain SFA. Replacing GS partitioning of trans-10,cis-12 CLA between different
with RCS in the diet decreased linearly the proportions tissues.
of several short- and medium-chain SFA in milk fat
18:1. Red clover silage in the diet resulted in the
(4:0–8:0, 14:0, and 16:0). Several studies have reported enrichment of several cis-18:1 (Δ11–16) and trans-18:1
that RCS decreases milk fat 14:0 and 16:0 concentra- (Δ6–10, 12–16) in milk fat that originate from bio-
tions (Dewhurst et al., 2003b, experiment 2; Vanhatalo hydrogenation of 18-carbon unsaturates in the rumen
Journal of Dairy Science Vol. 97 No. 6, 2014

3774
HALMEMIES-BEAUCHET-FILLEAU ET AL.
(Shingfield et al., 2010), but the magnitude of these support measurements of milk OBCFA as a marker of
increases were rather small, consistent with earlier microbial protein supply in lactating cows, with the
observations (Vanhatalo et al., 2007). Comparison of possible exception of iso 15:0.
the flow at the omasum and secretion in milk offered
no support that the efficiency of trans (Δ4–8, 10–16)
or cis (Δ11, 12, and 16) 18:1 isomer transfer from the
CONCLUSIONS
omasum into milk of 39 to 88% differed due to forage
species. However, the efficiency of transfer of trans-9
18:1, cis-13 18:1, and cis-15 18:1 were much higher (94,
170, and 98%, respectively) suggesting that chain elon-
gation or oxidation, or both, in nonmammary tissues
may contribute to the secretion of these FA in milk fat.
OBCFA. Forage species altered the relative distri-
bution and abundance of OBCFA in milk fat. Inclusion
of RCS in the diet enhanced linearly milk fat 15:0 and
17:0 concentration, whereas concentrations of iso 15:0
and iso 17:0 were decreased, as demonstrated in earlier
studies (Dewhurst et al., 2003b; Vanhatalo et al., 2007;
Moorby et al., 2009). However, differences in the flow
of 15:0 and 17:0 at the omasum were not reflected as
changes in the secretion of these FA in milk. In ad-
dition to rumen microbes, 15:0 and 17:0 in milk may
also originate from synthesis de novo in adipose and
the mammary glands using propionate as a precursor
(Vlaeminck et al., 2006). Consistent with synthesis
in ruminant tissues, the secretion of 15:0 and 17:0 in
milk exceeded flow at the omasum, on average, by 67
and 53%, respectively, which is in agreement with ear-
lier investigations (Dewhurst et al., 2007; Halmemies-
Beauchet-Filleau et al., 2013a,b).
Across treatments, a close positive association ex-
isted between the flow of microbial NAN and the sum
of 13- to 18-carbon OBCFA at the omasum. However,
secretion of total 13- to 18-carbon OBCFA in milk was
poorly correlated with the flows of NAN or OBCFA at
the omasum, although a positive relation was identified
between the flow and secretion for iso 15:0 and iso 17:0
in milk. It has been suggested that the amount of iso
15:0 and iso 17:0 in ruminal contents could serve as a
marker of Ruminococcus flavefaciens and Butyrivibrio
fibrisolvens populations (Vlaeminck et al., 2006), the
latter exhibiting phospholipatic activity and thought
responsible for the biohydrogenation of unsaturated
FA in the rumen (Lourenço et al., 2010). Recent stud-
ies have shown that replacing GS with RCS alters the
diversity and abundance of ruminal bacteria capable
of lipid metabolism (Huws et al., 2010), which may
explain, at least in part, differences in milk fat OBCFA
concentrations due to forage species. However, the out-
put of iso 17:0 in milk exceeded the flow at the omasum
by, on average, 92%, confirming substantial postrumi-
nal synthesis (Vlaeminck et al., 2006; Dewhurst et al.,
2007). Overall, the strength of the associations between
flows at the omasum and secretion in milk did not
Diets based on a mixture of RCS and GS stimulated
higher DMI and milk yields compared with either for-
age alone when offered as part of a TMR with a 60:40
forage-to-concentrate DM ratio. Forage species had no
effect on NAN at the omasum, with increases in rumi-
nal escape of dietary protein compensating for lowered
microbial protein synthesis when RCS replaced GS.
Furthermore, RCS increased the partitioning of dietary
N in feces and urine and lowered N secretion in milk. A
less efficient utilization of N for milk protein synthesis
on diets containing RCS appeared to be associated with
lower bioavailability of AA and a possible imbalance
between AA supply and requirements for milk protein
synthesis. Substituting GS for RCS in the diet enriched
milk fat 18:2n-6 and 18:3n-3 concentrations, with no
evidence of differences in the bioavailability of PUFA
or synthesis of 4- to 16-carbon SFA de novo in the
mammary glands due to forage species. For all diets,
a substantial proportion of 15:0, 17:0, and iso 17:0 se-
creted in milk was synthesized endogenously.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the contribu-
tion of staff at the Metabolism Unit of MTT Agrifood
Research Finland (Jokioinen, Finland) for the care of
experimental animals and the collection of experimen-
tal samples under the supervision of Laila Hakkarainen
and chemical analysis undertaken in the MTT labora-
tory under the guidance of Taina Jalava. This work was
supported in part by the Finnish Funding Agency for
Technology and Innovation (Helsinki, Finland), Valio
Ltd. (Helsinki, Finland), Raisio Feed Ltd. (Raisio,
Finland), and Kemira Ltd. (Helsinki, Finland), and a
PhD studentship from the Raisio Science Foundation
(Raisio, Finland).
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