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Harstad and K. A. Beauchemin
Y.-H. Chung, E. J. Mc Geough, S. Acharya, T. A. McAllister, S. M. McGinn, O. M.
fed sainfoin or alfalfa
Enteric methane emission, diet digestibility, and nitrogen excretion from beef heifers
doi: 10.2527/jas.2013-6498 originally published online August 13, 2013
2013, 91:4861-4874.J ANIM SCI
http://www.journalofanimalscience.org/content/91/10/4861
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4861
Enteric methane emission, diet digestibility,
and nitrogen excretion from beef heifers fed sainfoin or alfalfa
1
Y.-H. Chung,* E. J. Mc Geough,* S. Acharya,*
T. A. McAllister,* S. M. McGinn,* O. M. Harstad,† and K. A. Beauchemin*
2
*Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Canada;
and †Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, Norway NO-1432 Aas
ABSTRACT: Effects of plant-bound condensed tannin
(CT)-containing sainfoin vs. CT-free alfalfa (or low-CT
alfalfa-sainfoin mixture), plant stage of maturity, and
their interaction on enteric methane (CH
4
) emissions,
diet digestibility, and N excretion were studied, using 8
ruminally cannulated beef heifers in 2 sequential short-
term experiments (Exp. 1 and 2). In Exp. 1, rst growth
legumes were harvested daily and offered fresh to heifers.
Heifers were assigned to 100% sainfoin or 80% alfal-
fa:20% sainfoin (as-fed basis). Responses were measured
at early (late vegetative to early bud; stage 2 to 3) and late
(early ower; stage 5) stage of maturity. In Exp. 2, the
same legumes were harvested from second growth (late
bud; stage 4) and offered to heifers as hay; 100% sain-
foin or 100% alfalfa. In both experiments, heifers were
fed once daily at 1× maintenance. When fed as fresh for-
age (Exp. 1), sainfoin, compared with the alfalfa-sainfoin
blend, had greater digestibility of OM (74.7 vs. 70.9%;
P = 0.02), yet tended to have lower CP digestibility (73.2
vs. 77.1%; P = 0.059). There was no difference between
fresh legumes for CH
4
emissions [25.9 g/kg DMI ± 4.02
SE; 8.5% of gross energy intake (GEI) ± 1.26 SE; or 36.8
g/kg digested OM ± 1.75 SE]. The fresh legumes were
more digestible at early, rather than at late, maturity and,
consequently, enteric CH
4
(27.4 vs. 24.4 g/kg DMI; P <
0.004; 8.9 vs. 8.1% GEI; P < 0.008) was greater at early,
rather than at later, growth. When fed as hay (Exp. 2),
sainfoin, compared with alfalfa, had greater digestibility
of OM (60.5 vs. 50.3%; P = 0.007), lower digestibility of
CP (64.2 vs. 68.8%; P = 0.004), yet there was no difference
between the legume hays for CH
4
emissions (22.4 g/kg
DMI ± 1.29 SD and 7.1% GEI ± 0.40 SD). However, on
the basis of OM digested, CH
4
emissions were lower for
sainfoin than alfalfa hay (44.3 vs. 59.0 g/kg; P = 0.008).
Percentage of total N excretion in urine was less for sain-
foin compared with alfalfa, both for fresh legumes in
Exp. 1 (74 vs. 78%; P = 0.017) or hay in Exp. 2 (64 vs.
72%; P < 0.001), and increasing maturity lowered uri-
nary N excretion. In conclusion, feeding CT-containing
sainfoin partially shifted N excretion from urine to feces,
but it had little impact on enteric CH
4
emissions from
beef cattle fed at maintenance as compared with feeding
either 80% alfalfa:20% sainfoin (fresh forages) or 100%
alfalfa (hay). Feeding fresh legumes harvested between
the late vegetative to early bud stage, compared with har-
vested at the early ower stage, increased N excreted in
urine as well as enteric CH
4
emissions from beef cattle
fed at maintenance.
Key words: alfalfa, beef heifer, enteric methane emission,
nitrogen excretion, plant-bound condensed tannin, sainfoin
© 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2013.91:4861–4874
doi:10.2527/jas2013-6498
INTRODUCTION
Enteric methane (CH
4
) from the fermentation of
feed by ruminants is the largest source of greenhouse
gas emissions from beef production (Beauchemin et al.,
2010). Pasture quality indices including species com-
position, stage of maturity, and content of condensed
tannins (CT) have been shown to inuence enteric CH
4
production from grazing cattle. For example, lactating
1
Funding for the study was from Agriculture and Agri-Food Canada,
Norway BILAT project, and Alberta Livestock and Meat Agency. We
thank F. Bonneau, F. Cloutier, B. Farr, L. Jancewicz, J. Peyrat, S. Rimbert,
C. Robin, R. Roth, and J. Wijngaarden for harvesting, sampling, and/or
laboratory analyses; T. Coates for chamber measurements, D. Vedres
for GC analyses, and staff at the Metabolism Unit and Controlled
Environment Building of the Lethbridge Research Center (Agriculture
and Agri-Food Canada, Lethbridge, AB, Canada) for animal care.
2
Corresponding author: karen.beauchemin@agr.gc.ca
Received March 20, 2013.
Accepted July 4, 2013.
Chung et al.
4862
dairy cows produced 13% less CH
4
per unit of DMI when
grazed on CT-containing pastures (birdsfoot trefoil) com-
pared with cows grazed on the same pastures, but with
CT from the pastures inactivated (Woodward et al., 2004).
Sainfoin, a CT-containing legume forage, can serve as
an alternative forage crop to alfalfa or alfalfa-grass mixed
pastures in climate-adapted environments (i.e., yields sim-
ilar to alfalfa, except under very dry conditions; Ominski
and Wittenberg, 2004; Iwaasa et al., 2006). Sainfoin was
reported to support similar animal performance compared
with alfalfa when offered as silage or hay to background-
ed steers (Bouchard, 2011). Additional ruminant health
benets of sainfoin include a marked reduction in pasture
bloat with as little as 10% sainfoin in fresh alfalfa diets
(McMahon et al., 1999). Due to its CT content, sainfoin,
when compared with alfalfa, partially redirected N excre-
tion from urine to feces in sheep and therefore could re-
duce ammonia (NH
3
) volatilization from cattle manure
(Aufrère et al., 2008). Sainfoin has also been shown to
reduce CH
4
production in vitro (Theodoridou et al., 2011).
However, reports of effects of sainfoin on CH
4
production
in vivo are limited (Bouchard, 2011).
We hypothesized that enteric CH
4
emissions from
cattle could be reduced through forage management by
feeding cattle CT-containing legume forage. The objective
of this study was to compare enteric CH
4
emissions, diet
digestibility, and N excretions from cattle fed sainfoin (a
CT-containing legume forage) or alfalfa (a CT-free legume
forage) as fresh-cut forages at 2 stages of growth or as hay.
MATERIALS AND METHODS
This study was conducted in accordance with the
guidelines established by the Canadian Council on
Animal Care (1997) and was approved by the Lethbridge
(Alberta) Research Centre Animal Care Committee.
Experimental Designs, Dietary
Treatments, and Animal Care
Two short-term experiments (designated as Exp. 1
and 2) were conducted sequentially in 2010 to evaluate
the effects of plant-bound CT on enteric CH
4
produc-
tion, diet digestibility, and N excretion, by offering beef
heifers sainfoin (a plant-bound CT containing legume
forage) or alfalfa (a CT-free legume forage) as fresh-cut
forages at early or late stage of maturity (Exp. 1), or as
hay (Exp. 2).
Experiment 1 was conducted with 2 fresh legumes
and 4 heifers assigned to each legume, with the for-
ages compared at 2 stages of maturity (in time) using
a repeated measures design. Heifers were crossbred
and previously ruminally cannulated (2C model; Bar
Diamond, Parma, ID) ~6 mo before starting the experi-
ment. Beginning BW (630 kg ± 49.9 SD) were similar
between the 2 legume treatments. The sainfoin treat-
ment contained 100% fresh-cut sainfoin, whereas the al-
falfa treatment contained 80% fresh-cut alfalfa and 20%
fresh-cut sainfoin (as-fed basis). The inclusion of sain-
foin with alfalfa was deemed necessary from an animal
care perspective to prevent legume bloat as documented
previously (McMahon et al., 1999). The early and late
stages of maturity corresponded to cutting at the veg-
etative and full bloom stages, respectively, according to
Kalu and Fick’s assessment (1981) of legume maturity.
Heifers were adapted to the fresh-cut, early-maturity le-
gume diets for 8 d before measurement of enteric CH
4
and CO
2
production, and ruminal fermentation param-
eters for 3 d (from d 9 to 11). Apparent total tract digest-
ibility was assessed over 3 d (from d 13 to 15). Heifers
remained on their respective diets for an additional 22 d
before repeating the measurements of enteric CH
4
and
CO
2
production, and ruminal fermentation from d 37 to
39, and apparent total tract digestibility from d 44 to 46
for the later maturity legumes. Ruminal degradability
of the fresh-cut legumes was evaluated using the in situ
technique (described later in this section) after comple-
tion of measurements for the late stage of maturity.
Experiment 2 was conducted as a completely random-
ized block design with 2 legume treatments (100% alfal-
fa or 100% sainfoin as hay) and 4 heifers per treatment.
With the use of hay, bloat was not a concern and hence
the alfalfa treatment was not diluted with sainfoin, which
permitted a direct comparison of the 2 legumes without
the confounding effects of the mixture. The 8 heifers used
in Exp. 1 were rerandomized before use in Exp. 2. Heifers
were paired by unfasted live BW (blocking factor). Within
each pair, they were randomly assigned to alfalfa or sain-
foin hay diets. Pre-experimental heifer BW were similar
(614 kg ± 36.9 SD) between the 2 legume treatments.
Heifers were adapted to their respective diets for 14 d be-
fore measurement of enteric production of CH
4
and CO
2
,
ruminal fermentation from d 15 to 17, and apparent total
tract digestibility from d 22 to 24. After completing total
tract digestibility measurements, ruminal degradability of
the hays was evaluated using the in situ technique.
In both experiments, heifers were fed once daily (at
1300 h) at 1× maintenance energy requirement (NRC,
2000) to minimize feed sorting and uctuations in intake
because of their potential effects on enteric CH
4
emis-
sions. During the in situ evaluation period, heifers re-
ceived their diets in 2 equal portions at 0800 and 1600 h.
Heifers were housed in a ventilated, individual tie-stall
barn with free access to water and were exercised in an
open dry lot daily. During measurement of CH
4
and
CO
2
, heifers were housed in respiration chambers (with-
out bedding) and metabolism tie stalls (without bedding)
during measurement of digestibility.
Legume forage and enteric methane emission
4863
Baseline enteric production of CH
4
and CO
2
, and
ruminal fermentation from heifers were measured for 3
consecutive days before starting Exp. 1. Heifers were
offered chopped, mixed-species grass hay for ad libitum
intake for 7 d before baseline measurements. Data ob-
tained from the baseline period were used as covariates
for CH
4
and CO
2
production (for both Exp. 1 and 2) to
reduce animal variability.
Forage Management and Sampling
Pure stands (15 m × 270 m for each legume) of sain-
foin (LRC 3519) and alfalfa (cv AC Blue J) established
in 2008 and maintained on site (49°38´ N, 112°48´ W
and elevation 900 m; Agriculture and Agri-Food Canada,
Research Center, Lethbridge, AB, Canada) were used.
In Exp. 1, both legumes were harvested daily from
2-yr-old pure stands of each crop and delivered to the
animals fresh. The legumes were cut daily at 4 cm above
ground level. The DM contents of the fresh-cut legumes
were measured daily (dried for 48 h at 55°C in a forced-
air oven) and daily subsamples were composited by
stage of maturity and stored frozen at –20°C. The sam-
ples were later thawed at room temperature and assessed
for ruminal degradability, using the in situ technique.
Morphological development of fresh-cut legumes
delivered to the animals was assessed 3 times weekly by
measuring the mean stage (dry weight) of the herbage
samples collected, using a sampling quadrant (50 cm ×
50 cm). Each stem of the herbages sampled was separated
into morphological stages (stage 0 to 9) and counted, as
described by Kalu and Fick (1981). Separated stem sam-
ples were dried in a forced-air oven for 48 h at 55°C to a
constant weight and weights of each stage were record-
ed. The dried herbage samples were then reconstituted,
ground to pass a 1-mm screen (Wiley mill; A. H. Thomas,
Philadelphia, PA), composited separately for the period
that CH
4
and digestibility was measured, and stored at 20
to 25°C until analyzed for chemical composition.
In Exp. 2, morphological development of the le-
gumes at harvesting was assessed at multiple locations
within each pure stand. Legumes were cut and sun cured
in the eld. Forages were baled into small rectangular
bales (~0.6 m width × 1.2 m length × 0.6 m height) at a
moisture level of <10%; representative core samples of
the 2 legume hays were taken from all bales and stored
at 25°C until analyzed for chemical composition.
Animal Measurements and Sampling
Intake and BW. The amount of forage offered and
refused was recorded for each heifer daily. Refusals were
sampled daily when present, composited by sampling pe-
riod, and stored at –20°C until analyzed. Dry matter intake
was calculated by subtracting the dry weight of refusals
from the dry weight of legumes offered. Body weight was
measured at the start of studies and again immediately after
chamber measurements to calculate mean BW.
Enteric Gas Production. Production of enteric CH
4
and CO
2
gases was measured from individual heifers us-
ing 4 climate-controlled, open-circuit chambers (1 heifer/
chamber), according to Beauchemin and McGinn (2006).
Before and after the studies, between-chamber differ-
ences were documented by releasing the same amount
of CH
4
and CO
2
gas into each chamber (with no ani-
mals) and then determining whole chamber ux when
the exhaust concentration reached steady state. Chamber
uxes during animal measurements were then adjusted
to eliminate differences among chambers, as described
by Beauchemin and McGinn (2006). Heifers were exten-
sively acclimatized to the chamber environment and sam-
pling procedures before the experiments. Each chamber
measured 4.4 m wide × 3.7 m deep × 3.9 m tall (63.5 m
3
volume; model C1330; Conviron Inc., Winnipeg, MB,
Canada), with heifers being accommodated within me-
tabolism stalls. Concentrations of CH
4
and CO
2
in the
intake and exhaust air ducts were monitored, using a CH
4
analyzer (model Ultramat 5E; Siemens Inc., Karlsruhe,
Germany) and CO
2
/H
2
O analyzer (model LI-7000; LI-
COR Environmental, Lincoln, NE). The analyzers were
calibrated daily using reference gases. The difference be-
tween the incoming and outgoing mass of CH
4
or CO
2
was used to calculate the amount of gases generated by
each heifer within the chamber.
Ruminal Variables. Ruminal pH was measured each
minute while the heifers were in the chambers, using the
Lethbridge Research Centre Ruminal pH Measurement
System (LRCpH; Dascor, Escondido, CA), as described
by Penner et al. (2006). The system was standardized at
the start and end of the measurement period.
A composite sample of ruminal contents was ob-
tained from 5 sites (cranial dorsal, cranial ventral, cen-
tral rumen, caudal dorsal, and caudal ventral locations)
within the rumen. Samples were obtained immediately
before heifers were moved into the chambers, daily dur-
ing CH
4
and CO
2
measurement, and again immediately
after they were removed from the chambers (total 4 ru-
minal samples per heifer per period; all collected before
feeding). Ruminal contents were ltered through 2 layers
of polyester monolament fabric (Pecap 7–255/47, mesh
opening-355 μm; Tetko Inc., Scarborough, ON, Canada).
Aliquots of the ltrates (5 mL) were stored at –20°C until
analyzed for VFA [by mixing 5 mL of the ltrate with
1 mL of 25% (wt/vol) HPO
3
] and NH
3
[by mixing 5 mL
of the ltrate with 1 mL of 1% (wt/vol) H
2
SO
4
]. Another
5 mL of the ltrate was mixed with 5 mL of methyl green-
formalin-saline solution for protozoal quantication, with
samples stored at 20 to 25°C in the dark.
Chung et al.
4864
Total Collection for Digestibility. Apparent total
tract digestibility of forage nutrients was estimated by
total collection of urine and feces. Heifers were tted
with urinary catheters (Bardex Lubricath Foley cathe-
ter, 75 c.c. and 26 Fr.; Bard Canada Inc., Oakville, ON,
Canada) to ensure separate collection of urine and feces.
Loss of NH
3
from urine was prevented by acidifying
urine with 4 N H
2
SO
4
(900 to 1000 mL as needed, such
that nal urine pH <2) during collection. Total output of
urine and feces was measured every 24 h and samples
were thoroughly mixed and subsampled. An aliquot of
the daily urine was diluted with distilled water at a ratio
of 1:5 to prevent precipitation of uric acid (Chen and
Gomes, 1992) and stored at –20°C, until analyzed for
concentrations of total N, urea N, and uric acid. A sub-
sample of the daily feces was dried for 48 h at 55°C in
a forced-air oven to a constant weight. A representative
composite fecal sample was obtained by pooling dried
daily feces based on DM content. The composited fe-
cal samples were analyzed for ash, N, NDF, and ADF.
Apparent digestibility of forage nutrients was calculated
by the difference between intake of nutrient and fecal
output of the nutrient. Retention of N was calculated by
the difference between digested N and urinary N output.
In Situ Ruminal Incubation. In Exp. 1, 4 heifers (2
from each treatment) were maintained on their respective
treatment after the measurement of CH
4
and digestibility,
and used for the in situ evaluation of forage degradability.
Fresh pooled samples of forage for each of the 2 stages
of maturity were evaluated in each individual heifer. The
materials were chopped using a wet grinder (Knifetec
1095 Sample Mill, Foss Tecator, Höganäs, Sweden) to
achieve a particle length similar to masticated forage.
In Exp. 2, 3 heifers were used for the in situ evalu-
ation. Heifers were offered a standard mixed hay diet
consisting of 50:50 alfalfa to sainfoin for 7 d before ini-
tiating the in situ evaluation. Particle sizes of the repre-
sentative core samples of the legume hays were reduced
by grinding the samples to pass a 6-mm screen to achieve
a particle length similar to that used during incubation of
the fresh forages (Wiley mill). Both legume hays were
incubated in each individual animal.
For both Exp. 1 and 2, legumes (~5.5 g DM) were
weighed into incubation bags (10 × 20 cm) made of polyes-
ter monolament fabric (pore size, 51 ± 2 µm; B. and S. H.
Thompson, Ville Mont-Royal, QC, Canada). The ratio of
sample size to surface area was 12.5 mg DM/cm
2
(Vanzant
et al., 1998). Triplicate bags for each legume were heat
sealed and placed in large (20 × 30 cm) mesh retaining sacs
with 3- × 5-mm pores that permit ruminal uid to percolate.
Bags were incubated in the rumen for 2, 4, 8, 12, 18, 24, 48,
72, and 96 h. Upon removal, bags were submerged in cold
water, hand washed under running cold tap water until the
efuent was clear, and then rinsed again (35 bags/run) in a
sieve basket (volume = 22 L) under running cold tap water
(ow rate = 5 L/30 s) for 2 h (Madsen et al., 1995). Bags
for 0 h (i.e., pre-incubation bags) were incubated in water
at 39°C for 30 min and then washed in the same manner as
the other bags. After the bags were cleaned, they were dried
for 48 h at 55°C to a constant weight and then weighed
cold. The residues were pooled according to forage species,
stage of maturity, and incubation time, and ground through
a 1-mm screen for analyses of DM, OM, N, and NDF.
Ruminal Degradation Models. In situ ruminal deg-
radation kinetics of OM, CP, and NDF for the legume
samples were tted to a nonlinear model with or without
lag time, according to Ørskov and McDonald (1979), by
using the nonlinear procedure of SAS (SAS Inst. Inc.,
Cary, NC). The Marquardt compromise was used as the
iterative method for model tting. Model parameters in-
cluded a soluble (or rapidly degradable) fraction (A, %),
slowly (or potentially) degradable fraction (B, %), which
was degraded exponentially, and the fractional rate con-
stant at which B was degraded (c, /h) with or without lag
time (h), as appropriate. Effective ruminal degradability
(%) of each forage component was calculated according
to Ørskov and McDonald (1979), assuming a passage
rate of 4% per hour (Yu et al., 2004).
Laboratory Analyses
Dried samples of refusals, grass hay, composite le-
gume forages, composite feces, and core samples of the
legume hays were ground in a Wiley mill to pass a 1-mm
screen. Analytical DM content of the ground sample was
determined by drying for 2 h at 135°C (method 930.15;
AOAC, 2005), followed by hot weighing. Ash was deter-
mined by method 942.05 of AOAC (2005) and OM was
calculated as the difference between DM and ash content.
Sample NDF and ADF contents were determined, accord-
ing to Van Soest et al. (1991), with heat-stable amylase
and sodium sulte used in the NDF procedure. Crude fat
was determined using ether extraction (method 2003.05,
AOAC, 2006; Extraction Unit E-816 HE; BŰCHI
Labortechnik AG, Flawil, Switzerland) and starch, ac-
cording to Chung et al. (2011). Gross energy was de-
termined using a bomb calorimeter (model E2k; Digital
Data Systems, Johannesburg, South Africa). Contents of
extractable CT in forage samples were measured using a
Butanol-HCL method, according to Terrill et al. (1992).
The 1-mm ground samples were reground using a ball
mill (Mixer Mill MM2000; Retsch, Haan, Germany) for
determination of N. The CP (N × 6.25) content was deter-
mined by ash combustion with gas chromatography and
thermal conductivity detection (Carlo Erba Instrumentals,
Milan, Italy). Chemical analyses of the grass hay and le-
gume forages are reported in Table 1. In situ residues were
ground and analyzed for DM, ash, and CP, as described
Legume forage and enteric methane emission
4865
previously. The NDF content of in situ residue was de-
termined using the ANKOM A200 Filter Bag Technique
(ANKOM Technology, Macedon, NY) with heat-stable
amylase and sodium sulte in the procedure.
Concentrations of VFA in ltered ruminal uid were
quantied using crotonic acid as the internal standard
and gas chromatography (model 5890; Hewlett Packard,
Little Falls, DE), with a capillary column (30 m × 0.25
mm i.d., 1 µm phase thickness, bonded PEG; Supelco
Nukol, Sigma-Aldrich Canada, Oakville, ON, Canada)
and ame ionization detection. Details of the oven setup
for gas chromatography were reported in Chung et al.
(2011). Concentrations of NH
3
in ltered ruminal uid
were determined by the salicylate-nitroprusside-hypo-
chlorite method, using a ow injection analyzer (Sims et
al., 1995). Ruminal protozoa in ltered ruminal uid were
counted, using a counting chamber (Neubauer Improved
Bright-Line counting cell, 0.1 mm depth; Hausser
Scientic, Horsham, PA) and light microscope (Ogimoto
and Imai, 1981). Duplicates (2 counts per preparation) of
each sample were counted (5 out of 9 cells were counted
per count) and if either value differed from the average by
>10%, a third sample was counted.
Concentrations of total N in acidied urine were de-
termined as described previously. Concentrations of uri-
nary urea N were measured using the diacetyl monox-
ime method, adapted for an auto analyzer (method A332,
Astoria 2 Analyzer; Astoria-Pacic, Inc., Clackamas,
OR), and concentrations of urinary uric acid were mea-
sured, according to the uricase/trinder method (Kalckar,
1947; Praetorius and Poulsen, 1953), using an assay kit
(Pointe Scientic, Inc., Canton, MI).
Statistical Analysis
Data were analyzed using the mixed model procedure
of SAS. Heifer was the experimental unit for all variables.
Data for live BW, DMI, enteric CH
4
, and CO
2
production,
and ruminal variables were covariate-adjusted by their
baseline measurements, whereas apparent digestibility, in
situ kinetics, total tract digestibility, and N excretion were
not covariate-adjusted as there were no baseline mea-
surements. For Exp. 1, the general linear mixed model
included the xed effects of forage species (sainfoin or al-
falfa), stage of maturity (early or late maturity), and their
interaction. Stage of maturity was treated as a repeated
effect (excluding the analysis of in situ kinetics). For Exp.
2, the general linear mixed model included the xed effect
of forage species. In both experiments, where appropriate,
time or day of sampling was considered a repeated effect
in the model. Animal nested within the legume treatment
(as well as blocking factor for Exp. 2) was used in the
random statement. In situ degradation parameters (A, B,
c, and effective ruminal degradability) were analyzed, us-
ing cow nested within forage type as the random term (4
heifers in Exp. 1, 3 heifers in Exp. 2) and forage type as
the xed term in the model (Exp. 1: species, stage of ma-
turity, and their interaction; Exp. 2: species). In all cases,
denominator degrees of freedom were estimated using
the Kenward-Roger option in the model statement. The
PDIFF option adjusted by the Tukey method was includ-
ed in the LSMEANS statement to account for multiple
comparisons. Time series covariance structure was mod-
eled, using various options, and the best time series cova-
riance structure was selected based on the lowest Akaike
and Bayesian information criteria. Data are presented as
means ± SD or LSM ± SEM. Differences are declared
signicant at P ≤ 0.05 and trends are discussed at P ≤ 0.10.
Table 1. Chemical composition (% DM, unless otherwise stated) of grass hay, fresh-cut legumes at 2 stages of matu-
rity, and legume hays
Variable
Mixed
species-grass
hay
Exp. 1 Exp. 2
Fresh alfalfa Fresh sainfoin
SEM
P-value
Alfalfa
hay
2
Sainfoin
hay
2
Early
1
Late
1
Early
1
Late
1
Species Stage Species × stage
DM, % 85.6 17.5 22.6 14.4 21.3 0.01 0.10 0.004 0.43 75.7 76.1
OM 92.4 88.5 91.0 91.0 92.9 0.65 0.027 0.027 0.62 90.3 91.4
CP 9.3 24.1 18.0 23.6 15.0 0.89 0.12 0.001 0.24 18.5 17.9
NDF 60.9 32.2
b
47.1
a
22.4
c
45.6
a
0.91 0.004 <0.001 0.010 53.7 42.1
ADF 39.2 26.8
b
38.9
a
20.4
c
39.2
a
1.00 0.038 <0.001 0.027 42.8 36.9
Crude fat (ether extraction) 2.2 2.6 2.0 2.4 1.9 0.20 0.53 0.051 0.98 1.4 2.3
Starch 1.4 1.1 1.4 0.9 2.1 0.22 0.32 0.029 0.11 0.7 2.0
GE, Mcal/kg DM 4.34 3.99 4.05 4.18 4.00 0.064 0.33 0.40 0.14 4.20 4.22
Extractable CT
3
0.00 0.00
b
0.00
b
2.45
a
0.66
b
0.236 0.003 0.019 0.019 0.00 0.55
a–c
Within a row (Exp. 1), means without a common superscript differ (P ≤ 0.05).
1
Early stage of maturity was estimated to be between late vegetative to early bud stage (stage 2 to 3) and late stage of maturity at early ower stage (stage 5),
according to Kalu and Fick (1981), from the rst growth of the season.
2
Alfalfa and sainfoin hay when harvested were estimated to be at late bud stage (stage 4), according to Kalu and Fick (1981), from the second growth in the same season.
3
CT = condensed tannin.
Chung et al.
4866
RESULTS
Forage Stage of Maturity
In Exp. 1, stage of maturity (i.e., stage number ac-
cording to Kalu and Fick, 1981; Fig. 1) did not differ
between fresh sainfoin and alfalfa cut at either an early
or late stage of maturity. Sainfoin and alfalfa were both
estimated to be at late vegetative to early bud at early
maturity (stages 2 and 3; average stage number = 2.6 ±
0.09 SEM) and at early ower at late maturity (stage 5;
average stage number = 5.1 ± 0.09 SEM). As expected,
the estimated morphological stages of maturity differed
between the early and late cuts (stage number 2.6 vs. 5.1,
P < 0.01). Total mass of sainfoin was estimated at 9.1
and 9.6, and alfalfa at 6.0 and 8.6 t DM/ha ± 1.1 SEM
for early and late maturity, respectively.
In Exp. 2, the estimated morphological stages of ma-
turity when harvesting sainfoin hay (average stage num-
ber = 4 ± 0.7 SD) and alfalfa hay (average stage num-
ber = 4 ± 0.2 SD) were both at late bud stage (stage 4).
Total mass was 7.0 t DM/ha for sainfoin hay and 5.3 t
DM/ha for alfalfa hay.
Forage Chemical Composition
For fresh-cut legumes (Exp. 1), contents of DM, CP,
crude fat, starch, and GE were similar (P ≥ 0.10) be-
tween sainfoin and alfalfa, but OM content was greater
(P = 0.027) for sainfoin than alfalfa (Table 1). There was
a species × stage interaction for NDF (P = 0.010) and
ADF (P = 0.027), because early-cut alfalfa had greater
ber content than early-cut sainfoin; yet, late-cut le-
gumes did not differ. With increased stage of maturity,
contents of DM, OM, NDF, ADF, and starch increased
(P < 0.03), and contents of CP (P = 0.001) and crude fat
(P = 0.051) decreased for both legumes.
The chemical composition of hays used in Exp. 2 is
descriptive because the analysis was performed on repre-
sentative core samples and no statistical analysis could be
performed. However, contents of DM, OM, and GE ap-
peared to be similar between sainfoin and alfalfa hay, with
CP content slightly lower and crude fat and starch slightly
higher for sainfoin than alfalfa hay. Sainfoin hay had con-
siderably less NDF and ADF than alfalfa, similar to that
observed for the fresh legumes harvested at early maturity.
As expected, extractable CT in alfalfa (Exp. 1 and 2)
was undetectable, whereas extractable CT in fresh sainfoin
Figure 1. Physiological stage of maturity of the fresh-cut alfalfa and sainfoin (Exp. 1) evaluated, using the Kalu and Fick (1981) method. For the sainfoin
treatment, measurements of enteric CH
4
production were conducted from 31 May to 3 June and 1 to 3 July, for early and late stages of maturity, respectively;
measurements of apparent total tract digestibility were conducted from 4 to 7 June and 5 to 8 July, for early and late stages of maturity, respectively. For the
alfalfa treatment, measurements of enteric CH
4
production were conducted from 8 to 11 June and 6 to 9 July, for early and late stages of maturity, respectively;
measurements of apparent total tract digestibility were conducted from 11 to 14 June and 12 to 15 July, for early and late stages of maturity, respectively. Sainfoin
and alfalfa were both estimated to be at late vegetative to early bud at early maturity (stages 2 and 3; average stage number = 2.6 ± 0.09 SEM) and at early ower
at late maturity (stage 5; average stage number = 5.1 ± 0.09 SEM). As expected, the estimated morphological stages of maturity differed between the early and
late cuts; stage number 2.6 vs. 5.1, respectively (stage effect: P < 0.01).
Legume forage and enteric methane emission
4867
ranged from 2.45% at early maturity to 0.66% at late matu-
rity (species × stage interaction: P = 0.019, Exp. 1). Sainfoin
hay had an extractable CT content of 0.55% DM (Exp. 2).
Body Weight, Dry Matter Intake,
and Apparent Total Tract Digestibility
Although the heifers were fed at maintenance energy
intake, they gained slightly over the study, such that mean
BW was greater when they were consuming late rather than
early-maturity legumes (Exp. 1, stage effect: P = 0.005;
Table 2). For Exp. 1, there tended (P = 0.079) to be a spe-
cies × stage interaction for DMI during digestibility mea-
surements. The DMI of sainfoin was less than that of alfalfa
(4.45 vs. 5.0 kg/d, P = 0.030), but increasing maturity in-
creased (P < 0.001) intake to a greater extent for alfalfa (4.0
vs. 6.0 kg/d) than sainfoin (3.7 vs. 5.2 kg/d). Digestibilities
of DM and OM were greater (P < 0.020), and digestibility
of CP tended to be less (P = 0.059) for sainfoin compared
with alfalfa. Increasing maturity decreased (P < 0.001) di-
gestibility of DM, OM, and CP, as expected. There was a
species × stage interaction for NDF (P = 0.019) and ADF
(P = 0.050) digestibility, because ber digestibility differed
with maturity for alfalfa but not sainfoin.
When offered as hay (Exp. 2), DMI tended to be
greater (P = 0.094) for sainfoin than alfalfa. Apparent
total tract digestibilities of DM and OM were greater for
sainfoin than alfalfa (P < 0.01), digestibilities of NDF
and ADF were similar (P ≥ 0.21), and digestibility of CP
was lower (P = 0.004) for sainfoin than alfalfa.
Total Output and Nitrogen Excretion
When legumes were offered fresh in Exp. 1, there was
a trend for a species × stage interaction (P = 0.078) for
total output of fecal DM (Table 3), because the increase in
fecal output with increasing maturity was slightly greater
for alfalfa than sainfoin. However, fecal output was con-
sistently less for sainfoin than alfalfa (P = 0.003). There
was also a trend for a species × stage interaction (P =
0.095) for total output of urine. Although urine output
did not differ between fresh-cut alfalfa and sainfoin (P =
0.55), the decline in urine output with increasing maturity
(P < 0.001) was greater for sainfoin than alfalfa.
In Exp. 1, there was a species × stage interaction
(P = 0.031) for intake of N. Heifers fed fresh alfalfa har-
vested late had greatest N intake, those fed early alfalfa
had intermediate N intake, whereas those fed early and
late sainfoin had least N intake. As a result, heifers fed
fresh sainfoin excreted less N than those fed alfalfa (P =
0.003), but stage of maturity had no effect on total N ex-
cretion (P = 0.59). However, there was a trend for a spe-
cies × stage interaction (P = 0.087) for the percentage of
total N excreted in urine; heifers fed alfalfa had greater
percentage urinary N (P = 0.017), but the decline in uri-
nary N percentage with increasing stage of maturity (P <
0.001) was greater for alfalfa than sainfoin. There was
also a species × stage interaction (P = 0.017) for fecal N
excretion; fecal N excretion was greatest for heifers fed
late alfalfa, intermediate for those fed late sainfoin, and
least for those fed early alfalfa and early sainfoin. As a
result, there was a trend for a species × stage interaction
(P = 0.087) for percentage fecal N excretion; the per-
centage of N in feces was greater (P = 0.017) for heif-
ers fed sainfoin as compared with alfalfa, but increas-
ing the stage of maturity increased the percentage of N
in feces (P < 0.001) to a greater extent for alfalfa than
sainfoin. Retained N and concentration of urinary uric
acid N did not differ (P ≥ 0.21) between legumes fed
fresh, but retained N was greater (P = 0.005) in heif-
Table 2. Body weights (live and unfasted), DMI during digestibility measurements, and apparent total tract digest-
ibility of beef heifers (n = 4/treatment), offered alfalfa or sainfoin fresh, or as hay
Variable
Experiment 1 Experiment 2
Fresh alfalfa
1
Fresh sainfoin
1
SEM
P-value
Alfalfa
hay
3
Sainfoin
hay
3
SEM
P-value
Early
2
Late
2
Early
2
Late
2
Species Stage Species × stage
Mean BW, kg 596 605 594 601 2.2 0.22 0.005 0.72 616 611 1.8 0.098
DMI, kg/d 4.0 6.0 3.7 5.2 0.19 0.030 <0.001 0.079 5.1 5.2 0.07 0.094
Digestibility, % DM
DM 75.2 63.8 79.3 67.1 1.04 0.020 <0.001 0.62 52.3 62.7 1.65 0.005
OM 77.4 64.4 81.6 67.8 1.04 0.016 <0.001 0.59 50.3 60.5 1.76 0.007
CP 84.1 70.1 78.0 68.4 1.62 0.059 <0.001 0.14 68.8 64.2 0.99 0.004
NDF 55.3
a
48.9
b
45.3
b
47.9
b
2.57 0.058 0.28 0.019 38.9 35.0 2.09 0.21
ADF 56.0
a
48.9
b
49.6
ab
48.9
b
2.26 0.20 0.021 0.050 35.4 33.1 2.09 0.44
a,b
Within a row (Exp. 1), means without a common superscript differ (P ≤ 0.05).
1
Alfalfa treatment contained 80% fresh-cut alfalfa and 20% fresh-cut sainfoin, and sainfoin treatment contained 100% fresh-cut sainfoin (as-fed basis).
2
Early stage of maturity was estimated to be between late vegetative to early bud stage (stage 2 to 3) and late stage of maturity at early ower stage (stage 5),
according to Kalu and Fick (1981), from the rst growth of the season.
3
Alfalfa and sainfoin hay when harvested were estimated to be late bud stage (stage 4), according to Kalu and Fick (1981), from the second growth of the
same season.
Chung et al.
4868
ers fed late vs. early-maturity legumes. Concentration of
urea N in urine was greater (P < 0.001) for fresh alfalfa
than sainfoin, and it decreased with maturity (P < 0.001).
However, concentrations of uric acid N in urine were not
affected (P ≥ 0.21) by legume species or maturity.
The results observed for N excretion in Exp. 1 with
fresh forages were generally conrmed in Exp. 2 using
hay, with some minor exceptions. In Exp. 2, outputs of
both fecal DM and urine were less for sainfoin hay than
alfalfa hay (P ≤ 0.040). As in Exp. 1, heifers fed fresh
alfalfa had greater (P = 0.043) N intake than those fed
sainfoin, so they had less total N excretion (P = 0.043).
Likewise, the percentage of total N excreted in urine was
less (P < 0.001), and the percentage in feces greater (P <
0.001), for heifers fed sainfoin as compared with alfalfa
when legumes were offered as hay. Retained N tended
to be greater for sainfoin (P = 0.063) than alfalfa hay, in
contrast with Exp. 1. Concentration of urinary urea was
less (P = 0.004) for sainfoin than alfalfa, and there were
no differences (P = 0.43) in urinary uric acid content
between the legumes, similar to observations in Exp. 1.
Ruminal Variables
In Exp. 1, there were species × stage interactions (P ≤
0.063) for mean ruminal pH, pH range, and minimum
pH (Table 4). In Exp. 2, mean pH tended (P = 0.091) to
be lower for sainfoin than alfalfa hay, with greater daily
uctuations (P = 0.043) for sainfoin. However, in both
studies, the pH remained above the desirable threshold
for ruminal ber digestion (i.e., 5.8), thus the differences
in pH variables due to treatments were relatively minor.
The impact of legumes on protozoa depended on
whether fresh forage or hay was fed. In Exp. 1, total
numbers of protozoa and proportions of Holotrichs and
Entodiniomorphs were not affected (P ≥ 0.30) by spe-
cies or maturity. However, in Exp. 2, numbers of pro-
tozoa were greater for sainfoin hay than alfalfa hay (P =
0.027), with a greater proportion being represented by
Holotrichs and a smaller proportion by Entodiniomorphs
(P ≤ 0.003).
In both experiments, concentrations of total VFA
were not affected by legume species, but there were,
however, changes in the individual proportions of VFA.
Proportion of acetate was consistently less for sainfoin
than alfalfa when fed fresh (P = 0.067) or as hay (P =
0.003). In Exp. 1, there was a species × stage interaction
(P < 0.001) for the proportion of propionate; propionate
proportion was only greater for sainfoin compared with
alfalfa at the late-maturity stage. In Exp. 2, propionate
proportion was similarly higher for sainfoin hay than al-
falfa hay (P = 0.005). Changes in the individual propor-
tions of VFA led to changes in the acetate to propionate
ratio. In Exp. 1, there was a species × stage interaction
(P = 0.009) for acetate to propionate ratio, with a lower
ratio for late sainfoin compared with the other treatments.
In Exp. 2, the ratio was lower for sainfoin than alfalfa
(P = 0.002). In addition, there was a species × stage in-
teraction for butyrate proportion (P < 0.001) in Exp. 1,
with early sainfoin having higher butyrate proportion
Table 3. Nitrogen intake, outputs, and nitrogen excretion from beef heifers (n = 4/treatment), offered alfalfa or sain-
foin fresh, or as hay
Variable
Exp. 1 Exp. 2
Fresh alfalfa
1
Fresh sainfoin
1
SEM
P-value
Alfalfa
hay
3
Sainfoin
hay
3
SEM
P-value
Early
2
Late
2
Early
2
Late
2
Species Stage Species × stage
Total outputs,
Feces, kg DM/d 0.99 2.12 0.75 1.70 0.074 0.003 <0.001 0.078 2.4 1.9 0.08 0.007
Urine, L/d 13.6 11.2 14.1 9.2 1.10 0.55 <0.001 0.095 10.2 7.7 0.69 0.040
N intake, g/d 136.5
b
156.8
a
123.2
bc
119.3
c
4.90 0.006 0.14 0.031 131.5 127.7 2.29 0.08
Excretion of N,
Total N, g/d 149 153 129 119 7.0 0.003 0.59 0.19 144 123 5.3 0.043
Urinary N, g/d 127 107 103 82 5.6 <0.001 <0.001 0.88 104 80 4.3 0.005
Urinary N, % total N excretion 86 70 80 68 1.3 0.017 <0.001 0.087 72 64 0.8 <0.001
Fecal N, g/d 21.7
c
45.6
a
26.3
c
37.8
b
2.60 0.55 <0.001 0.017 40.8 44.5 1.86 0.22
Fecal N, % total N excretion 14 30 20 32 1.3 0.017 <0.001 0.087 28 36 0.9 <0.001
Retained N,
4
g/d –12.2 4.2 –5.6 0.2 4.3 0.79 0.005 0.12 –13.0 2.6 5.36 0.063
Urinary urea N, g/d 51.9 42.5 36.4 30.0 1.84 <0.001 <0.001 0.28 44.6 31.1 2.00 0.004
Urinary uric acid N, g/d 0.38 0.44 0.29 0.29 0.064 0.21 0.21 0.27 0.34 0.27 0.061 0.43
a–c
Within a row (Exp. 1), means without a common superscript differ (P ≤ 0.05).
1
Alfalfa treatment contained 80% fresh-cut alfalfa and 20% fresh-cut sainfoin, and sainfoin treatment contained 100% fresh-cut sainfoin (as-fed basis).
2
Early stage of maturity was estimated to be between late vegetative to early bud stage (stage 2 to 3) and late stage of maturity at early ower stage (stage 5),
according to Kalu and Fick (1981), from the rst growth of the season.
3
Alfalfa and sainfoin hay when harvested were estimated to be late bud stage (stage 4), according to Kalu and Fick (1981), from the second growth of the same season.
4
Retained N = digested N – urinary N; Digested N = intake N – fecal N.
Legume forage and enteric methane emission
4869
compared with the other treatments, which were simi-
lar. In Exp. 2, butyrate proportion was higher for sainfoin
than alfalfa (P = 0.016). There was a trend for a species ×
stage interaction for valerate proportion (P = 0.059), as
well in Exp. 1, and a trend (P = 0.067) in Exp. 2 for higher
valerate for sainfoin vs. alfalfa. The branched-chain VFA
and NH
3
concentrations were not affected by species in
Exp. 1 or Exp. 2, but increasing maturity in Exp. 1 (P <
0.001) lowered their concentrations.
In Situ Ruminal Degradability
In Exp. 1, sainfoin and alfalfa both harvested at early
and late maturity were incubated in the rumen of 4 heif-
ers, with 2 heifers receiving sainfoin and 2 heifers receiv-
ing alfalfa (both forages fed were at late maturity). There
was no effect of diet on in situ ruminal disappearance of
legumes and therefore the data were pooled. Sainfoin
OM was more ruminally degradable, but its CP was less
degradable than that of alfalfa (Table 5; P < 0.001). For
both fresh legumes, OM and CP were more ruminally de-
gradable at early than at late maturity (P < 0.001). There
was a species × stage interaction (P = 0.041) for ruminal
degradability of NDF; NDF degradability was greatest for
early sainfoin, intermediate for early alfalfa, and least for
late alfalfa and late sainfoin. In Exp. 2, OM (P < 0.001)
was more degradable and NDF tended (P = 0.066) to be
more degradable for sainfoin than alfalfa, but CP was less
degradable (P = 0.015) for sainfoin than alfalfa.
Enteric Methane Production
In Exp. 1, compared with alfalfa, sainfoin did not re-
duce CH
4
emission (P ≥ 0.38), even after correction for
intake (26.1 vs. 25.7 g/kg DMI), OM digested (36.7 vs.
36.9 g/kg OMI), or GEI (8.5 vs. 8.4% of GEI) for fresh-
cut sainfoin vs. fresh-cut alfalfa (Table 6). Compared
with late maturity, feeding fresh legumes at early matu-
rity increased enteric CH
4
emissions from restrictively
fed beef heifers: 27.4 vs. 24.4 g CH
4
/kg DMI and 8.9
vs. 8.1% of GEI, for early vs. late stage of maturity, re-
spectively (P < 0.01). Production of CO
2
was less for
Table 4. Ruminal fermentation variables in ruminal uid from beef heifers (n = 4/treatment), offered alfalfa or sain-
foin fresh, or as hay
Variable
1
Exp. 1 Exp. 2
Fresh alfalfa
2
Fresh sainfoin
2
SEM
P-value
Alfalfa
hay
4
Sainfoin
hay
4
SEM
P-value
Early
3
Late
3
Early
3
Late
3
Species Stage Species × stage
Ruminal pH,
Mean 6.69
a
6.61
ab
6.52
b
6.64
a
0.047 0.26 0.55 0.005 6.81 6.70 0.035 0.091
Range
5
0.99 1.06 1.29 1.14 0.077 0.054 0.51 0.063 0.66 0.93 0.063 0.043
Maximum 7.25 7.15 7.21 7.17 0.045 0.78 0.02 0.27 7.15 7.19 0.026 0.38
Minimum 6.25
a
6.09
ab
5.92
b
6.03
b
0.083 0.046 0.65 0.028 6.54 6.34 0.061 0.094
Total protozoa,
6
×10
5
cell/mL 3.4 2.7 3.4 2.7 0.38 0.97 0.30 0.74 1.2 2.1 0.19 0.027
Holotrichs,
7
% total 4.9 3.7 1.5 3.9 1.91 0.34 0.68 0.24 0.6 3.4 0.55 0.002
Entodiniomorphs,
7
% total 95.1 96.3 98.5 96.1 1.95 0.34 0.68 0.24 99.4 96.6 0.56 0.003
Total VFA, mM
66.1 66.2 64.3 57.3 6.41 0.53 0.29 0.27 58.7 54.0 4.79 0.54
Individual VFA, mol/100 mol
Acetate (A) 72.5 73.4 70.4 70.8 0.77 0.067 0.057 0.45 72.5 70.9 0.37 0.003
Propionate (P) 12.8
c
13.9
b
12.5
c
15.8
a
0.45 0.051 <0.001 <0.001 14.0 14.5 0.18 0.005
A:P ratio 5.7
a
5.3
a
5.7
a
4.5
b
0.22 0.055 <0.001 0.009 5.2 4.9 0.08 0.002
Butyrate 7.0
b
7.2
b
9.5
a
7.7
b
0.48 0.056 0.004 <0.001 6.2 7.3 0.21 0.016
Valerate 0.98 0.84 1.11 0.79 0.070 0.51 <0.001 0.059 0.91 1.12 0.057 0.067
BCVFA
8
6.8 4.6 6.4 4.8 0.33 0.82 <0.001 0.26 6.4 6.3 0.35 0.82
NH
3
, mM
10.3 7.4 12.7 7.1 1.06 0.19 <0.001 0.102 8.7 9.1 0.74 0.73
a–c
Within a row (Exp. 1), means without a common superscript differ (P ≤ 0.05).
1
Corresponding ruminal fermentation variables determined immediately before and after the heifers were moved into the chambers, and daily while the heifers
were in the chambers (sample no. = 4/animal).
2
Alfalfa treatment contained 80% fresh-cut alfalfa and 20% fresh-cut sainfoin, and sainfoin treatment contained 100% fresh-cut sainfoin (as-fed basis).
3
Early stage of maturity was estimated to be between late vegetative to early bud stage (stage 2 to 3) and late stage of maturity at early ower stage (stage 5),
according to Kalu and Fick (1981), from the rst growth of the season.
4
Alfalfa and sainfoin hay when harvested were estimated to be late bud stage (stage 4), according to Kalu and Fick (1981), from the second growth of the same season.
5
Range = maximum ruminal pH – minimum ruminal pH.
6
Probabilities of effects on protozoal population were calculated based on log10 conversion of the population density, which is reported as inverse log10 LSM in the table.
7
Holotrich ciliates = Isotricha + Dasytricha; Entodiniomorphid protozoa = Entodinium + Diplodinium + Ostracodinium + Polyplastron + Eudiplodinium +
Epidinium + Ophryoscolex.
8
Branched-chain VFA (BCVFA) = isobutyrate + isovalerate.
Chung et al.
4870
early vs. late stage of maturity legumes (4.8 vs. 8.9 kg/d,
respectively; P < 0.001)
For Exp. 2, heifers offered sainfoin hay exhibited
greater (P = 0.010) CH
4
emissions per day than those fed
alfalfa hay, with emissions of CH
4
as grams CH
4
/kg OM
digested being greater for alfalfa (P = 0.008). However,
legume species did not affect CH
4
emissions on a DMI
or percentage of GE intake basis (P ≥ 0.80). Emissions
of CO
2
per animal per day were not affected by legume
species when offered as hay (P = 0.84).
DISCUSSION
This study examined the effects of feeding plant-
bound CT as a means of reducing enteric CH
4
emis-
sions and urinary N excretion from cattle. Hence, CT-
containing sainfoin was compared with CT-free alfalfa
when offered fresh (Exp. 1) at early (late vegetative to
early bud, stage 2 to 3) and late (early ower; stage 5)
maturity, or as hay (Exp. 2). Although the hay was har-
vested at a physiological stage of maturity (late bud;
stage 4) intermediate to the stages used in Exp. 1, it was
from regrowth material and wilted before baling, which
may have caused some leaf loss. Hence, NDF and ADF
concentrations were higher and digestibility lower for
the hays, compared with the late-maturity fresh forages
used in Exp. 1.
While our objective was to evaluate whether feeding
the CT-containing legume sainfoin, rather than alfalfa,
to cattle would reduce enteric CH
4
emissions, it should
be stated that these 2 legumes differed in chemical com-
position, in addition to CT content. As CH
4
is a function
of DMI and chemical composition of feed (Johnson and
Johnson, 1995), we attempted to control these sources
of variation by harvesting the material at a similar stage
of maturity and by limit feeding the animals. It should
also be emphasized that in Exp. 1, the alfalfa treatment
consisted of 80% alfalfa:20% sainfoin to prevent bloat.
Furthermore, because the fresh forages were harvested
at an early stage of maturity from the rst cutting of the
season in Exp. 1, the adaptation time of the animals to
fresh forages was very short. To minimize variability
among animals, baseline measurements conducted be-
fore starting Exp. 1 were used as covariates.
Table 5. In situ ruminal disappearances of alfalfa and sainfoin forage components, fresh-cut forage or as hay
Variable
1
Exp. 1 (n = 4) Exp. 2 (n = 3)
Fresh alfalfa
2
Fresh sainfoin
2
SEM
P-value
Alfalfa
hay
4
Sainfoin
hay
4
SEM
P-value
Early
3
Late
3
Early
3
Late
3
Species Stage Species × stage
OM,
A, % 34.7 26.3 36.2 27.4 0.69 0.033 <0.001 0.64 15.8 22.4 0.51 <0.001
B, % 48.8 42.4 55.2 46.8 1.10 <0.001 <0.001 0.30 45.4 50.6 0.66 0.005
c, /h 0.095 0.083 0.093 0.070 0.0055 0.051 <0.001 0.17 0.063 0.067 0.0053 0.68
Effective ruminal
degradability, %
68.8
b
55.0
d
74.3
a
56.9
c
0.88 <0.001 <0.001 <0.001 44.0 53.8 0.50 <0.001
CP,
A, % 49.1
a
46.4
b
32.6
d
38.7
c
0.78 <0.001 0.019 <0.001 34.3 26.4 1.35 0.015
B, % 45.4
c
40.6
d
62.3
a
48.6
b
1.06 <0.001 <0.001 <0.001 45.5 59.0 0.91 <0.001
c, /h 0.115
a
0.108
a
0.088
b
0.068
c
0.0063 <0.001 <0.001 0.044 0.060 0.047 0.0047 0.12
Effective ruminal
degradability, %
82.7 76.1 75.3 69.5 0.79 <0.001 <0.001 0.36 60.8 57.7 0.53 0.015
NDF,
A, % - - - - - - - - 2.2 0.2 0.59 0.074
B, % 57.5
b
48.2
c
73.3
a
51.5
bc
1.63 <0.001 <0.001 0.002 45.0 53.3 1.39 0.013
c, /h 0.105 0.075 0.108 0.078 0.0119 0.79 0.009 1.00 0.053 0.060 0.0062 0.49
Lag time, h 3.3 1.7 3.7 2.9 0.38 0.053 0.011 0.29 - - - -
Effective ruminal
degradability, %
42.3
b
30.6
c
50.3
a
34.3
c
1.72 <0.001 <0.001 0.041 27.6 31.4 1.06 0.066
a–d
Within a row (Exp. 1), means without a common superscript differ (P ≤ 0.05).
1
A = soluble fraction; B = slowly degradable fraction; c = fraction rate constant at which B is degraded; effective ruminal degradability was calculated as-
suming a passage rate of 0.04/h.
2
Both fresh-cut (100%) alfalfa and sainfoin were incubated in the rumen of 4 beef heifers, with which 2 heifers received the alfalfa treatment (80% fresh-cut
alfalfa and 20% fresh-cut sainfoin; as-fed basis) and 2 heifers received the sainfoin treatment (100% fresh-cut sainfoin). There was no effect of dietary treat-
ment on in situ ruminal disappearance of legume forages and therefore data from the 4 beef heifers were pooled.
3
Early stage of maturity was estimated to be between late vegetative to early bud stage (stage 2 to 3) and late stage of maturity at early ower stage, accord-
ing to Kalu and Fick (1981), from the rst growth of the season.
4
Alfalfa and sainfoin hay when harvested were estimated to be late bud stage (stage 4), according to Kalu and Fick (1981), from the second growth of the same season.
Legume forage and enteric methane emission
4871
Effects of Sainfoin on Enteric Methane Emission, Diet
Digestibility, and Nitrogen Excretion
Contrary to our hypothesis, feeding sainfoin vs. alfal-
fa did not reduce enteric CH
4
emissions from beef heifers
offered fresh (Exp. 1) or dried (Exp. 2) forages, even when
these legumes were offered at an early stage of maturity
when CT content of sainfoin was greatest. Sainfoin has
been proposed as an alternative forage crop to alfalfa or
alfalfa-grass mixed pastures to reduce enteric CH
4
emis-
sions from ruminants, in addition to its benets for reduc-
ing pasture bloat and N excretion in urine (Ominski and
Wittenberg, 2004; Iwaasa et al., 2006). Sainfoin contains
CT, a plant secondary metabolite that has been shown
to reduce ruminal methanogenesis and decrease ruminal
protozoa numbers in some studies (Tavendale et al., 2005;
Bhatta et al., 2009). In addition to the lack of effect on
CH
4
emissions, an inhibitory effect of CT-containing sain-
foin on ruminal protozoa was not evident in our studies.
In vitro studies that directly compared the CH
4
-mitigating
potential of sainfoin with alfalfa report inconsistent results
(McMahon et al., 1999; Guglielmelli and Calabro, 2011;
Theodoridou et al., 2011) and animal studies are lacking
in the literature. In support of our ndings, one in vivo
study also reported no difference in enteric CH
4
emissions
for alfalfa and sainfoin, preserved as either hay or silage,
when fed ad libitum to beef steers (Bouchard, 2011).
We expected lower enteric CH
4
emissions from heif-
ers fed sainfoin compared with those fed alfalfa because
of the greater CT content in sainfoin, especially for early-
maturity legumes. Content of extractable CT in sainfoin
was reduced with advancing stage of maturity in Exp. 1
(from 2.45 to 0.66% DM of CT for early to late maturity,
respectively) and there is likely a positive relationship
between CT content and its bioactivity in the rumen. In
contrast to our expectations, CT in sainfoin, even when
present at high concentrations in early-maturity fresh
sainfoin, did not reduce enteric CH
4
emissions. Emissions
of enteric CH
4
observed in our study ranged from 7.1 to
9.1% GEI, which is in the range typically observed for
cattle fed forages at maintenance (Blaxter and Clapperton,
1965). For cattle fed ad libitum, the percentage of GEI lost
as CH
4
would be expected to decrease by ~1.6% units per
level of intake (Johnson and Johnson, 1995).
It might be argued that the CT contained in the small
amount of sainfoin offered with the fresh alfalfa treat-
ments could have suppressed CH
4
production from the
animals fed fresh alfalfa, thereby partially negating treat-
ment effects. However, the difference between the sain-
foin contents of the fresh legume diets (20% vs. 100%
sainfoin) should have provided adequate separation in
CT content of the forages to evaluate the potential effects
of plant-bound CT in sainfoin on CH
4
production. If the
CH
4
reduction effect of CT in sainfoin is concentration
dependent, the greater CT concentrations in early vs. late
sainfoin should have lowered CH
4
production from early
sainfoin compared with late sainfoin, which was not the
case. Furthermore, compared with alfalfa, sainfoin great-
ly reduced total tract CP digestibility, effective ruminal
degradability of CP, and urinary N excretion, especially
when sainfoin was offered at early maturity at which time
the CT content in sainfoin was highest. These differences
in N metabolism were seen to be a direct effect of the CT
content of sainfoin, indicating the CT present were bio-
logically active. Moreover, a direct comparison between
sainfoin and alfalfa on CH
4
production was conducted in
Exp. 2, using 100% of each legume as hay and no CH
4
-
suppressing effect of sainfoin was observed. However, it
should be noted that the CT concentration in sainfoin hay
was lower than that of fresh sainfoin (0.55 vs. 2.45% for
early or 0.66% for late).
Relatively low extractable CT content in sainfoin
may partially explain why sainfoin did not reduce CH
4
compared with alfalfa in our study (0.55 to 2.45% DM of
extractable CT in sainfoin) and in the study of Bouchard
Table 6. Emissions of enteric CH
4
and CO
2
from beef heifers (n = 4/treatment) offered alfalfa or sainfoin fresh, or as hay
Variable
Exp. 1 Exp. 2
Fresh alfalfa
1
Fresh sainfoin
1
SEM
P-value
Alfalfa
hay
3
Sainfoin
hay
3
SEM
P-value
Early
2
Late
2
Early
2
Late
2
Species Stage Species × stage
DMI in chambers, kg/d 3.6 4.9 3.3 4.8 0.18 0.18 <0.001 0.57 5.1 5.1 0.07 0.81
Methane intensity,
CH
4
, g/d 95 120 90 113 5.3 0.38 <0.001 0.69 113 119 1.3 0.010
CH
4
, g/kg DMI
4
26.6 24.8 28.2 24.0 1.28 0.69 0.004 0.21 22.3 22.5 0.49 0.80
CH
4
, g/kg OM digested 37.8 35.9 35.7 37.7 2.14 0.95 0.93 0.107 59.0 44.3 2.95 0.008
CH
4
, % GE intake 8.6 8.2 9.1 8.0 0.40 0.63 0.008 0.18 7.1 7.1 0.16 0.87
CO
2
, kg/d 5.1 9.4 4.5 8.5 0.40 0.21 <0.001 0.50 8.3 8.5 0.66 0.84
1
Alfalfa treatment contained 80% fresh-cut alfalfa and 20% fresh-cut sainfoin; sainfoin treatment contained 100% fresh-cut sainfoin (as-fed basis).
2
Early stage of maturity was estimated to be at between late vegetative to early bud stage (stage 2 to 3) and late stage of maturity at early ower stage (stage 5),
according to Kalu and Fick (1981), from the rst growth of the season.
3
Alfalfa and sainfoin hays when harvested were estimated to be late bud stage (stage 4), according to Kalu and Fick (1981; from the second growth of the same season).
4
Absolute CH
4
or CO
2
production (grams per animal per day), corrected for DMI determined on the days of CH
4
and CO
2
measurement.
Chung et al.
4872
(2011; 1.1 to 1.2% DM of extractable CT in sainfoin).
However, the levels of CT in sainfoin in our study were
enough to depress apparent CP digestibility and reduce
urinary N excretion. The contents of extractable CT
in sainfoin in our study were within the lower end of
the CT content range (1.63 to 9.44% DM) reported for
sainfoin grown in Western Canada (Berard et al., 2011).
However, the extractable CT contents in our study are
comparable to those reported by others for sainfoin har-
vested at similar stages of maturity (Aufrère et al., 2008;
Theodoridou et al., 2011).
Sainfoin has been consistently shown to reduce uri-
nary N losses without affecting (i.e., reducing or increas-
ing) N retention in ruminants. When compared with alfal-
fa, sainfoin as fresh forage or hay both reduced urinary N
excretion without affecting N retention, an outcome sup-
ported by Aufrère et al. (2008) using sheep. When com-
pared with temperate grass-clover mixtures, sainfoin as
dehydrated and ensiled forages both also reduced urine-
N loss and had no effect on body N-retention in lambs
(Scharenberg et al., 2007). The reduction in urinary N loss
was associated with lower CP digestibility and lower ef-
fective ruminal degradability of CP for sainfoin compared
with alfalfa, although, surprisingly, rumen NH
3
concen-
trations were not affected by forage species.
The physiological development of both legumes
over the growing season was very similar (Fig. 1) and
care was taken to compare the legumes at similar stages
of maturity. Nevertheless, apparent DM and OM digest-
ibility was higher for fresh sainfoin than fresh alfalfa
(Exp. 1), which was conrmed in situ where the effec-
tive ruminal degradability of OM was also higher for
fresh sainfoin than fresh alfalfa. In addition, in situ effec-
tive ruminal degradability of NDF was greater for fresh
sainfoin than alfalfa, although there was less difference
in total tract digestibility of ber (NDF and ADF) be-
tween the two fresh legumes, possibly because the fresh
forages were fed as a mixture for the alfalfa treatment
(i.e., 80% alfalfa:20% sainfoin, as-is basis). The lack of
treatment effects on total VFA concentration in both ex-
periments was inconsistent with the observed difference
in ruminal degradability of OM.
Despite lower acetate to propionate ratio and higher
CT content, sainfoin did not reduce enteric CH
4
produc-
tion of cattle fed fresh legumes or hay. One possible ex-
planation may be the higher nutritive value of sainfoin
compared with alfalfa, as evidenced by greater OM di-
gestibility in the total tract and higher effective ruminal
degradability of OM. Expressing CH
4
emissions on the
basis of OM digested revealed that in Exp. 2, emissions
were in fact lower for sainfoin than alfalfa hay, although
this difference was not observed in Exp. 1.
Effects of Legume Maturity on Enteric Methane
Emission, Diet Digestibility, and Nitrogen Excretion
Improving forage quality has been suggested as a
means of mitigating ruminant CH
4
emissions (Beauchemin
et al., 2011). However, our study demonstrates that heif-
ers fed at maintenance produce more CH
4
(as a percentage
of GEI, grams CH
4
/kg DMI, and grams CH
4
/kg NDF di-
gested) when offered freshly cut legumes at early maturity
compared with late maturity. The impact of forage quality
on enteric CH
4
emissions has been inconsistent in the lit-
erature. For example, Wims et al. (2010) reported a 14%
reduction in CH
4
emissions (as a percentage of GEI) from
dairy cows grazed on more digestible swards (indicated as
lower herbage mass) than cows grazed on less digestible
swards (greater herbage mass), but this may have reected
differences in DMI. Boadi et al. (2002) reported a 44% re-
duction in CH
4
production per unit forage DMI or GEI from
steers grazed on early-season pastures, as compared with
late-season pastures. In contrast, Hart et al. (2009) reported
no impact of sward maturity (71% and 83% for low and
high apparent OM digestibility, respectively) on CH
4
emis-
sions from beef cattle offered freshly cut herbage. Boadi
and Wittenberg (2002) reported a lower CH
4
emission (as
liters CH
4
/kg digestible OM intake but not as a percentage
of GEI) from dairy and beef heifers when they were fed
ad libitum, but not when they were fed restrictively. It has
been suggested that greater fractional CH
4
losses can oc-
cur when cattle are fed highly digestible diets at restrictive
intakes (Johnson and Johnson, 1995). Although daily CH
4
emissions were greater for early vs. late-maturity forages,
highly digestible forages promote improvements in animal
performance, which reduces emissions per unit of product
(milk or meat), something that was not possible to estimate
in this study. Furthermore, it must be recognized that the
experimental periods in this study were relatively short and
it is possible that a longer feeding period is necessary to
cause substantial shifts in CH
4
production from cattle.
As expected, the legumes harvested at early maturity
were more digestible than those harvested at late matu-
rity. The lower DMI of heifers consuming early vs. late
forages was thought to be related to a combination of
factors including very low DM content (alfalfa, 16.2%
DM; sainfoin, 13.3% DM), high OM digestibility (al-
falfa, 77.4% DM; sainfoin, 81.6% DM), and the short
adaptation time used (8 d) to adjust the heifers from hay
to fresh forages. As a result of the lower DMI for heifers
fed early-maturity forages compared with late-maturity
forages, N retention was negative.
Urinary N excretion is considered to have a greater im-
pact on environmental pollution than fecal N excretion. In
our study, beef heifers offered early-maturity, fresh legumes
excreted a greater percentage of N in urine, than in feces,
than when offered late-maturity legumes. The estimated
ruminal and apparent total tract digestibility of CP were
Legume forage and enteric methane emission
4873
greater for fresh legumes at early maturity and hence, more
forage N was degraded and absorbed from the digestive
tract, increasing urinary as compared with fecal N excre-
tion. Others have also reported an inverse relationship be-
tween urinary N excretion and forage maturity (Vanhatalo et
al., 2009; Theodoridou et al., 2010), with digestibility of N
greater for less-mature forages, leading to greater excretion
of N in urine. On the contrary, forage maturity did not affect
the partitioning of N between urine and feces in Aufrère et
al. (2008), likely due to a lack of difference in N digestibility
in the rumen and total tract at different growth stages.
In conclusion, CT-containing sainfoin repartitioned N
excretion from urine to feces, but it had minimal impact on
enteric CH
4
emissions from beef heifers when compared
with alfalfa (or 80% alfalfa, 20% sainfoin, as-fed basis, for
fresh forages). Despite a lack of effect on enteric CH
4
, the
observed reduction in urinary N excretion, together with
bloat-resistant properties with similar animal production
performance to alfalfa, makes sainfoin an attractive, al-
ternative forage crop to alfalfa or alfalfa-grass mixtures.
However, the persistency of sainfoin under grazing pres-
sure may need to be improved for it to be widely accepted
for pastures. Decreasing the stage of maturity of fresh le-
gumes increased enteric CH
4
emissions, adjusted for DMI,
and increased the proportion of N excreted in urine in non-
growing beef cattle fed at maintenance. However, feeding
legumes harvested at an early stage of maturity would be
expected to decrease emission intensity (i.e., grams CH
4
/
kg gain) in growing beef cattle fed ad libitum.
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