Animal Feed Science and Technology 175 (2012) 7684

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Animal Feed Science and Technology

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Methane emissions by goats consuming Sericea lespedeza at different

feeding frequencies
R. Puchala a , G. Animut a , A.K. Patra a , G.D. Detweiler a , J.E. Wells b , V.H. Varel b , T. Sahlu a ,
A.L. Goetsch a,
a
b

American Institute for Goat Research, P. O. Box 730, Langston University, Langston, OK 73050, USA
USDA, ARS, U.S. Meat Animal Research Center, P. O. Box 166, Clay Center, NE 68933, USA

a r t i c l e

i n f o

Article history:
Received 8 November 2011
Received in revised form 10 March 2012
Accepted 17 March 2012

Keywords:
Condensed tannins
Goats
Methane

a b s t r a c t
Twenty-four yearling Boer (87.5%) Spanish wethers (32.5 0.36 kg body weight) were
used in a 32 d experiment to assess effects of frequency of feeding condensed tannin (CT)containing Sericea lespedeza (SL; Lespedeza cuneata) on ruminal methane emission. Fresh
SL (153 g/kg CT) was fed at 1.3 times the metabolizable energy requirement for maintenance
every day (1SL), other day (2SL), fourth day (4SL), and eighth day (8SL), with alfalfa (Medicago sativa) offered at the same level on other days. Ruminal uid for microbial assays was
collected 1 d after SL feeding and at the end of the feeding interval (short and long interval
samples, respectively). Dry matter intake was not affected by frequency of SL feeding. Daily
ruminal methane emissions increased at a decreasing rate (Linear and Quadratic; P<0.01) as
frequency of SL feeding decreased (6.3, 7.4, 10.5, 12.0 g/d for 1SL, 2SL, 4SL, and 8SL, respectively), but emissions on days when SL was fed were not affected by SL feeding frequency
(6.3, 6.4, 6.7, 7.0 g/d, respectively). There were carryover effects of feeding SL on ruminal
methane emissions. For example, with 8SL ruminal methane emission did not reach a maximum until day 56, or 45 days after SL was rst fed. Energy in ruminally emitted methane
relative to digestible energy intake increased linearly (P<0.05) as frequency of SL feeding
decreased (49, 48, 66, 81 kJ/MJ for 1SL, 2SL, 4SL, and 8SL, respectively). The number of protozoa in the short interval sample was not affected by frequency of feeding SL (5.2, 5.3,
5.7, 6.5 105 /ml), whereas the number in the long interval sample increased at a decreasing rate (Linear P<0.01; Quadratic P=0.02) as frequency of SL feeding decreased (6.5, 10.4,
18.4, 20.5 105 /ml for 1SL, 2SL, 4SL, and 8SL, respectively). In vitro methane emissions (3
wk incubation in serum bottles for methanogens; indicative of methanogen presence and
activity in ruminal uid) were lower for short than for long samples (19.0 and 24.2 ml,
respectively) and increased linearly (P<0.05) as frequency of SL feeding decreased (19.3,
19.3, 23.0, 24.8 for 1SL, 2SL, 4SL, and 8SL, respectively). In conclusion, the inuence of CT
containing SL on ruminal methane emission was immediate and short-lived, and the effect
appeared attributable to activity of methanogenic bacteria and possibly ciliate protozoa.
2012 Elsevier B.V. All rights reserved.

Abbreviations: BW, body weight; CP, crude protein; CT, condensed tannins; DE, digestible energy; DM, dry matter; GE, gross energy; HE, heat energy;
IVTDMD, in vitro true DM; ME, metabolizable energy; NDF, neutral detergent ber; OM, organic matter; PEG, polyethylene glycol; RE, recovered energy;
SL, Sericea lespedeza.
Corresponding author. Tel.: +1 405 466 6164; fax: +1 405 466 6180.
E-mail address: goetsch@langston.edu (A.L. Goetsch).
0377-8401/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2012.03.015

R. Puchala et al. / Animal Feed Science and Technology 175 (2012) 7684

77

1. Introduction
Methane emitted by ruminants represents a loss of energy that otherwise could be used for productive purposes, and
contributes to global methane emission. Animut et al. (2008a) noted that the condensed tannin (CT)-containing forage Kobe
lespedeza (Lespedeza striata) reduced ruminal methane emissions by goats compared with sorghum-sudangrass (Sorghum
bicolor). Ruminal methane emissions decreased at a decreasing rate as the dietary level of lespedeza increased, with a greater
effect per unit of CT at low versus high levels. Effect of CT on ruminal methane emissions appeared immediate, as assessed by
ruminal emission on the rst 2 d of feeding lespedeza after consuming grass on preceding days. In a related study (Animut
et al., 2008b), CT of Kobe lespedeza, Sericea lespedeza (Lespedeza cuneata), and Kobe lespedeza plus quebracho affected
ruminal methane emissions by a similar magnitude, with a doubling when polyethylene glycol (PEG), a non-ionic detergent
that has high afnity for CT (Frutos et al., 2004), was fed. Factors responsible for effects of CT on ruminal methane emissions
have not been clearly established, although studies of Animut et al. (2008a,b) indicated that CT appeared to have direct effect
on activity of methanogenic bacteria and some inuence on presence of ciliate protozoa.
Because effects of CT on ruminal methane emission are rapid after consumption and higher per unit of CT at low versus
high dietary levels, infrequent feeding and/or supplementation of CT sources might be useful to lessen ruminal methane
production. Therefore, objectives of this study were to assess effects of feeding the CT-containing forage Sericea lespedeza
at different frequencies and a legume (i.e., alfalfa) very low in CT on forage intake and digestion, ruminal methane emissions,
and heat production by goats.
2. Materials and methods
2.1. Animals and treatments
The experimental protocol was approved by the Langston University Animal Care Committee. Twenty-four yearling Boer
(87.5%) Spanish goat wethers with an initial body weight (BW) of 32.5 0.36 kg were used. Treatments entailed feeding
fresh Sericea lespedeza (original variety; SL), high in CT, every day (1SL), other day (2SL), fourth day (4SL), or eighth day
(8SL), with fresh alfalfa (Medicago sativa Cimarron) fed on the other days, at 1.3 times the metabolizable energy requirement
for maintenance (Luo et al., 2004; Sahlu et al., 2004). Soil in which the alfalfa was grown was Lawrie silt loam (i.e., ne-silty,
mixed, superactive, thermic Pachic Argiustolls) and that for SL was Piedmont silty clay loam (i.e., ne, mixed, superactive,
thermic Udertic Argiustolls). First growth forages were harvested daily using a small self-propelled Troy-Bilt sickle bar
mower (Garden Way Incorporated, Troy, NY, USA) at an approximate height of 6 cm. Forage samples were analyzed for
in vitro true dry matter (DM) digestibility (IVTDMD), which was used to predict digestible energy (DE) assuming a gross
energy (GE) concentration of 18.447 MJ/kg DM. The metabolizable energy (ME) content of forage was then calculated as
820 kJ/MJ of DE (AFRC, 1998).
2.2. Measurements
The experiment was 32 d, with 24 for adaptation and 8 for measurements. On the rst day of the collection period,
termed day 1, all wethers were fed SL. Ruminal methane and CO2 emission and O2 consumption were measured throughout
the 8 d period. The indirect open-circuit respiration calorimetry system had four head-boxes (Sable Systems International,
Las Vegas, NV, USA), allowing simultaneous gas exchange measurements on four wethers. Thus, six groups of four wethers
each, with one assigned randomly from each treatment were formed and began the experiment sequentially 8 d apart.
For adaptation to diets, wethers were placed in elevated 1.2 m 2 m pens with plastic-coated expanded metal oors. Feces
and urine collections and respiratory measurements occurred while wethers were in the 0.7 m 1.2 m metabolic crates.
During the entire experimental period, wethers were fed diets in equal portions at 08:00 and 15:00 h and had free access to
water and trace mineralized salt blocks (Big 6 Mineral Salt, American Stockman, Overland Park, KS, USA; 2400 mg/kg Mn,
2400 mg/kg Fe, 260380 mg/kg Cu, 320 mg/kg Zn, 70 mg/kg I, and 40 mg/kg Co fresh weight basis).
Feedstuffs were sampled each day and weekly composite samples were formed. Feed refusals were weighed daily and
sampled during the 8 d measurement period. Feces was collected in wire-screen baskets placed under the oor of the
metabolism crates in order to keep feces and urine separate, and urine was collected with a funnel sloping or draining into
plastic buckets containing 10 ml of 100 ml/l sulfuric acid. Aliquots of feces and urine (150 g/kg) and 40 g of orts were sampled
daily and used to form composite samples for each wether. All samples were stored at 20 C until analyses.
The calorimetry system is based on measures of gas levels in outside air and that exiting head-boxes. All gas arising from
the head of animals ows from the box to the analysis system. Head-boxes allow outside air to enter from areas such as
between the neck and the impermeable sock situated around the neck and connected to the box as well as small openings
between the removable drawer in which feed and water are placed.
Oxygen concentration was analyzed using a fuel cell FC-1B O2 analyzer (Sable Systems International), and CH4 and CO2
concentrations were measured with infrared analyzers (CA-1B for CO2 and MA-1 for CH4 ; Sable Systems International).
Prior to gas exchange measurements for each wether group, analyzers were calibrated with gases of known concentrations.
Ethanol combustion tests were completed to assure complete recovery of O2 and CO2 produced with the same ow rates as

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R. Puchala et al. / Animal Feed Science and Technology 175 (2012) 7684

used during measurements; recovery averaged 997 and 1003 ml/l of O2 and CO2 . Heat energy (HE) was determined according
to Brouwers (1965) equation.
Approximately 50 ml of ruminal digesta (i.e., one sample/goat) was sampled via stomach tube 17 h after the last meal at
15:00 h of day 1 (short interval samples) and 8 (long interval samples) of the sampling period. A subsample was collected in
a sterilized O2 -free container within 1 h of sampling for microbial measurements. The pH of the remaining ruminal uid was
measured immediately, followed by placement of 1 ml into a tube containing 4 ml of a solution of methyl green, formalin,
and saline (0.06 g methyl green, 0.85 g sodium chloride, 10 ml of 700 ml/l formaldehyde solution, and 90 ml deionized water)
for protozoa enumeration (Kamra et al., 1991) and 3 ml into a tube with 2 ml of 3 M HCl for ammonia analysis. Samples for
ammonia were frozen at 20 C until analysis. Wethers were weighed at the start of the experiment and at the beginning
and end of the measurement period.
2.3. Laboratory analyses
Samples of forage sources, forage refusals, and feces were ground to pass a 1 mm screen after drying in a forced air oven
at 55 C for 48 h. Samples were analyzed for dry matter (DM; ID 967.03), ash (ID 942.05), and N (ID 976.06) of AOAC (2006).
Samples were also analyzed for GE using a bomb calorimeter (Parr 6300; Parr Instrument Co., Inc., Moline, IL, USA) and
neutral detergent ber (aNDF; Van Soest et al., 1991) with the addition of a heat stable alpha amylase and sodium sulte.
The NDF was determined using an ANKOM200 Fiber Analyzer (lter bag technique; ANKOM Technology Corp., MacEdon,
NY, USA) and expressed inclusive of residual ash. The IVTDMD of forage samples was determined by placing 0.25 g samples
in ANKOM F57 lter bags and incubation in a DaisyII incubator (ANKOM Technology Corp.) for 48 h. Bags were heat sealed
and incubated in buffered ruminal uid using a previously described method (Wilman and Adesogan, 2000), with aNDF as
the end-point measure. Ruminal uid for IVTDMD was collected from four Boer crossbred wether goats grazing native grass
pasture, consisting primarily of common bermudagrass (Cynodon dactylon), Indiangrass (Sorghastrum nutans), Little Bluestem
(Andropogon scoparium), and Big Bluestem (Andropogon gerardi), and supplemented with approximately 7.5 g/kg BW (DM
basis) of a pelleted concentrate containing 203 g/kg crude protein (CP) on a DM basis. The ingredient composition (DM basis)
of the supplemental concentrate was 150 g/kg cottonseed hulls, 200 g/kg dehydrated alfalfa meal, 134 g/kg wheat middlings,
250 g/kg ground corn grain, 188.5 g/kg soybean meal (540 g/kg CP, Preston, 2011; solvent extracted), 50 g/kg pelleting agent
(Pellet Partner; Westway Feed Products LLC, New Orleans, LA, USA), 4 g/kg dicalcium phosphate, 11 g/kg calcium carbonate,
5 g/kg salt, 5 g/kg ammonium chloride, and 2.5 g/kg trace mineral and vitamin premix (49.5 mg/kg methionine, 10.7 mg/kg K,
7.9 mg/kg Mg, 12 mg/kg S, 37.7 mg/kg Zn, 35.5 mg/kg Fe, 19 mg/kg Mn, 90 mg/kg Co, 480 mg/kg I, 26 mg/kg Se, 2,646,000 IU/kg
vitamin A, 882 IU/kg vitamin D3 , 3308 IU/kg vitamin E, 11.0 mg/kg vitamin B12 , 364 mg/kg menadione, 2426 mg/kg riboavin,
4057 mg/kg d-pantothenic acid, 11,025 mg/kg niacin, 163 mg/kg vitamin B6 , 44.1 mg/kg folic acid, 27,672 mg/kg choline, and
2.2 mg/kg biotin, fresh weight basis; Nutra Blend Corp., Neosho, MO, USA).
Urine samples were analyzed for DM (lyophilization), and N and GE concentrations in lyophilized urine samples were
determined as described above. Forage subsamples were also lyophilized for the analysis of CT with the butanolHCl colorimetric procedure of Terrill et al. (1992) using CT extracted (Sephadex LH-20, Sigma Chemical Co., St. Louis, MO, USA) from
SL as the standard (Jackson et al., 1996). Ruminal uid was analyzed for ammonia N (Broderick and Kang, 1980).
For ruminal microbial analysis, serial 10 fold dilutions of ruminal uid were prepared for each sample using the anaerobic
dilution solution of Bryant and Burkey (1953). The dilution range used was from 108 to 1010 for counts of total viable bacteria
and 107 to 109 for cellulolytic bacteria. Total viable counts were determined in roll-tubes using the complete medium of
Leedle and Hespell (1980). The cellulolytic medium used was that described by Halliwell and Bryant (1963). Cellulolytic
bacterial counts were determined by the Most Probable Number method (Morvan et al., 1994). All tubes were incubated at
39 C for 2 wk. There was 50 ml of culture media for methanogens (Morvan et al., 1994) dispensed into serum bottles and
inoculated with 1 ml of 104 diluted ruminal uid and incubated for 3 wk for estimation of CH4 gas production. Methanogenic
cultures were pressurized to 202 kPa with 800 ml/l H2 and 200 ml/l CO2 . Total viable counts were determined by direct
count. Methane produced in serum bottles was analyzed using an infrared analyzer (MA-1, Sable Systems International).
The gas mixture from the 150 ml bottles used for incubation of methanogens was transferred into 250 ml glass syringe and
injected at a rate of 400 ml/min into the infrared analyzer through a 5 cm 1.5 cm column lled with granules of calcium
sulfate as a desiccant (WA Hammond Drierite Company, Xenia, OH, USA). Methane production is indicative of presence and
activity of methanogens in ruminal uid. Ciliate protozoa were enumerated microscopically using a 0.1-mm deep Neubauer
hemocytometer counting chamber (Hausser Scientic, Horsham, PA, USA) after xing with methyl green formalin saline
solution.
2.4. Calculations and statistical analysis
Intake of DE was calculated as the difference between GE intake and fecal GE, energy lost as methane was total methane
emitted in l/day 39.5388 kJ/l (Brouwer, 1965), and ME was the difference between DE and the sum of GE in urine and
methane. Recovered energy (RE) was the difference between ME and HE.
Data were analyzed using mixed model procedures of SAS (1990Version). Data were analyzed as a completely randomized
design with the model: Yij =  + Ti + eij, where Yij is the dependent variable,  the overall mean, Ti the xed treatment effect,
and eij the residual. In addition to statistical analysis of average daily variables over the 8 day period of measurements,

R. Puchala et al. / Animal Feed Science and Technology 175 (2012) 7684

79

Table 1
Composition of forage consumed by yearling Boer goat wethers.
Sericea lespedeza

Dry matter (g/kg)

Ash (g/kg DM)
Nitrogen (g/kg DM)
a
Neutral detergent ber (g/kg DM)
Gross energy (MJ/kg DM)
In vitro true DM digestibilitya (g/kg)
Condensed tanninsb (g/kg DM)
a
b

Alfalfa

Mean

SE

Mean

SE

378
71
21.2
393
18.7
716
153

21.2
4.6
1.6
11.7
0.14
14.1
3.6

314
131
27.0
386
17.1
769
2

12.1
11.9
1.36
10.2
0.25
7.1
0.2

Filter bag technique, with NDF as the end-point measure at 48 h of incubation.

Analyzed using extracted condensed tannins from Sericea lespedeza as the standard.

Table 2
Effects of frequency of feeding Sericea lespedeza on body weight and intake and digestion of dry matter, organic matter, and nitrogen by yearling Boer goat
wethers.
Treatmenta
1SL
Body weight (kg)
Dry matter
Intake (g/d)
Digestion (g/kg)
Digestion (g/d)
Organic matter
Intake (g/d)
Digestion (g/kg)
Digestion (g/d)
Nitrogen
Intake (g/d)
Digestion (g/kg)
Digestion (g/d
a
b

32.3

SEM
2SL
32.7

4SL
32.3

8SL
32.5

Pb
Linear

1.29

Quadratic

0.99

0.95

881
493
437

904
532
483

957
579
560

906
568
521

50.5
24.7
45.1

0.76
0.05
0.22

0.31
0.09
0.13

796
485
389

827
531
440

799
576
466

826
567
474

45.2
25.2
40.2

0.76
0.04
0.19

0.92
0.08
0.39

19.1
436
8.4

22.5
586
13.2

24.9
690
17.3

23.7
705
16.9

1.33
22.3
1.29

0.05
<0.01
<0.01

0.03
<0.01
<0.01

1SL, 2SL, 4SL, and 8SL = Sericea lespedeza fed every day, other day, fourth data, and eighth day, respectively, with alfalfa fed on other days.
Linear and quadratic effects of frequency of feeding Sericea lespedeza.

a mixed model (Littell et al., 1998) consisting of treatment, day of sampling (i.e., short and long), and their interaction,
with a measure for day and random effect of animal within treatment, was used to address potential effects of time after
consuming CT-containing forage. For variables with a signicant (P<0.05) interaction between day and treatment, an analysis
was conducted by day. Means separation was by orthogonal contrasts for linear and quadratic cubic effects of frequency of
SL feeding with polynomials determined by the IML procedure of SAS. For a mixed model to determine effects of treatment
and day on daily ruminal methane emission and HE within the 8 day measurement period, means separation was by least
signicant difference when the treatment effect was signicant (P<0.05). For observations of 2SL, 4SL, and 8SL, averages of
days of feeding alfalfa or SL were subjected to mixed model analysis with a model consisting of treatment, feeding of alfalfa
versus SL, and their interactions, with a repeated measure of feeding of the different forages and random effect of animal
within treatment.
3. Results
The composition of forages (Table 1) was generally similar to previous reports from this research site (Puchala et al.,
2005, in press; Animut et al., 2008a,b). Although, the level of CT in SL was less in fresh SL but similar to that SL hay used by
Puchala et al. (in press). The concentration of CT in alfalfa was negligible as expected. The level of NDF in alfalfa was slightly
lower than in SL, and in accordance IVTDMD was greater for alfalfa. The GE content was greater for SL, which was primarily
attributable to the lower level of ash.
Intakes of DM and organic matter (OM) were not affected by frequency of feeding SL (Table 2). Digestibilities of DM
and OM increased linearly as frequency of SL feeding deceased, with a tendency (P0.10) to a maximum at 4SL. Nitrogen
intake and digested N intake increased at a decreasing rate (Linear P<0.05; Quadratic P<0.03) with decreasing frequency of
SL feeding.
Frequency of feeding SL did not affect GE intake (Table 3), but DE (kJ/MJ) differed among treatments in accordance with
DM and OM digestibilities. However, DE in MJ/d was not affected by frequency of SL feeding. Urinary energy tended to
increase linearly (P=0.06) as frequency of feeding SL decreased.
Ruminal methane emissions in g/d and MJ/d increased at a decreasing rate (Linear P<0.01; Quadratic P<0.01) as frequency
of SL feeding deceased (Table 3) because of differences between days when SL and alfalfa were fed (Fig. 1). For example,
ruminal methane emission on days when SL was fed was 6.3, 6.4, 6.7, and 7.0 g/d for 1SL, 2SL, 4SL, and 8SL, respectively.

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R. Puchala et al. / Animal Feed Science and Technology 175 (2012) 7684

Table 3
Effects of frequency of feeding Sericea lespedeza on energy utilization by yearling Boer goat wethers.a
Treatmentb

GE intake (MJ/d)
DE (kJ/MJ)
DE (MJ/d)
Urinary energy (MJ/d)
Methane
g/day
MJ/d
kJ/MJ GE
kJ/MJ DE
g/kg dry matter intake
g/kg digestible OM intake
ME (MJ/d)
ME (kJ/MJ)
Heat energy (MJ/day)
Heat energy (kJ/kg BW0.75 )
Recovered energyd (MJ/day)
a
b
c
d

SEM

Pc

1SL

2SL

4SL

8SL

16.21
503
8.26
0.50

16.60
567
9.43
0.51

17.11
601
10.41
0.66

15.71
610
9.70
0.65

0.913
32.5
0.913
0.061

0.61
0.05
0.36
0.06

0.33
0.18
0.18
0.21

6.3
0.38
24.1
49.4
7.3
17.1
7.38
888
6.04
444
1.34

7.4
0.45
26.9
48.0
8.2
17.0
8.48
896
6.17
453
2.31

10.5
0.63
37.7
65.8
11.2
24.2
9.11
866
7.19
532
1.93

12.0
0.73
47.1
80.6
13.6
27.0
8.32
847
7.19
530
1.13

0.30
0.018
2.59
8.86
0.76
2.78
0.911
18.2
0.257
14.1
0.771

<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.61
0.06
<0.01
<0.01
0.56

<0.01
<0.01
0.28
0.87
0.24
0.47
0.23
0.93
0.07
0.01
0.43

Linear

Quadratic

GE = gross energy; DE = digestible energy; OM = organic matter; ME = metabolizable energy.

1SL, 2SL, 4SL, and 8SL = Sericea lespedeza fed every day, other day, fourth data, and eighth day, respectively, with alfalfa fed on other days.
Linear and quadratic effects of frequency of feeding Sericea lespedeza.
Difference between ME intake and heat energy.

18
1

CH4 (g/d)

16
14
12
10
8
6
1

4
5
Day of the feeding cycle

Fig. 1. Daily methane emission by yearling Boer goat wethers fed Sericea lespedeza every day (), other day (), fourth day (), or eighth day (), with
fresh alfalfa fed on other days. [All goats received Sericea lespedeza on d 1.].
650
1

HE (kJ/ kg MBW)

600
550
500
450
400
1

Day of the feeding cycle

Fig. 2. Daily heat energy by yearling Boer goat wethers fed Sericea lespedeza every day (), other day (), fourth day (), or eighth day (), with fresh
alfalfa fed on other days. [All goats received Sericea lespedeza on day 1. MBW = kg BW0.75 .].

Ruminally emitted methane energy relative to intake of DM, digestible OM, and DE increased linearly (P<0.05) as frequency
of SL feeding decreased. Metabolizable energy intake in MJ/d not affected by frequency of feeding SL, although ME in kJ/MJ
DE tended to decrease linearly (P=0.06) as frequency of SL feeding decreased.
Average daily HE in MJ/d (Linear P<0.01; Quadratic P=0.07) and kJ/kg BW0.75 (Linear P<0.01; Quadratic P<0.02) increased
at a decreasing rate as frequency of SL feeding decreased (Table 3). Heat energy for 2SL on most days when alfalfa was fed
was higher than for 1SL and lower when SL was offered (P<0.05; Fig. 2). Conversely, for 4SL and 8SL, on days when SL was
fed HE was similar to or slightly higher (i.e., d 5 of the 8 d sampling periods for 4SL) than that for 1SL, but was not lower.
Nonetheless, RE was not inuenced by frequency of SL feeding.

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81

Table 4
Effects of frequency of feeding Sericea lespedeza on ruminal pH and ammonia N concentration, numbers of bacteria and protozoa, and in vitro methane
production for yearling Boer goat wethers.
Samplea

Mean

SEM

Treatmentb
1SL

Ruminal pH
Short
Long
Ruminal ammonia
nitrogen (mg/100 ml)

6.46 a
6.74 b

Bacteria (1010 /ml)

4.8
4.1

Ciliate protozoa
(105 /ml)

Short
Long

Cellulolytic bacteriad

Short
Long

8.75
8.92

Short
Long

19.0 a
24.2 b

In vitro methane
productione (ml)

2SL

4SL

8SL

Pc
Linear

6.67

6.63

6.55

6.55

0.062

2.6
3.1

2.3
3.4

2.5
6.8

2.5
5.9

5.1

4.8

4.3

5.2
6.5

5.3
10.4

19.3

19.3

Quadratic

0.19

0.41

0.42
0.44

0.96
<0.01

0.87
<0.01

4.2

1.34

0.75

0.79

5.7
18.4

6.5
20.5

0.98
1.85

0.31
<0.01

0.99
0.02

23.0

24.8

1.73

0.02

0.58

0.036

Short
Long
Short
Long

SEM

0.74

0.132

1.07

a
Short = 1 d after lespedeza was fed; long = at the end of the interval of feeding lespedeza (i.e., nearly 1, 2, 4, and 8 days for 1SL, 2SL, 4SL, and 8SL,
respectively.
b
1SL, 2SL, 4SL, and 8SL = Sericea lespedeza fed every day, other day, fourth data, and eighth day, respectively, with alfalfa fed on other days.
c
Linear and quadratic effects of frequency of feeding Sericea lespedeza.
d
Most Probable Number.
e
Incubation of 0.0001 ml ruminal uid in a methanogenic medium for 3 wk, with methane production indicative of initial methanogen presence.
Sample means without a common letter (a,b) differ (P<0.05).

Ruminal pH was higher (P<0.05) for long than for short samples (Table 4). There was no effect of frequency of feeding SL
on ruminal ammonia N concentration for short samples, but for long samples the level increased at a decreasing rate (Linear
P<0.01; Quadratic P<0.01) as frequency of SL feeding decreased. Neither the number of total nor cellulolytic bacteria was
affected by frequency of SL feeding or day of sampling. The number of ciliate protozoa in short samples was not affected by
frequency of SL feeding. Conversely, the number in long samples increased at a decreasing rate (Linear P<0.01; Quadratic
P<0.02) as SL feeding frequency decreased. In vitro methane production was less for short than for long samples (P<0.05)
and increased linearly (P<0.02) as frequency of SL feeding decreased.
4. Discussion
4.1. Intake and digestion
The lack of effect of frequency of feeding SL on intake of DM, OM and GE agrees with recent ndings with SL and alfalfa
fed as fresh forage and hay (Puchala et al., in press). However, in other studies intake of diets of various forages containing
CT has been less and greater than that of forages very low in CT (Barry and McNabb, 1999; Landau et al., 2000; Bhatta et al.,
2002; Carulla et al., 2005; Puchala et al., 2005, in press; Animut et al., 2008a,b).
Based on IVTDMD, slightly higher DM and OM digestibilities (i.e., g/kg) were expected for treatments with infrequent
feeding of SL compared with daily or every other day feeding, as occurred. It is not possible to determine if observed in vivo
differences were attributable to effects of CT or factors such as level of NDF or wall structure. Based on supplementation
with PEG, CT did not affect DM or OM digestibility with fresh SL, but with SL hay feeding it decreased OM digestibility by
10% (Puchala et al., in press). The lack of treatment effects on daily intake of digestible OM, DE, and ME agrees with previous
research with lespedeza (Animut et al., 2008a,b; Puchala et al., in press). Intake of DE was not inuenced by frequency of
SL feeding, despite the linear effect on DE concentration, because of similar GE intake among treatments and relatively high
variability in DE concentration and daily intake compared with values for OM.
In close accordance with results of Puchala et al. (in press) relative to PEG supplementation of SL as fresh forage and
hay, effects of SL CT on apparent total tract N digestion were substantial. Effects of PEG of a similar magnitude as in our
experiment were noted by Animut et al. (2008b) with fresh Kobe lespedeza and SL. Based on estimates of metabolic fecal CP
and true protein digestibility of Moore et al. (2004), predicted N digestion was 670, 700, 710, and 710 g/kg for 1SL, 2SL, 4SL,
and 8SL, respectively. Actual values for 4SL and 8SL were similar to predictions, but those for 1SL and 2SL were much lower.
To ascertain the major site of effects of CT on total tract N digestion, Animut et al. (2008a,b) and Puchala et al. (in press)
viewed ruminal ammonia concentration as an indicator of binding of protein in the rumen by CT and, thereby, decreased
degradation by ruminal microbes. On this basis, it was postulated that major effects of CT in those studies were postruminal
through decreased rebinding of CT to proteins (Arienti et al., 1974) and/prevention of incomplete release of protein initially

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bound to CT in the rumen (Min et al., 2003). However in our experiment ruminal ammonia concentration in long samples
of 4SL and 8SL was greater than for 1SL and 2SL and compared with all short samples, suggesting that CT binding of protein
in the rumen for 1SL and 2SL lessened ruminal microbial protein degradation. This probably also occurred with 4SL and 8SL
on days of SL feeding, but without effect on ruminal ammonia concentration near the time of subsequent feeding of SL. It
appears that CT affected ruminal microbial protein digestion for at least 1 day after the day of SL feeding, with dissipation of
the effect between 2 and 3 days. However it is not possible to elucidate the magnitude or contribution of any postruminal
effects to treatment differences in total tract N digestion.

4.2. Methane
Ruminal methane emissions relative to DE intake for 1SL and 2SL were 74 and 60 of that of 4SL and 8SL, respectively.
Magnitudes of effect were slightly greater than observed by Puchala et al. (in press) with SL in fresh and hay forms (199
and 153 g/kg CT, respectively; 6889% of that with consumption of alfalfa and SL supplemented with PEG) but less than for
fresh Kobe lespedeza (151 g/kg CT) compared with sorghum-sudangrass (52%; Animut et al., 2008a) and fresh SL (140 g/kg
CT), Kobe lespedeza (151 g/kg), a mixture of these forages, and Kobe lespedeza plus quebracho tannin fed without and with
PEG (55%; Animut et al., 2008b). Smaller effects of CT on methane emission have been previously observed. For example,
Woodward et al. (2001) reported that sheep methane emission relative to digestible DM intake was 2429% lower when
the CT-containing forage Greater Birds-foot Trefoil (Lotus pedunculatus) was fed compared with ryegrass or alfalfa. A similar
difference relative to DM intake (i.e., 23%) occurred for cows fed silage of Birds-foot Trefoil (Lotus corniculatus) compared
with ryegrass silage. Such differences among studies may be attributable to CT level, with a much higher concentration in
SL of our experiment compared with L. pedunculatus (8% CT) or L. corniculatus (26 g/kg CT) in these and other studies.
Ruminal methane emissions within the 8 d sampling period indicates immediate and maximal, or near maximal, impact
on the day of feeding SL. On all but one day ruminal methane emissions for 2SL on the day of SL consumption was similar
to that for 1SL. Likewise, on one of the 2 d SL feeding for 4SL, ruminal methane emissions were similar to that for 1SL and
2SL and, on the other day, the value was similar to that for 2SL and only slightly higher than for 1SL. There were carryover
effects of feeding SL on ruminal methane emission. For 8SL, ruminal methane emission did not reach a maximum until day
56, or 45 days after SL was fed. Therefore, for 4SL, SL was fed before maximum ruminal methane emission was achieved.
Furthermore, there appeared to be a somewhat different pattern of change in methane emission for 4SL on the last 12d
before feeding SL compared with 8SL, suggesting a different long-term change in microbial activity for 4SL versus 8SL.
Based on methane emissions relative to DE intake, in order to achieve a sizable reduction in daily ruminal methane
emission by feeding SL, feeding either every day or other day seems necessary. However, the linear effect of frequency of SL
feeding raises the possibility that the effect with feeding every third day might not have been markedly different from that
of the 2SL treatment, which warrants further experimentation. Another area in need of study is the impact of level of CT in
SL as well as other sources of CT when fed infrequently.
Previous research with different sources of CT suggested that effects on ruminal methane emission are direct on
methanogenic bacteria and also via impacts on activity of ciliate protozoa, such as in numbers or associations with
methanogens (Animut et al., 2008a,b; Puchala et al., in press). In vitro methane production and the number of ciliate protozoa
in this experiment seem supportive of these effects, but do not provide adequate information for partitioning causal factors.

4.3. Energy
Even though RE was not affected by treatment, considerably higher HE for 4SL and 8SL versus and 2SL and temporal
patterns in HE during the 8 day measurement period suggest effects of CT on energy metabolism. Heat energy for 2SL
changed markedly from day to day, being lower on days of feeding SL compared with alfalfa, as was also evident for 4SL and
8SL. These effects would not seem attributable to differences in feed intake, as there was no effect on DM intake of forage
being fed on a particular day for 2SL, 4SL, and 8SL treatments. A small portion of the effect of SL feeding frequency on HE
could have been due to higher energy use in hepatic ureagenesis as indicated by the linear increase in urinary energy as
frequency of SL feeding decreased. Also, CT and other plant secondary metabolites in SL could have affected feeding behavior,
although not characterized in our experiment. Condensed tannins have decreased the length and increased the number of
feeding bouts (Perevolotsky et al., 2006, as cited by Estell, 2010), suggesting that eating time could have differed among
treatments in our experiment. It has been postulated that HE increases with increasing time spent eating due to associated
physiological processes (Osuji, 1974). Likewise, Berhan et al. (2005) and Beker et al. (2009) observed a relationship between
time spent grazing and HE by meat goat does, but similar relationships did not occur with meat goat wethers grazing while
tethered or given free movement (Patra et al., 2008a,b) or with meat goat does given continuous pasture access compared
with does conned at night without feed access (Tovar-Luna et al., 2011). In addition, with different animal species, other
plant secondary metabolites have lessened locomotor activity and HE, perhaps as a coping mechanism for increased energy
use in toxin elimination (Estell, 2010). But, relevance of these ndings to CT and goats is unknown and, specically, how
such inuences might be mediated, such as direct pharmacological effects of toxins in the central nervous system, which
has only been suggested (Boyle and Dearing, 2003).

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83

5. Conclusions
The inuence of SL CT on ruminal methane emissions by goats was immediate and short lived. The effect appeared
attributable to changes in activity of methanogenic bacteria, with probable involvement of activity of ciliate protozoa. Feeding
forages, such as SL, high in CT may provide a means of reducing ruminal methane emission by goats, with benets of increased
efciency of energy utilization. Although potential adverse effect of forage CT on N digestion warrants consideration, its
importance will likely vary with dietary N level.
Disclaimer
Mention of trade names or commercial products in this article is solely for the purpose of providing specic information
and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.
Acknowledgement
This experiment was supported by USDA Project Number 2004-38814-15045.
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