Grouping and classification of Paspalum genotypes
activity and carbon gain for a longer time period (Givnish
according to tolerance to drought
1987; Lamont et al. 2002; Escudero et al. 2008).
Reduction in water loss and protection of meristems can
Among the accessions there is genetic diversity, as seen
also ensure regrowth and survival of plants when drought
in the PCA. However, there was no grouping per species
conditions occur, and thus represent a strategy that some
or per botanical group, which reflects the high genetic
plants use to tolerate drought (Volaire and Lelièvre 2001;
variability that may be present not only among species but
Munne-Bosch and Alegre 2004; Volaire et al. 2014).
also within each species of this genus. Our results suggest
Besides decreasing the production of plant biomass,
that these Paspalum accessions can be grouped according
drought can change the photoassimilate partitioning in
to response strategies to stress caused by water restriction.
plants. Studies with Urochloa and Paspalum subjected to
The first group comprised of P. regnellii BGP 215, 248
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
160 C.G . Pezzopane, A.G. Lima, P.G. Cruz, T. Beloni, A.P. Fávero and P.M. Santos
and 397, P. malacophyllum BGP 293, P. dilatatum BGP
On the other hand, little change in root biomass (Figure
234 and P. atratum BGP 308 showed the best values in
1C), along with the other observed results, suggests that it
variables of development; the second group made up of
may use stomatal control mechanisms to reduce water
accessions P. malacophyllum BGP 289, P. quarinii BGP
loss and delay tissue dehydration.
229, P. regnellii BGP 112 and P. conspersum BGP 402
Accession P. urvillei x P. dilatatum BGP 238 behaved
were characterized by the greatest number of days to lose
similarly to accession P. regnellii BGP 215 because it also
turgor in the predawn period and genotype P. urvillei x
has high values of biomass of dead matter and roots but,
P. dilatatum BGP 238 was not grouped with the others,
unlike BGP 215, the moisture stress had a negative effect
forming a specific group. Apparently, there was no
on root biomass, with 35% reduction compared with the
correlation between collecting site and strategy used by
control (Figure 1C). Time to wilting of BGP 238 was rela-
plants to overcome water deficit.
tively long, being similar to that of genotypes that were
Accessions that stood out in terms of development
grouped by this characteristic ( P. regnellii BGP 289,
variables can be further divided into 3 subgroups:
P. quarinii BGP 229, P. regnellii BGP 112 and
P. regnellii BGP 215 (group 1); P. malacophyllum BGP
P. conspersum BGP 402; Figures 2 and 1G), but the bio-
293 (group 2); P. atratum BGP 308, P. regnellii BGP 397,
mass of green matter was higher, suggesting that this ac-
P. dilatatum BGP 234 and P. regnellii BGP 248 (group 3).
cession has good potential for use under conditions where
According to PCA and the mean values of variables
there is risk of severe drought (Figures 2 and 1B).
represented in it, accession P. regnellii BGP 215 was the
Accessions P. malacophyllum BGP 289, P. quarinii
first to wilt, despite increased root system biomass and
BGP 229, P. regnellii BGP 112 and P. conspersum BGP
reductions in leaf area. This result suggests that this
402, which were characterized by the greatest number of
accession is able to maintain productivity under mild
days to wilting (Figures 2 and 1G), presented a more
water stress by expanding the root system and exploration
conservative strategy of use of natural resources, which
of a greater volume of soil, but is not tolerant of severe
provided high tolerance to conditions of severe water
drought. Pérez-Ramos et al. (2013) found that accessions
stress. More conservative genotypes in the use of
with a more aggressive survival strategy based on
resources have smaller leaf area, maintain turgor and
increased acquisition of resources, when in deep soils,
activate osmoregulation mechanisms at the leaf blade
reduce the rate of dehydration of the meristem by
level during moderate drought, and under reduced water
deepening the root system and increasing the absorption
availability, they prioritize meristems and tips of the
of water.
roots, ensuring the recovery of plants after the elimination
Santos et al. (2013) studied forage plants of the genus
of stress (Volaire and Lelièvre 2001; Volaire et al. 2014).
Urochlo a under water stress and also observed different
This is because meristems exhibit higher osmotic
behavior among the cultivars, which presented different
adjustment than other tissues during drought (Munns et
strategies of survival. Urochloa brizantha cv. Piatã
al. 1979; Matsuda and Riazi 1981; West et al. 1990) and
decreased vegetative development, consequently reduc-
therefore have potential for regeneration when the aerial
ing production, indicating a conservative strategy,
part of the plant is dead (Van Peer et al. 2004).
lowering metabolism for its survival; U. brizantha cv.
The Paspalum accessions evaluated in this study can
Marandu presented a more aggressive strategy, which did
be categorized according to their strategies in response to
not reduce productive development, but maintained high
abiotic stress due to imposed water restriction.
productivity, which, according to the authors, promoted
Knowledge of these survival strategies, which may focus
advantages under mild stress, but under conditions of
on reduced development or maintenance of productivity,
severe stress, survival may be compromised because there
will contribute to the creation and selection of genotypes
was no reduction of metabolism.
for use in the Paspalum breeding program. This assumes
Accession P. malacophyllum BGP 293 presented a
greater importance as more severe global climate change
distinct response (Figure 1); even though wilted at 14
scenarios are forecast.
days, it maintained a relatively high leaf area and little
Under the conditions of this experiment, where the
biomass of dead matter at the time of harvest (Figures 1A
evaluation assessment was interrupted when the
and 1D). The high leaf water potential (Figure 1E)
genotype’s shoots wilted in the predawn period and no
indicates that osmotic adjustment is not among the main
recovery period was allowed, it is suggested that
mechanisms of tolerance to water stress of this genotype,
accessions be separated into 2 groups so that they can be
because early stomatal closure helps control water loss.
used in breeding programs aimed at tolerance to drought.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Water stress tolerance in Paspalum 161
For environments subjected to the occurrence of
Cruz CD. 2006. Programa Genes: Análise multivariada e
prolonged droughts, the most promising candidates
simulação. Editora UFV, Viçosa, MG, Brazil.
appear to be: P. malacophyllum BGP 289, P. quarinii
Escudero A; Mediavilla S; Heilmeier H. 2008. Leaf longevity
and drought: Avoidance of the costs and risks of early leaf
BGP 229, P. regnellii BGP 112, P. conspersum BGP 402
abscission as inferred from the leaf carbon isotopic
and P. urvillei x P. dilatatum BGP 238, as they adopt
composition. Functional Plant Biology 35:705–713. DOI:
strategies in which survival under adverse conditions is
prioritized. On the other hand, for cases of mild-moderate
Garcez Neto AF; Gobbi KF. 2013. Características morfo-
water stress, priority should be given to accessions P.
anatômicas e fisiológicas de gramíneas associadas à
atratum BGP 308, P. regnellii BGP 215, BGP 248 and
tolerância à seca. In: Souza FHD de; Matta FP; Fávero AP,
BGP 397, P. dilatatum BGP 234 and P. malacophyllum
eds. Construção de ideótipos de gramíneas para usos
BGP 293, where productivity losses during water
diversos. Embrapa, Brasília, DF, Brazil. p. 175–189.
Givnish TJ. 1987. Comparative studies of leaf form: Assessing
restriction are lower.
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Acknowledgments
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The authors are grateful to FAPESP and CAPES for
five Brachiaria species. I. Biomass production, leaf growth,
financial support.
root distribution, water use and forage quality. Plant and
Soil 243:229–241. DOI: 10.1023/A:1019956719475
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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Tropical Grasslands-Forrajes Tropicales (2017) Vol. 5(3):163–175 163
Research Paper
Screening of common tropical grass and legume forages in Ethiopia
for their nutrient composition and methane production profile in
vitro
Composición nutricional y producción de metano in vitro de algunas
gramíneas y leguminosas forrajeras comunes en Etiopía
ABERRA MELESSE1,2, HERBERT STEINGASS2, MARGIT SCHOLLENBERGER2 AND MARKUS
RODEHUTSCORD2
1 School of Animal and Range Sciences, Hawassa University, Hawassa, Ethiopia. www.hu.edu.et
2 Institute of Animal Science, University of Hohenheim, Stuttgart, Germany. www.uni-hohenheim.de
Abstract
A study was conducted to assess the nutrient composition, in vitro gas production (GP) characteristics and methane
(CH4) production potential of some common Ethiopian grass and legume forages. Crude protein (CP) concentration was
lower in grasses than in legumes, while the reverse was observed for neutral detergent fiber (aNDFom) and acid detergent
fiber (ADFom) concentrations. Within the 9 grasses tested, Cynodon dactylon had the highest CP concentration (187
g/kg DM), while Panicum coloratum and Cenchrus ciliaris had the lowest (70 and 82 g/kg DM, respectively) values.
Chloris gayana contained the highest aNDFom (651 g/kg DM) concentration, while Avena sativa had the lowest (484
g/kg DM). Among the 3 legumes tested, Vicia sativa had the highest CP concentration (346 g/kg DM). The aNDFom
and ADFom concentrations were highest in V. sativa and lowest in Medicago sativa. In grasses, Brachiaria mutica had the highest calcium, magnesium, iron and manganese concentrations, while in legumes the highest concentrations of
phosphorus, potassium and zinc were observed in V. sativa. Methane production was generally higher (P<0.05) in grasses
than in legumes. Panicum coloratum produced the lowest (P<0.05) CH4 levels within the grasses followed by
B. mutica, while Desmodium intortum produced the lowest (P<0.05) CH4 levels within the legumes. Panicum coloratum and D. intortum appear to have potential as suitable forage species for ruminants, resulting in reduced CH4 emissions.
Studies with animals are needed to verify these in vitro findings.
Keywords : In vitro gas production, minerals, nutrient profiles, tropical pastures.
Resumen
En el laboratorio de Hawassa University, Etiopía, se realizó un estudio para evaluar la composición nutricional, la
producción de gas (PG) in vitro y el potencial de producción de metano (CH4) de 9 gramíneas y 3 leguminosas forrajeras
comunes en Etiopía. Como era de esperar, la concentración de proteína cruda (PC) fue menor en las gramíneas que en
las leguminosas, mientras que las concentraciones de fibra detergente neutro (FDN) y fibra detergente ácido (FDA)
fueron más altas en las primeras. Entre las gramíneas evaluadas, Cynodon dactylon presentó la mayor concentración de
PC (187 g/kg de MS), mientras que Panicum coloratum y Cenchrus ciliaris presentaron los valores más bajos (70 y 82
g/kg de MS, respectivamente). Chloris gayana presentó los valores más altos de FDN (651 g/kg de MS) y Avena sativa
los más bajos (484 g/kg de MS). Entre las leguminosas, Vicia sativa presentó las mayores concentraciones de PC (346
___________
Correspondence: Aberra Melesse, School of Animal and Range
Sciences, Hawassa University, P.O. Box 05, Hawassa, Ethiopia.
Email: a_melesse@uni-hohenheim.de
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
164 A. Melesse, H. Steingass, M. Schollenberger and M. Rodehutscord
g/kg de MS) y de ambas fibras, mientras que las concentraciones más bajas de FDN y FDA se registraron en Medicago
sativa. Respecto a minerales, Brachiaria mutica presentó las mayores concentraciones de calcio, magnesio, hierro y manganeso entre las gramíneas, mientras que en las leguminosas se observaron en V. sativa las mayores concentraciones
de fósforo, potasio y zinc. La producción de CH4 fue generalmente mayor (P<0.05) en las gramíneas que en las
leguminosas. Entre las gramíneas, P. coloratum presentó los niveles de CH4 más bajos (P<0.05), seguido por B. mutica, mientras que entre las leguminosas Desmodium intortum produjo los niveles de CH4 más bajos (P<0.05). Con miras a
emisiones reducidas de CH4, P. coloratum y D. intortum parecen tener potencial como especies forrajeras amigables con el medio ambiente. Se requieren estudios con animales rumiantes para verificar estos hallazgos obtenidos in vitro.
Palabras clave : Minerales, pastos tropicales, perfiles nutricionales, producción de gas in vitro.
Introduction
scale specialized facilities and resources, there has been
growing interest in using in vitro techniques to simulate
Developing countries in general and African nations in
the in vivo process (Blümmel et al. 2005; Bhatta et al.
particular are increasingly becoming victims of climate
2008; Soliva et al. 2008; Melesse et al. 2013). Use of in
change as global temperatures rise. The Intergovern-
vitro gas-production techniques allows the screening of
mental Panel on Climate Change (IPCC) has attributed
significant numbers of species rapidly and at relatively
the temperature increases to human activities, including
low cost (Soliva et al. 2008; Singh et al. 2012).
releases of the greenhouse gases, carbon dioxide, methane
We used in vitro techniques to assess: a) the chemical
and nitrous oxide into the atmosphere. They have
and mineral compositions; and b) ruminal fermentation
requested nations to quantify the amounts of gases they
characteristics and CH4 emission potentials, of some
produce and to develop research to limit further gaseous
common Ethiopian green forages (9 grasses and 3
emissions (Moss et al. 2000).
legumes) for their subsequent use in formulating diets for
Ruminants are a major source of methane (CH4)
ruminants with lower potential emissions of CH4.
emissions, and France et al. (1993) estimated that the
world’s cattle emit about 100 Mt of CH4 into the
Materials and Methods
atmosphere annually, constituting 12.5‒20% of the total
global CH4 emissions. More than half of the global cattle
Feed sample collection
population are located in the tropics (McCrabb and
Hunter 1999), a large proportion of which are supported
Samples of grasses and legumes were collected during
on relatively low-quality, highly fibrous feed resources.
the small rainy season (March‒May) in 2013. Samples of
This constitutes a significant source of global CH4
Avena sativa and Vicia sativa were collected from the first
emissions. Moreover, enteric CH4 emissions in ruminants
stage of growth on the forage farms of College of
represent a loss of 2‒12% of gross energy of feeds
Agriculture, Hawassa University, Hawassa (7°03'43.38"
(McCrabb and Hunter 1999). As a result, CH4 emissions
N, 38°28'34.86" E; 1,700 masl). Samples of Pennisetum
from livestock have become a focus of research activities,
purpureum, Chloris gayana, Panicum maximum,
especially in countries where agriculture is an important
Panicum coloratum, Hyparrhenia cymbaria, Desmodium
economic sector.
intortum and Medicago sativa were collected at the pre-
A wide diversity of forage sources are used in feeding
flowering stage of plants from ILRI’s (International
livestock in the tropics. Improving the feed resource base
Livestock Research Institute) Forage Seed Multiplication
by identifying alternative and more nutritious feeds with
Center located at Debre-Zeit (8°45'8.10" N, 38°58'42.46"
low CH4 production would both reduce greenhouse gas
E; 2,006 masl). Samples of Brachiaria mutica, Cenchrus
emissions and increase the efficiency of energy utilization
ciliaris and Cynodon dactylon were collected at the pre-
in forage. There is a lack of data describing and
flowering stage of plants from ILRI’s Forage Seed
identifying those tropical grass and legume forages with
Multiplication Center located at Zeway (7°55'59.99" N,
low CH4 production potential when fed to ruminant
38°43'0.01" E; 1,640 masl). All samples were dried on
animals.
plastic sheets kept in shade, ground to pass a 1 mm sieve
Since in vivo studies of methanogenesis by ruminants
and transported in air-tight plastic containers to the
are time-consuming and expensive, and require large-
University of Hohenheim, Germany, for analyses.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Nutrient composition and methane production of forages in Ethiopia 165
Chemical analyses
sample was weighed and transferred into 100 ml
calibrated glass syringes, fitted with white Vaseline-
Chemical analyses of proximate nutrients, fiber fractions
lubricated glass plungers.
and minerals were performed as outlined by Verband
A buffer solution was prepared and maintained in a
Deutscher Landwirtschaftlicher Untersuchungs- und
water bath at 39 °C under continuous flushing with CO2.
Forschungsanstalten (VDLUFA 2007). The samples were
Rumen fluid was collected before the morning feeding
analyzed at the Institute of Animal Science, University of
from 2 rumen-cannulated, lactating Jersey cows, fed a
Hohenheim, for dry matter (DM, method 3.1), ash (method
total mixed ration consisting (DM basis) of 20% maize
8.1), crude protein (CP, method 4.1.1; N x 6.25), petroleum
silage, 20% grass silage, 20% hay and 40% dairy
ether extract (EE, method 5.1.1) and crude fiber (CF,
concentrate. The rumen fluid from both cows was mixed,
method 6.1.1). Neutral detergent fiber (aNDFom) was
filtered and added to the buffer solution (1:2 v/v) under
assayed on an organic matter basis after amylase treatment
constant stirring. Thirty mL of buffered rumen fluid was
(method 6.5.1) and acid detergent fiber on an organic
injected into each syringe, which was then immediately
matter basis (ADFom, method 6.5.2). Acid detergent lignin
placed into a rotating disc and oven-incubated at constant
(ADL) was analyzed according to method 6.5.3. Cellulose
temperature of 39 °C. Three syringes with only buffered
and hemicellulose were computed as ADFom minus ADL
rumen fluid, termed as blanks, plus 3 syringes with hay
and aNDFom minus ADFom, respectively. Non-fiber
standard and 3 with concentrate standard with known GP
carbohydrate (NFC) concentration was calculated as 100 ‒
were included in each run. The GP of samples, blanks and
(aNDFom + CP + crude fat + ash) according to NRC
standards was recorded at 2, 4, 6, 8, 12, 24, 32, 48, 72 and
(2001). Nitrogen free extract (NFE) was computed as OM
96 hours of incubation. The plunger of the syringe was re-
– (CF + EE + CP). Minerals [Ca, P, magnesium (Mg),
set to 30 ml after the 6 and 24 hour readings. For
potassium (K), sodium (Na), iron (Fe), copper (Cu), man-
metabolizable energy (ME) estimation, the GP of the feed
ganese (Mn) and zinc (Zn)] were determined according to
samples was recalculated as 24 h GP on 200 mg DM using
methods 10 and 11 of VDLUFA (2007) using an Induc-
results from the blanks, with the corrections determined
tively Coupled Plasma spectrometer (ICP-OES).
by the standards of hay and concentrate, the sample
Four species with possible anti-nutritional factors
weight and its DM concentration.
( Chloris gayana, Desmodium intortum, Medicago sativa
The estimations of organic matter digestibility (OMD)
and Vicia sativa) were selected from the collection and
and ME were carried out according to Menke et al. (1979)
analyzed for concentrations of total phenols and non-tannin
and Menke and Steingass (1988) by using the following
phenols using the Folin-Ciocalteu method [Jayanegara et
equations:
al. (2011) with modifications as described by Wischer et al.
ME (MJ/kg DM) = 1.68 + 0.1418*GP + 0.0073*CP +
(2013)]. Extractable condensed tannins were analyzed
0.0217*XL – 0.0028 XA
according to Jayanegara et al. (2011). Concentrations of
OMD (%) = 14.88 + 0.889*GP + 0.0448*CP +
tannin phenols were then calculated as differences between
0.0651*XA
total phenol and non-tannin phenol concentrations. The
where: GP, CP, XL and XA are 24 h gas production
absorbance of total phenols and non-tannin phenols was
(ml/200 mg DM), crude protein, crude fat and ash (g/kg
recorded at 725 nm using a UV-VIS spectrophotometer
DM) of the incubated feed samples, respectively.
(Perkin Elmer Instruments, Norwalk, CT, USA). Con-
The corrected GP recorded between 2 and 96 h of
densed tannins were analyzed by the butanol-HCl-iron
incubation and the kinetics of GP were described by using
method according to Jayanegara et al. (2011). The
the exponential equation: y = b*(1−e(−c(t−lag))), which
absorbance was read at 550 nm using the same UV-VIS
assumed one pool of asymptotic GP (b, ml/200 mg DM)
spectrophotometer as for total phenols and non-tannin
with a constant fractional rate of GP (c, per hour) with a
phenols and was expressed as leucocyanidin equivalents.
lag phase (lag, hours) in the onset of GP; parameter “y” is
All analyses were run in duplicate and were averaged.
GP at time “t” (Blümmel et al. 2003; 2005).
If deviation between duplicates was above the level
specified for each analysis, the analyses were repeated.
Methane production
In vitro gas production
For CH4 determination, 6 separate in vitro runs were
performed. Based on the previous in vitro GP results for
Gas production (GP) was determined according to the
each feed sample, we calculated the quantity of each feed
VDLUFA official method (VDLUFA 2007, method 25.1)
sample to be incubated for 24 h without having to remove
(Menke and Steingass 1988). About 200 mg of feed
the gas produced in the syringes during the incubation
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
166 A. Melesse, H. Steingass, M. Schollenberger and M. Rodehutscord
period. After 24 h of incubation, total GP was recorded,
material and eij is the residual error. All multiple
and the incubation liquid was carefully decanted, while
comparisons among means were performed with
leaving the gas inside the syringes. The CH4 content of
Duncan’s multiple range tests.
the total gas in the syringes was then analyzed using an
infrared methane analyzer (Pronova Analysentechnik,
Results
Berlin, Germany) calibrated with a reference gas
(13.0% CH4 by volume, Westfalen AG, Münster,
Crude nutrients and anti-nutritional factors
Germany). Syringes were directly connected to the
analyzer and about 20 ml of gas was injected for about 20
Crude nutrient concentrations in the studied grass and
seconds until the displayed CH4 concentration was
legume plants are presented in Table 1. The CP
constant. The CH4 produced by each sample was
concentrations in grass species ranged from 70 g/kg DM
corrected by the amount of CH4 produced by blank
( Panicum coloratum) to 220 g/kg DM ( Avena sativa),
syringes (containing only the rumen fluid) and by the
while those in legumes ranged from 257 g/kg DM
factors of reference hay and concentrate feed, which were
( Medicago sativa) to 346 g/kg DM ( Vicia sativa). For
included in each run.
grasses, CF concentrations ranged from 281 g/kg DM
( A. sativa) to 322 g/kg DM ( Cenchrus ciliaris and Chloris
Statistical analyses
gayana), while values for legumes ranged from 213 g/kg
DM ( Desmodium intortum) to 249 g/kg DM ( M. sativa).
Results of chemical and mineral composition are
expressed as means of duplicate analyses of a bulked
Similarly, grasses contained more aNDFom (484‒651
g/kg DM) than legumes (364‒404
sample. Model fitting for gas production kinetics and
g/kg DM). Concen-
parameter estimation was done according to Blümmel et
trations of ADL in grasses (25.3‒41.3 g/kg DM) were
al. (2003) by using the computer program GraphPad
lower than those in legumes (48.2‒89.8 g/kg DM).
Prism 5.0 (2007) for Windows (GraphPad Software Inc.,
As shown in Table 2, concentrations of total phenols
La Jolla, CA, USA). Data on 24 h gas and methane
were comparable for M. sativa, V. sativa and C. gayana,
productions were subjected to the GLM of the Statistical
while those for D. intortum were higher by a factor of 10.
Analysis System (SAS 2010). Analysis of variance was
No tannin phenols or extractable condensed tannins were
conducted according to the following model: yij = μ + Pi
detected in either M. sativa or V. sativa, while C. gayana
+ Rj + eij, where: yij is the independent variable, μ is the
contained very low concentrations of these compounds.
overall mean, Pi is the effect of the i th plant material, Rj
Both tannin phenols and extractable condensed tannins
is the effect of the j th experimental run of the i th plant
were at high concentrations in D. intortum.
Table 1. Crude nutrient concentrations (g/kg DM) in some common grass and legume forages grown in Ethiopia.
Forage species
Ash
CP1
EE
CF
NFE aNDFom ADFom ADL Cellulose Hemi-
NFC
cellulose
Grasses
Avena sativa
133
226
31.2
281
263
484
326
25.3
301
159
126
Brachiaria mutica
167
159
12.2
229
382
504
277
29.8
247
227
158
Cenchrus ciliaris
154
82
14.5
322
380
601
373
26.7
346
228
149
Chloris gayana
131
135
13.3
322
341
651
370
41.1
329
281
70
Cynodon dactylon
125
187
14.7
272
357
609
323
41.3
282
286
64
Hyparrhenia cymbaria
105
156
11.6
299
375
605
333
33.7
299
272
122
Panicum coloratum
102
70
21.0
292
461
633
322
26.3
296
312
174
Panicum maximum
140
140
12.5
284
373
566
344
27.1
317
222
142
Pennisetum purpureum
173
121
10.9
315
325
599
372
33.4
339
227
96
Legumes
Desmodium intortum
91.7
258
9.3
213
365
396
319
89.8
229
77.2
245
Medicago sativa
151
257
13.3
249
282
364
302
48.2
254
61.7
215
Vicia sativa
147
346
17.2
218
214
404
336
56.6
279
67.9
86
1CP = crude protein; EE = crude fat; CF = crude fiber; NFE = nitrogen free extract; aNDFom = neutral detergent fiber on organic
matter basis after amylase treatment; ADFom = acid detergent fiber on organic matter basis; ADL = acid detergent lignin; NFC =
non-fiber carbohydrates.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Nutrient composition and methane production of forages in Ethiopia 167
Table 2. Concentrations (g/kg DM) of total phenols, tannin phenols and extractable condensed tannins in some selected forage species in Ethiopia.
Species
Type of forage
Total phenols
Tannin phenols
Extractable condensed tannins
Medicago sativa
Legume
6.7
nd1
nd
Desmodium intortum
Legume
77.7
57.6
77.6
Vicia sativa
Legume
7.6
nd
nd
Chloris gayana
Grass
6.8
1.3
0.2
1nd = not detected.
Minerals
in legumes from 8.1 MJ/kg DM in D. intortum to 10.2 MJ/
kg DM in V. sativa. Organic matter digestibility in grasses
As presented in Table 3, among the grasses Brachiaria
ranged from 56.1% in P. coloratum to 79.6% in A. sativa,
mutica had the highest concentrations of Ca, Mg, Fe and
and from 64.6 to 82% in legumes. The highest asymp-
Mn, while P concentration was highest in A. sativa,
totic GP (parameter b) values for grasses were observed
Pennisetum purpureum and Panicum maximum. In
in
Hyparrhenia.
cymbaria
(58.6
ml)
and
leguminous forages, M. sativa and V. sativa had Ca
P. maximum (59.2 ml) with the lowest in B. mutica (49.2
concentrations of about 9 g/kg DM, while V. sativa had
ml). Values for legumes were generally lower with
the highest P concentration (5.6 g/kg DM). Sodium
a range of 39.0‒45.2 ml. The fractional rates of GP per
concentrations varied widely in both legumes and grasses,
with ranges of 0.06‒8.01 g/kg DM for grasses and 0.10‒
hour (parameter c) for grasses ranged from 0.0387
( P. coloratum) to 0.0667 ( A. sativa). The range for
4.26 g/kg DM for legumes.
legumes was 0.0537 ( D. intortum) to 0.0851 ( V. sativa).
In vitro gas production profiles and fermentation kinetics
As shown in Table 4, the values for the goodness of fit
(R2) of the exponential model were above 94% for all
As shown in Table 4, metabolizable energy (ME)
species.
concentrations in grasses ranged from 7.4 MJ/kg DM
Patterns of gas production for the grasses are shown in
in Panicum coloratum to 10.6 MJ/kg DM in A. sativa and
Figure 1 and for legumes in Figure 2.
Table 3. Mineral composition of some common grass and legume forages grown in Ethiopia.
Forage species
Major (g/kg DM)
Trace (mg/kg DM)
Ca1
P
Mg
K
Na
Fe
Cu
Mn
Zn
Grasses
Avena sativa
3.69
5.24
2.18
38.0
8.01
104
2.44
46.9
78.4
Brachiaria mutica
8.55
3.49
4.63
26.0
1.83
716
7.02
84.7
29.9
Cenchrus ciliaris
4.30
2.54
2.95
24.3
0.45
430
4.21
31.7
18.6
Chloris gayana
3.88
2.94
1.98
35.6
0.50
210
4.75
68.7
28.9
Cynodon dactylon
5.11
2.07
2.46
24.9
0.15
181
5.09
57.5
35.0
Hyparrhenia cymbaria
4.27
1.40
2.41
20.6
0.06
150
5.87
44.3
28.1
Panicum coloratum
3.20
2.78
3.66
11.0
1.82
191
3.98
21.7
15.2
Panicum maximum
4.14
4.56
4.38
23.9
3.54
420
8.27
40.6
26.3
Pennisetum purpureum
3.09
4.49
3.49
38.9
0.16
267
7.50
27.5
25.5
Legumes
Desmodium intortum
6.85
2.37
6.25
18.8
0.10
486
7.14
58.7
34.8
Medicago sativa
9.32
3.24
3.02
45.9
4.26
494
2.42
55.2
65.3
Vicia sativa
9.05
5.57
3.12
46.0
2.64
441
3.37
61.3
388
1Ca = calcium; P = phosphorus; Mg = magnesium; K = potassium; Na = sodium; Fe = iron; Cu = copper; Mn = manganese; Zn =
zinc.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
168 A. Melesse, H. Steingass, M. Schollenberger and M. Rodehutscord
Table 4. In vitro estimates of metabolizable energy (ME), organic matter digestibility (OMD) and kinetics of gas production (ml/200
mg DM) in some common grass and legume forages grown in Ethiopia.
Forage species
ME (MJ/kg DM)
OMD (%)
b1
c
Lag time (h)
R2
Grasses
Avena sativa
10.6
79.6
54.7
0.0667
1.17
97.3
Brachiaria mutica
8.0
66.7
49.2
0.0448
0.20
97.3
Cenchrus ciliaris
8.5
66.9
57.7
0.0469
0.94
98.2
Cynodon dactylon
9.7
75.2
56.9
0.0537
1.47
97.7
Chloris gayana
9.0
70.3
56.4
0.0511
1.53
97.7
Hyparrhenia cymbaria
10.0
75.6
58.6
0.0613
1.39
97.7
Panicum coloratum
7.4
56.1
55.4
0.0387
0.81
96.2
Panicum maximum
9.7
75.2
59.2
0.0548
0.96
97.8
Pennisetum purpureum
8.4
68.8
54.4
0.0475
1.25
97.4
Legumes
Desmodium intortum
8.1
64.6
39.0
0.0537
0.37
94.7
Medicago sativa
9.5
77.2
45.2
0.0839
0.94
97.9
Vicia sativa
10.2
82.0
45.0
0.0851
0.89
96.1
1b = total asymptotic gas production (ml/200 mg DM); c = the rate at which b is produced per hour with a lag phase in the onset of
gas production.
Methane production
gas production followed a similar pattern with highest
values for H. cymbaria, P. maximum, A. sativa and
Most grass species produced significant amounts of
Cynodon dactylon and lowest for P. coloratum (P<0.05).
methane during digestion, but P. coloratum produced
Methane:total gas ratios (CH4:GP) ranged from 0.18:1
about half that of other species (P<0.05) (Table 5). Total
( B. mutica) to 0.11:1 ( P. coloratum) (P<0.05).
Figure 1. Patterns of gas production of some tropical grass forages during in vitro incubation for 96 h.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Nutrient composition and methane production of forages in Ethiopia 169
Figure 2. Patterns of gas production of some tropical legume forages during in vitro incubation for 96 h.
Desmodium intortum produced much less CH4
ratio was observed in D. intortum (0.12:1), which
than V. sativa and M. sativa (P<0.05) differing
differed significantly from those for the other 2
significantly from these other legumes (Table 5).
legumes (0.16:1). In general, grasses produced
While differences between species for total gas
comparatively higher (P<0.05) GP, CH4 and CH4:GP
production were not so marked, the lowest CH4:GP
ratios than legume forages.
Table 5. In vitro methane and total gas production profiles (±SD) in some common grass and legume forages grown in Ethiopia.
Forage species
CH4 (ml/200 mg DM)
GP (ml/200 mg DM)
CH4:GP (v:v)
Grasses
Avena sativa
6.16±1.00ab1
44.0±5.34abc
0.140d
Brachiaria mutica
5.80±0.53b
33.5±1.41e
0.178a
Cenchrus ciliaris
6.92±0.45a
40.9±1.62cd
0.169b
Chloris gayana
6.44±0.68ab
41.8±3.20bcd
0.159bc
Cynodon dactylon
7.01±0.53a
43.6±4.38abc
0.161bc
Hyparrhenia cymbaria
6.78±0.68a
47.0±2.10a
0.145d
Panicum coloratum
3.18±0.47c
31.4±1.02e
0.107e
Panicum maximum
7.01±1.06a
44.9±2.00ab
0.159bc
Pennisetum purpureum
6.94±1.06a
39.7±2.15d
0.165bc
Legumes
Desmodium intortum
3.67±0.39b
29.9±1.03b
0.123b
Medicago sativa
5.90±0.72a
37.5±2.86a
0.157a
Vicia sativa
5.73±0.51a
36.1±3.25a
0.159a
Grasses vs. legumes
Grasses
6.23±1.42a
40.3±5.52a
0.156a
Legumes
5.34±1.21b
36.9±6.15b
0.144b
1Means within columns and plant types followed by different letters differ significantly (P<0.05).
CH4 = methane production; GP = total gas production at 24 h incubation of feed samples.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
170 A. Melesse, H. Steingass, M. Schollenberger and M. Rodehutscord
Discussion
where a cut-and-carry system operates, they become quite
relevant. All leguminous forages contained less fiber than
Crude nutrient and mineral concentrations
grasses, which might be explained in part by lower
hemicellulose concentration in the legumes at comparable
The generally lower CP concentrations in the grasses than
levels of cellulose (Table 1). Cellulose and hemicellulose
in the legumes were consistent with the reports of Singh
in forages represent the main sources of energy to
et al. (2012) for Indian green forages. Consistent with the
ruminants (Merkel et al. 1999).
reports of Tessema and Baars (2006), all forages studied
The aNDFom, ADFom and ADL concentrations in
had protein concentrations above 8%, suggested by Van
V. sativa were comparable with those reported by Berhane
Soest (1982) as the critical level, below which intake may
et al. (2006) from the lowlands of northern Ethiopia.
fall due to lack of sufficient nitrogen for effective
While the CP concentration in P. coloratum was similar
proliferation of rumen micro-organisms. Higher CP
to the observation of the same authors, they reported
values in leguminous forages than in grasses might be
higher NDF, ADF and ADL values than those we found.
related to the N-fixing abilities of the legumes. Our
Such variations might be induced by the stage of maturity
current findings are in good agreement with those of
of the forage at harvest as grasses increase stem
Tessema and Baars (2006) from Ethiopia, that pure
proportions with age, resulting in higher NDF, ADF and
legume stands and grass-legume mixtures produced
lignin and lower CP values (Mero and Udén 1997; 1998).
forage with higher CP and lower fiber concentrations than
Except for P. coloratum, lipid concentrations in the
pure stands of grass. The CP concentrations in C. gayana
forages investigated here were much lower than
and P. maximum in the present study are lower than those
observations reported by Singh et al. (2012) and Pamo et
reported by Tessema and Baars (2006). However, those
al. (2007). These variations might be attributed mainly to
authors also reported lower CP for M. sativa than found
stage of maturity of the forage at the time of sampling and
in the present study. These differences in CP
different environmental conditions. While lipids do not
concentrations could be explained due to stage of
constitute a major source of energy from forages, forages
maturity, N profile of the soils where they had been grown
with high lipid concentrations may be a tool to modify
and differences in efficiency of protein accumulation
milk fatty acid profile towards more long-chain and
during growth. Moreover, differences in nutrient
unsaturated fatty acids (Elgersma 2015).
concentrations in the feeds may be due to variations in the
Phosphorus is one of the most important minerals for
stage of growth and plant parts (i.e. twigs, leaves, soft
many metabolic processes in animals and a deficiency of
stem) when sampled.
P in the diet can retard growth and reproductive
All leguminous forages had higher lignin con-
performance of livestock (Paterson et al. 1996). While
centrations than grasses as reported by Singh et al. (2012).
V. sativa and A. sativa were found to be the richest sources
This might be explained by the fact that the leguminous
of P in our study (>0.5% P), all forages had P concen-
forages synthesize lignin for strength and rigidity of
trations above 0.2%. Brachiaria mutica, V. sativa and
plant cell walls. Singh et al. (2012) reported 310 and 58.8
A. sativa proved to be the richest sources of Ca, which is
g/kg DM for ADF and ADL concentrations, respectively,
closely related to P metabolism in the formation of bones.
in M. sativa, which are comparable with the present
The calculated average Ca:P ratio for legume forages in
findings.
the present study was 2.0:1, while for grasses it was 1.7:1,
Except for B. mutica, grass species in the present study
both of which fall within the recommended range for Ca:P
had higher aNDFom, ADFom, cellulose and hemi-
ratio in feedstuffs of 1:2 to 2:1 (NRC 2001), indicating
cellulose concentrations than legumes, which is in
that the studied forages are likely to be a well-balanced
accordance with the findings of Tessema and Baars
source of both minerals.
(2006) and Singh et al. (2012). The threshold level of
NDF in tropical grasses, beyond which DM intake of
In vitro gas and methane production
cattle is affected, is suggested to be 600 g/kg DM
(Meissner et al. 1991) and all legumes and some grasses
The study has shown that methane production from all
( B. mutica, P. maximum, P. purpureum and C. ciliaris)
forages tested was relatively uniform, with the exception
had lower NDF values than this critical level. Since
of P. coloratum and D. intortum, which produced much
animals, when allowed to selectively graze, can select a
less CH4 than the remaining species. The observed low in
better quality diet than feed on offer, these issues may not
vitro GP pattern in D. intortum (Figure 2) might be
be a major problem under a grazing situation. However,
explained by the presence of high concentrations of total
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Nutrient composition and methane production of forages in Ethiopia 171
phenols (77.7 g/kg DM) and condensed tannins (77.6 g/kg
Fermentation of cell wall carbohydrates produces
DM), which have the ability to complex with protein and
more CH4 than fermentation of soluble sugars, which
are a major cause of the resistance of this legume to
produce more CH4 than fermentation of starch (Johnson
bacterial decomposition.
et al. 1996) and legume forages are digested more quickly
Consistent with the current observations, Mero and
than grasses. This was demonstrated for M. sativa and
Udén (1998) reported in vivo OMD values between 61.5
V. sativa in the current study, which means that intake and
and 64.8% for C. ciliaris hay harvested at 6 weeks of
productivity on leguminous pasture can be higher than on
age. They also reported comparable OMD values for
grasses. In tannin-containing forages, excess plant
P. coloratum harvested at 6 weeks of age. Berhane et al.
proteins that become bound to tannins leave the rumen
(2006) reported values of 65.5 and 68.3 ml for in vitro GP
without being digested. However, some leguminous
(parameter b) of fresh-cut V. sativa and P. coloratum,
forages containing tannins, such as D. intortum, can
respectively, which were higher than those observed in
release these proteins in the abomasum in response to low
the current study. Panicum coloratum in the present study
pH. This allows the protein to be digested and absorbed
had the lowest ME and OMD values. Except for
in the small intestine (Waghorn et al. 1987), resulting in
D. intortum, legumes produced more gas than grasses
high productivity in both sheep (Douglas et al. 1995) and
within 96 h of incubation, which is consistent with the
cattle (Wen et al. 2002).
findings of Singh et al. (2012).
Legumes contain higher CP than grasses at the same
The observed variations in CH4 production among the
stage of maturity and protein fermentation in vitro has
investigated forages may be due to variations in their
been shown to be associated with lower CH4 production
chemical composition. Such variations in in vitro CH4
than fermentation of carbohydrates (Cone and Van Gelder
production have been observed in straws, forages and
1999). In vitro studies conducted by Soliva et al. (2008),
food industry byproducts (Santoso and Hariadi 2009;
Tiemann et al. (2008), Bekele et al. (2009) and Archimède
Singh et al. 2012). In the current study, except for
et al. (2011) have shown that a large portion of the
P. coloratum and B. mutica, all investigated grasses had
variability of CH4 production in legumes can be
higher CH4 values than leguminous forages, which is in
associated with the presence of secondary metabolites
agreement with the findings of Boadi et al. (2004) and
(condensed tannins, saponins) in some legume species,
Navarro-Villa et al. (2011). At 12 h fermentation,
which can inhibit CH4 formation (Beauchemin et al. 2007;
Widiawati and Thalib (2007) found that in grasses CH4
Jouany and Morgavi 2007). In the present study,
production per unit of OM degraded was twice that in
D. intortum had the highest phenols and extractable
legume forages. Moreover, hydrolysis of legumes such as
condensed tannins, which possibly contributed to the
lucerne and red clover generates less CH4/g DM than
reduction of CH4 production in this species. In other
hydrolysis of grasses (Ramirez-Restrepo and Barry
studies, prolonged feeding of tanniniferous forage
2005). The lower CH4 values in legumes vs. grasses might
legumes showed that animals receiving D. intortum had
be attributed to less extensive in vitro rumen fermentation
the lowest total worm burdens, the lowest female:male
of legumes as suggested by Navarro-Villa et al. (2011).
parasite ratios, the lowest numbers of eggs in the uterus
When CH4 emissions are expressed as a proportion of
of each female worm and the lowest per capita fecundity
gross energy intake (Waghorn et al. 2006), values are
(Debela et al. 2012). There is high variability among
lower for animals fed forage legumes (Waghorn et al.
legumes, particularly regarding the presence of secondary
2002) than for those receiving a predominantly grass diet.
metabolites such as tannins, which are more common in
Beauchemin et al. (2008) proposed that the lower CH4
tropical legumes (Waghorn 2008).
emissions of legume-fed animals is a result of a
Tropical legumes show promise as a means of
combination of factors including the presence of
reducing CH4 production, partly because of their lower
condensed tannins, lower fiber concentration, higher DM
fiber concentration and faster rate of passage than grasses,
intake and an increased passage rate from the rumen. In
and in some cases, the presence of condensed tannins as
the current study, no extractable condensed tannins were
observed in D. intortum in this study. Various studies
detected in M. sativa and V. sativa. Beauchemin et al.
have reported that condensed tannins in legume forages
(2008) also reported that, although differences in CH4
are able to suppress ruminal methanogenesis directly
emissions reflect compositional differences between
through their antimethanogenic activity and indirectly
grasses and legumes, stage of maturity at the time of
through their antiprotozoal activity (Goel and Makkar
harvest can be a confounding factor.
2012). Patra and Saxena (2010), Pellikaan et al. (2011)
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
172 A. Melesse, H. Steingass, M. Schollenberger and M. Rodehutscord
and Goel and Makkar (2012) indicated that condensed
could be fed alone or in combination to supplement
and hydrolyzable tannins extracted from a diverse array
tropical feed resources for practical mitigation of CH4
of plant materials reduced CH4 production in vitro.
emissions from ruminants. We recommend animal-based
Similarly, Puchala et al. (2005) demonstrated that the
experiments to validate the actual feeding values of these
presence of condensed tannins in forages can decrease
forages, which showed reduced CH4 production in vitro,
CH4 production in vivo. This was confirmed by Animut
and to assess their production potential.
et al. (2008), who observed decreased CH4 emissions in
sheep fed a ration supplemented with different condensed
Acknowledgments
tannin sources.
The CH4 values measured at 24 h in vitro for M. sativa,
This research work was sponsored by Alexander von
P. purpureum and P. maximum reported by Singh et al.
Humboldt Foundation (Germany) under grant category
(2012) are generally higher than those obtained from the
‘Research Fellowship for Experienced Researchers’, for
current study. These variations could be due to quality and
which the authors are highly grateful. We acknowledge
maturity stage of the forages, soil type and climate in
the Southern Agricultural Research Institute, Holleta
which forages have been grown.
Agricultural Research Center and Forage Seed Production
Enteric CH4 production could be influenced by the
Center (Genebank) of ILRI for allowing the collection of
nature of carbohydrates fermented, such as cellulose,
some feed samples used in this study. We very much
hemicelluloses and soluble residues of the diets. In the
appreciate the excellent technical support provided by
present study, grasses had higher aNDFom, ADFom,
Mrs. Sibylle Rupp and Miss Julia Holstein in chemical
cellulose and hemicellulose concentrations than legumes
and methane gas analyses, respectively.
and produced more CH4 per unit weight. Similarly, Moss
et al. (1994) reported that digestible ADF, cellulose and
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(Received for publication 23 March 2016; accepted 15 May 2017; published 30 September 2017)
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