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

10.1071/fp08037

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.

the relative roles of selective pressures and phylogenetic

constraints. New Phytologist 106:131‒160. DOI: 10.1111/

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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

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(Received for publication 10 February 2017; accepted 15 July 2017; published 30 September 2017)

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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Tropical Grasslands-Forrajes Tropicales (2017) Vol. 5(3):163–175 163

DOI: 10.17138/TGFT(5)163-175

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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