Trop Anim Health Prod (2012) 44:1097–1104

DOI 10.1007/s11250-011-0045-5

ORIGINAL RESEARCH

Feeding of tropical trees and shrub foliages as a strategy

to reduce ruminal methanogenesis: studies conducted
in Cuba
Denia Caridad Delgado & Juana Galindo & Rogelio González &
Niurca González & Idania Scull & Luís Dihigo & Juan Cairo &
Ana Irma Aldama & Onidia Moreira

Accepted: 8 December 2011 / Published online: 29 December 2011

# Springer Science+Business Media B.V. 2011

Abstract The aim of this paper was to present the main results methanogenesis from animals fed with low-quality forage diets
obtained in Cuba on the effects of feeding tropical trees and and for improving their productivity.
shrubs on rumen methanogenesis in animals fed with low
quality fibrous diets. More than 20 tree and shrub foliages Keyword Methane mitigation . Rumen . Supplementation .
were screened for phytochemicals and analyzed for chemical Tropical trees . Shrubs
constituents. From these samples, seven promising plants
(Samanea saman, Albizia lebbeck, Tithonia diversifolia, Leu-
caena leucocephala, Trichantera gigantea, Sapindus sapona-
Introduction
ria, and Morus alba) were evaluated for methane reduction
using an in vitro rumen fermentation system. Results indicated
Methane production is an inevitable consequence of carbohy-
that the inclusion levels of 25% of Sapindo, Morus, or Tri-
drate fermentation in the rumen. It is favored when animals are
chantera foliages in the foliages/grass mixtures (grass being
fed low quality forages, which is a general characteristic of
Pennisetum purpureum) reduced (P<0.01) methane produc-
tropical forages in a number of countries, including Cuba.
tion in vitro when compared to Pennisetum alone (17.0, 19.1,
Nevertheless, it is possible to manipulate fermentation to
and 18.0 versus 26.2 mL CH4/g fermented dry matter, respec-
reduce methane production. A number of methods have been
tively). It was demonstrated that S. saman, A. lebbeck, or T.
evaluated for reducing enteric methane production by means
diversifolia accession 23 foliages when mixed at the rate of
of chemical and biotechnological approaches, including the
30% in Cynodon nlemfuensis grass produced lower methane
use of additives (Attwood and McSweeney 2008; Lascano
compared to the grass alone. Inclusion levels of 15% and 25%
and Cárdenas 2010) and supplementation with concentrates
of a ruminal activator supplement containing 29% of L. leuco-
(Beauchemin et al. 2008). It is well established that inclusion
cehala foliage meal reduced methane by 37% and 42% when
of concentrates in the diet reduces the proportion of dietary
compared to the treatment without supplementation. In vivo
energy converted to CH4 and improves animal performance.
experiment with sheep showed that inclusion of 27% of L.
However it is clear that this method is impractical for devel-
leucocephala in the diet increased the DM intake but did not
oping country situations due to high cost of cereals and their
show significant difference in methane production compared
competition with human feed. In addition, chemicals and
to control diet without this foliage. The results of these experi-
additives are expensive and it has been found that ruminal
ments suggest that the feeding of tropical tree and shrub
microorganism population adapts to these compounds, result-
foliages could be an attractive strategy for reduction of ruminal
ing in lack of sustained beneficial effects. In such situations, it
is more appropriated to use strategic supplementation and
D. C. Delgado (*) : J. Galindo : R. González : N. González : management practices to shift ruminal fermentation to propi-
I. Scull : L. Dihigo : J. Cairo : A. I. Aldama : O. Moreira onate production, with the final result of reduced methane
Instituto de Ciencia Animal,
production per unit output.
Carretera Central Km 47 ½,
San José de las Lajas, Mayabeque, Cuba Strategic supplementation can reduce methane emissions by
e-mail: ddelgado@ica.co.cu 10% to 40% (Carmona et al. 2005). In addition it is possible to
1098
increase efficiency of production by up to fivefold with proper
dietary supplementation. This has been done without changing
the basal feed resources and by identifying and providing
critical nutrients that are otherwise deficient in the diet. In this
way nutrient availability is balanced with animal requirements
and forage intake is stimulated at the same time. Consequently,
substantial increase in animal productivity can be achieved
(Colman and Beever 2009).
Tree and shrub foliages can be used as catalytic supple-
ments to provide rumen soluble protein and minerals. In addi-
tion, plant secondary metabolites such as tannins and saponins
present in some tree leaves and shrubs appear to have anti-
methanogenic properties (Goel et al. 2008; Jayanegara et al.
2009). The use of leaves of trees and shrubs and plant extracts
as rumen manipulators, with the aim to reduce methane pro-
duction, has also been reported (Kamra et al. 2008; Delgado et
al. 2010). This communication presents and discusses main
results obtained in Cuba from different experiments directed to
manipulate rumen fermentation and reduce rumen methano-
genesis by the strategic supplementation of tropical trees and
shrubs foliages to ruminants fed with fibrous diets of low
quality.
Materials and methods
The studies were conducted at the Institute of Animal Sci-
ence located at 22º 53′ latitude N, 82º 02′ longitude W, 92 m
above mean sea level.
Assessment of protein-rich plants with respect to reduction
in methane production and methanogen population
Experiment 1 The potential of three tropical plants (Tri-
chantera gigantea, Sapindus saponaria, and Morus alba)
to reduce methane production was evaluated. The plants
were sown manually using stalks of 40 cm at a density of
0.40 cm and 1-m apart. For preparation of the plant foliages,
leaves with petioles and young stems were collected, simu-
lating animal selection; stem diameter was not greater than
0.5 cm, according to Paterson et al. (1983). The foliage was
mixed to obtain a representative sample of approximately
10 kg for each type of the plants. Pennisetum purpureum cv
Cuba CT-115 was used as a basal diet in mixtures with the
foliages. It was cut manually in grazing areas, dried in an
oven at 60°C for 48 h and ground to pass through a sieve of
1.0 mm for further analysis.
Seven treatment were studied: T1, Pennisetum; T2, Tri-
chantera; T3, Sapindus; T4, Morus; T5, T6, and T7 were
mixtures of foliage and grass in the ratio 25:75 of Trichan-
tera/Pennisetum; Sapindus/Pennisetum, and Morus/Pennise-
tum, respectively. The foliage/grass ratio of 25:75 was taken
assuming as the appropriated level of foliage to provide
Trop Anim Health Prod (2012) 44:1097–1104
essential nutrients in the basal grass, according to the principle
of strategic supplementation where efficiency of feed utiliza-
tion from low quality forages would be achieved with
amounts of supplements not exceeding 30% in the diet.
The in vitro gas production of Theodorou et al. (1994)
was used. The samples (500 mg of ground feed) were put in
100-mL bottles with 30 mL of a mixture of rumen/buffer
(10 mL of rumen/20 mL of buffer as in Menke's syringe
method). The bottles were sealed in anaerobic conditions
and incubated in water bath at 39°C. For the in vitro gas
method, the ruminal inoculum was obtained before morning
feeding, from two rumen cannulated crossed zebu steers
grazing star grass. One kilogram per day of commercial
concentrate was also provided every day to these animals.
Four replicates were conducted.
The mixture of gases from the fermentative vessel was
collected after 4, 8, 12, and 24 h of incubation by displacing
the volume in 50-mL syringes. At each time, the gaseous
content in the syringes was injected in a gas chromatograph
to determine methane production. Total methane emitted
after 24 h of incubation was obtained by summing methane
produced at each fermentation time. The dry matter (DM)
fermented after 24 h of incubation was determined and
results expressed as milliliters per gram DM fermented.
The foliages used were screened for secondary metabolites
(Table 1).
Experiment 2 Other 13 plants were supplemented with grass
and defauning capacity, in vitro rumen methanogenesis and
populations of methanogens, cellulolytic bacteria, total viable
bacteria, and protozoa were evaluated. The ratio of grass for-
age/foliages mixtures were 70:30. The plants selected were
Leucaena leucocephala, Gliricidia sepium, Samanea saman,
Albizia lebbeck, Azadirachta indica, Moringa olifera, Pithece-
lobium dulce, Cordia alba, Guazuma ulmifolia, Enterolobium
cyclocarpum, Tithonia diversifolia accession 10, and T. diver-
sifolia accession 23. The selection of these plants was based on
a previous study that showed the presence of secondary metab-
olites, particularly tannins and saponin in these plants. The
procedure for collection of the plant foliages was similar to
that mentioned in experiment 1. The screening for some plant
secondary metabolites was also realized (Table 2). Star grass
(Cynodon nlemfuensis) was the basal pasture. It was obtained
from a non-grazed area of the Institute of Animal Science. The
sample was dried in an oven at 60°C for 48 h and ground to
pass through a sieve of 1.0 mm. The rumen inoculum was
obtained (using a vacuum pump) from two rumen cannulated
buffalo grazing star grass supplemented with 1 kg day−1 of
commercial concentrate. It was filtered through a Muslin cloth.
The rumen liquor obtained from the two animals was pooled.
The amount of sample incubated was 500 mg. Four replicates
were conducted. The gas and methane production were deter-
mined after 0, 4, 8, 12 and 24 h of incubation
Trop Anim Health Prod (2012) 44:1097–1104 1099

Table 1 Chemical composition

of the experimental plant mate- Experimental diets Parameters, (% dry matter) Secondary metabolites
rials and presence of secondary
metabolites Dry matter Crude protein NDF Ash Tannins Saponins

P. purpureum 90.8 7.4 78.0 13.6

S. saponaria 95.3 19.6 52.6 16.2 ++ +
M. alba 92.6 18.7 31.8 16.5 ++ +++
T. gigantea 93.8 19.8 45.3 21.8 ++ −
Sapindus/forage, 25:75 95.3 17.3 62.4 15.7
Morus/forage, 25:75 94.2 14.6 62.5 15.8
+++ high, ++ moderate, + low, Trichantera/forage, 25:75 95.4 11.9 63.9 17.8
− not detected

Experiment 3 Based on the results of experiment 2, the tree Assessment of using a rumen activator (a supplement)
and shrub foliages that substantially reduced methane produc- on in vitro methane production
tion were selected with the aim to study their effects on the
populations of methanogens, cellulolytic bacteria and fungi, Experiment 4 The effect of different supplementation levels
total viable bacteria, and protozoa. The foliages studied were of a ruminal fermentation activator supplement on methano-
S. saman, A. lebbeck, and T. diversifolia accession 23. genesis reduction in ruminants fed fibrous diets was evalu-
Microbiological studies on the populations of methano- ated. The concentrate-based supplement, including L.
gens, cellulolytic bacteria and fungi, total viable bacteria, leucocephala, was used in order to give necessary nutrients
and protozoa were determined. Total viable bacteria, cellulo- for microorganisms, to increase microbial activity and opti-
lytic bacteria, and cellulolytic fungi were counted according to mize rumen fermentation. The rumen activator contained
the anaerobic technique of Hungate (1970). The culture me- soybean meal, 16%; leucaena foliage meal, 29%; rice,
dium of Caldwell and Bryant (1966), modified by Elías 10%; sugar cane molasses, 30%; urea, 3.0%; sunflower
(1971) was used for total viable and cellulolytic bacteria. oil, 4.0%; zeolite, 1.5%; magnesium sulfate, 0.6%; ammo-
The culture medium for fungi included 100,000 IU of peni- nium sulfate, 2.0%; agglutinant (cement), 2.0%; and sodium
cillin and 0.1 g of streptomycin/100 mL. Protozoa were pre- chloride, 2.0%. The moisture content of this activator was
served in formaldehyde at 10% (v/v) and counted in an optical 18.5% and the chemical composition as percent DM was:
microscope using Neubauer chamber after dying protozoa organic matter (OM), 63.9; Ash, 17.6; crude protein (CP),
with a solution of gentian violet (0.01% in glacial acetic acid). 45.9; phosphorus, 0.49; calcium, 0.68; and crude fat, 6.6%.
The methanogens were counted according to the anaerobic The basal feed was grass, P. purpureum Cuba CT-115.
technique of Hungate (1970) but using a mixture of hydrogen Treatments consisted of different ratios of the supplement/
and carbon dioxide (60:40) in the gaseous phase. The culture grass: 0:100, 5:95, 15:85, and 25:75 in a completely ran-
media used was that described by Anderson and Horn (1987). domized design. Rumen fluid as inoculum was obtained
before morning feeding from three rumen-fistulated cross-
breed Zebu steers fed kingrass forage, and incubations were
done in vessels as described above. Gas was collected by
Table 2 In vitro methane emissions from the experimental diets
displacing the volume in syringes after 2, 4, 6, 8, 12, 18, 24,
Treatments Methane emission 36, 48, and 72 h. Methane was analyzed as described above.
The concentration of methane produced was calculated by
mL mL/g DM g/kg DM the general gases equation. For calculating the loss of ener-
incubated incubated
gy by means of methane, the heat of methane combustion
Pennisetum purpureum (forage) 13.4a 26.18a 17.0a was considered.
Sapindo foliage 7.3c 14.0cd 9.1de
Trichantera foliage 5.6c 10.8d 7.0e
Morus foliage 7.5c 13.7cd 8.9e
In vivo studies
Sap/forage 25:75 9.1c 17.0bc 11.4d
Mor/forage 25:75 10.3ab 19.1b 12.4b
The objective of this study was to determine the effect of
supplementing L. leucocephala with a low quality forage on
Trich/forage 25:75 8.0c 18.0bc 11.6d
methane production in sheep. The tunnel method (Lockyer
SEM 1.2 1.5 0.9
and Jarvis 1995) was used to quantify the methane produc-
Means with different lowercase letters in a column differ at P<0.05 tion from sheep.
1100
Experiment 5 Four male rumen-fistulated Pelibuey sheep,
aged 8 months (live weight 25 kg±3.5), individually kept in
metabolism cages, were used in a crossover design with two
treatments and two replicates by treatment (two animals per
treatment). The treatments consisted of a control diet: P.
purpureum clone CT-169, 63% and concentrate, 37% (on
DM basis); and an experimental diet: P. purpureum 63%,
Leucaena 27%, and concentrate 10% (on DM basis). Pen-
nisetum was harvested in dry season (November–December
of 2008), after 4 months of establishment. Feeds were of-
fered twice a day in equal portions. The feed intake and
refusal were daily measured. Chemical composition of con-
centrate (percent on DM basis) was OM 95.1%, crude
protein 19.8%, and ash 4.9%.
Methane determination It was determined using a gas chro-
matograph Philips PU-4400 fitted with a flame ionization
detector. One-milliliter gas mixture obtained in syringes
from the tunnel was injected in the chromatograph. Pure
methane (99.9%) was used for preparation of the calibration
curve. Helium gas was used as a carrier gas (1 m/min), oven
temperature was 60°C, attenuation to 200°C, and detector
temperature was 200°C.
Chamber method for in vivo methane determination Four
metabolism cages (200×82×147 cm) were adapted to obtain
individual chambers. Each chamber was covered on its sides
with white polyethylene sheet (polyethylene sheet that is used
for making green house). The chambers had one inlet opening
(5-cm diameter) in the front and one outlet opening in the rear
(5-cm diameter) in which an exhaust pump was connected to
remove the inside air. A variable speed domestic fan was used
inside the chamber to keep the air circulated. During the
adaptation periods, the polyethylene sheets on the walls of
the chamber were kept open to maintain free air flow. After
adaptation for 17 days, the experimental chambers were
sealed with the animals inside. Gas was manually taken every
hour using a 100-mL capacity syringe for two consecutive
24-h periods. Two air samples were taken, one from inside and
another from outside the chamber. The mean temperature
inside the chamber was 22°C. The airflow was determined
using a hand anemometer (Extech Instruments Corporation
series 451126, MA, USA) at the same time when the sample
was taken for methane measurement. Samples were stored in
evacuated flasks of 60-mL capacity for later analysis. Before
keeping the animals inside the chambers, a known amount of
methane was released (a balloon containing known amount of
methane was released) and recovery of this methane was 95%.
Chemical analysis DM, CP, and ash were determined by
the AOAC method (1995). Neutral detergent fiber (NDF)
was determined by the method of Goering and Van Soest
(1970).
Trop Anim Health Prod (2012) 44:1097–1104
Phytochemical screening of plant materials to qualitative
assess the presence of secondary metabolites was realized
according to Rondina and Coussio (1969). The assays used
to detect the secondary metabolites were: tannins by means
of the Jello test, triterpenoides and steroids by means of the
Lieberman–Bourchard reaction, alkaloids by the Dragen-
dorff tests, flavonoids by the Shinoda reaction, and saponins
by the foam test.
Statistical analysis Data were subject to ANOVA using
SPSS system for Windows (Visauta 1998). Differences be-
tween means were detected by Duncan test (1955). For the
studies on effects of foliages on microbial populations,
factorial design was used to evaluate treatments. The count-
ing of microorganisms was transformed to Log N, to guar-
antee the normality conditions in the growth curve. The
formula was (K + N). Applied for the analysis was 10×,
where K is the constant representing the logarithm of the
dilution in which the microorganisms were inoculated; N is
the logarithm of the colony forming units as colony forming
units per milliliters, tfu × mL-1, or cells per milliliters; 10 is
the base of the logarithms and X is the dilution
In experiment 5, general lineal model, SSPS system, was
used to control the effect of treatment, animals, and periods.
In the necessary cases, the differences between means were
tested according Duncan (1955).
Results and discussion
Experiment 1 Table 1 shows the presence of a moderate
level (++) of tannins in all plants while M. alba contained
a high level (+++) of saponins. Pennisetum produced
26.2 mL CH4/g DM fermented and when it was mixed with
foliages of Sapindus, Morus, or Trichantera at 25%, it not
only improved the nutritional value of the ration by increas-
ing the protein value (Table 1), but also reduced (P<0.01)
the amount of methane produced: 17.0, 19.1 and 18.0 mL/g
of DM incubated, respectively (Table 2).
These results are attributed, mainly, to the presence of
secondary compounds that seem to have antimethanogenic
properties, specifically tannins and saponins. Both tannins
and saponins have received attention for their ability to reduce
methane formation (Hess et al. 2003; Rochfort et al. 2008, and
Soliva et al. 2008) found that S. saponaria fruit decreased
methane production and ruminal protozoa. Reduced CH4 pro-
duction from rumen fermentation by saponins has also been
demonstrated in various studies (Guo et al. 2009; Holtshausen
et al. 2009; Martin et al. 2010). Tropical plants rich in con-
densed have been shown to reduce rumen methanogenesis
(Beauchemin et al. 2008; Soliva et al. 2008) probably due to
both direct effects of the tannins on methanogen activity and
Trop Anim Health Prod (2012) 44:1097–1104 1101

Table 3 Effect of tree and shrub foliages when mixed at 30% level in star grass (C. nlemfuensis) on methane concentration in vitro and presence of
secondary metabolites in the plant foliages

Foliages mixed at 30% level in grass CH4, mL/100 mL gas Secondary metabolites

Tannins Flavonoids Saponins Triterpenes Steroids Anthcyanidins Alkaloids

S. saman 0.43a ++ + + ++ ++ + +++

A. lebbeck 0. 57a + + ++ + + + +++
A. indica 0.86a +++ + + + + + +++
T. diversifolia accession 23 0.92a ++ ++ + + ++ + ++
T. diversifolia accession 10 − ++ − ++ ++ ++ + ++
C. alba 1.18a ND ND ND ND ND ND ND
L. leucocephala 1.64a +++ + ++ ++ ++ + +++
P. dulce 2.03a + − + + + − ++
M. olifera 2.53a ++ − + +++ − − ++
G. sepium 2.90ab + + ++ + + + −
G. ulmifolia 3.80ab ++ − + +++ − − ++
E. cyclocarpum 6.42b +++ + ++ + + − ++
C. nlemfuensis 6.51b ND ND ND ND ND ND ND
SEM 0.12

Means with different lowercase letters in a column differ at P<0.05

+++ high, ++ moderate, + low, − not detected, ND not determined

indirect effects via decreasing fiber digestion. Jayanegara et al. a source of the referred secondary metabolites for their use
(2011) studied the relationships among various phenolic frac- as additives. This plant is not accepted by cattle.
tions and methane emissions from tropical plants. Condensed It was demonstrated that most of the foliages assessed, as
and hydrolysable tannins contributed to the decrease in CH4/ mixtures with star grass, produced less methane compared
digestible OM. to star grass alone. The promising foliages were: S. saman,
The supplementation with these foliages also increased A. lebbeck, A. indica and T. diversifolia ecotype 23, C. alba,
the nutritional quality of the diet by increasing the protein L. leucocephala, P. dulce, and M. olifera (Table 3).
content (Table 1) and a reduction in methane production E. cyclocarpum is a protein-rich foliage and it has been
could also be due to increased rumen fermentation efficien- assessed as a modifier of the ruminal fermentation. Navas-
cy as a result of provision of nutrients deficient in the basal Camacho et al. (1994) reported that E. cyclocarpum reduced
diet (P. purpureum). From the results of the present study, it total protozoa population in the rumen. However, Delgado
is evident that the three foliages studied have the capability et al. (2010) reported that Enterolobium did not exert defau-
to reduce methane production when supplemented to low nating effects but increased the ruminal cellulolytic fungi
quality forage, P. purpureum. population. In the present study, it did not show any effect
on protozoal population although it contained high levels of
Experiments 2 and 3 The secondary metabolites, qualitative-
ly evaluated, indicated the presence of high amounts of tan-
nins (+++) in A. indica, L. leucocephala, and E. cyclocarpum
foliages and moderate amounts of tannins (++) in S. saman, G.
ulmifolia as well as in two of the ecotypes of T. diversifolia
assessed. None of the plants examined contained high
amounts of saponins. A. lebeck, L. leucocephala, and G.
sepium foliages contained moderate level (++) of saponins.
The presence of flavonoids, triterpens, steroids, alkaloids, and
anthocyanidins was variable in different plants while steroids
and anthocyanidins were not detected in M. olifera (Table 3)
The inclusion of A. indica in the assessment was, only,
due to its high content of tannins and some saponins that Fig. 1 Effect of the fermentation time (in hours) on the methane
may reduce methane production and, strategically, could be production (milliliters per liters of gas produced)
1102 Trop Anim Health Prod (2012) 44:1097–1104

Table 4 Effect of mixtures of foliage/grass on rumen microbial pop- Table 6 Effects of Leucaena on intake, apparent digestibility of dry
ulation (transformed data as log×) matter and neutral detergent fiber, and methane production in sheep fed
low quality grass
Control 30:70 30:70 30:70 SEM
Cynodon Albizia/grass Saman/grass Tithonia/grass
Indicators Control Control +
Leucaena
Total viable 1.55 (36.0) 1.62 (42.0) 1.60 (40.0) 0.54 (35.0) 0.23
bacteria,
1011 cfu mL−1 Dry matter ingestion, kg 0.86±0.02 0.903±0.02
Methanogenic 1.85a (71.27) 1.54b (35.05) 1.28c (19.16) 1.24c (17.27) 0.25*
bacteria, 1010 Dry matter ingestion, % live weight 2.79±0.11* 3.32±0.11*
cfu mL−1 Organic matter ingestion, kg 0.59±0.02* 0.70±0.02*
Protozoa, 105cells 0.95a (8.94) 0.85b (7.05) 0.81c (6.51) 0.82bc (6.64) 0.13**
mL−1 Dry matter digestibility, % 45.01±2.73 47.79±2.37
NDFD, % 39.36±2.98 40.67±2.58
Means with different lowercase letters in a row differ at P<0.05; Organic matter digestibility, % 48.99±2.87 52.39±2.49
parentheses contain number of colonies without transformation to log×
Methane production
*P<0.001; **P<0.05
L/day 5.93±1.63 6.55±1.63
Methane, (L/kg DMI) 9.04±2.06 7.63±2.06
tannins and moderate of saponins. Plant age, soil type, and CH4 (L/MW) 0.53±0.14 0.59±0.14
the presence of others secondary compounds may have been
NDFD neutral detergent fiber apparent digestibility, MW metabolic
responsible for these differences in results obtained.
weight
The effect of fermentation time on methane production is
*P<0.01
presented in Fig. 1. It was found that the highest production
of this gas was at 8 h (39.59 mL/L of total gas produced)
and decreased at 12 and 24 h (30.91 and 29.85 mL/L of total 37% of the methane production in the rumen (Hook et al.
gas produced, respectively). 2010). Elimination of protozoa or defaunation is known to
In relation to control (star grass alone), no effect on popu- reduce methane emission by ruminants.
lation of total viable bacteria was found on using S. saman, A. This is a need to the study of intrinsic factors of the plants
lebbeck, and T. diversifolia accession 23 (Table 4). This effect studied that may have depressive effects on the cellulolytic
is important and indicates the possibility of using these plants fungi population. Further studies on the effect of the sec-
without causing depression in the total bacterial population. ondary metabolites of these plants on the microbial popula-
The inclusion of foliages in the diets decreased (P<0.001) tions are warranted.
metanogens with respect to star grass. The reduction by S. No effect on the population of total viable bacteria on
saman and T. diversifolia was threefold and by A. lebeck 1.8- using S. saman, A. lebbeck, and T. diversifolia was found in
fold compared to the control. relation to control, P. purpureum. These results confirm the
Table 4 shows that S. saman, Albizia, and Tithonia re- hypothesis that certain secondary metabolites in the trees are
duced (P<0.0 5) protozoa population and S. saman is the capable of reducing methanogenic archea and protozoa pop-
most promising one. Rumen protozoa, as stated previously, ulation (Galindo et al. 2005; Beauchemin et al. 2008) and
share a symbiotic relationship with methanogens, participat- could explain the reduction in the methane emission ob-
ing in interspecies hydrogen which provides methanogens served in this experiment.
with the hydrogen they require to reduce carbon dioxide to
methane. It has been estimated that the methanogens asso- Experiment 4 Results indicated that different levels of sup-
ciated with the ciliate protozoa are responsible for 9% to plementation to Pennisetum grass had no effect on the

Table 5 Effect of different supplementation levels on methane emissions in in vitro conditions at 24 h of fermentation

Rate supplement/forage

0/100 5/95 15/85 25/75 SEM±

Gas production, mL 119.18 130.11 131.27 119.38 11.21

Fermentation rate, c (% h−1) 1.03 0.92 0.81 0.95 0.09
Lag phase, h 6.39a 8.87a 5.27ab 3.06b 1.02*
Volume CH4 produced, mL/g fermented DM 30.76a 27.69a 19.20b 16.54b 1.75*
Energy losses as CH4, J/g fermented DM 17.45a 16.73a 11.52b 9.95b 1.11*

Media with different lowercase letters in the same line differ P<0.05 (Duncan 1955)
*P<0.01
Trop Anim Health Prod (2012) 44:1097–1104
potential gas production. However, the lag phase decreased
(P < 0.01) with the increase in the level of supplement
(Table 5). Inclusion levels of 15% and 25% reduced the
methane (in milliliters per gram DM fermented) by 37%
and 42% with respect to the treatment without supplemen-
tation. The energy losses by means of the methane de-
creased from 17.45 J/g DM fermented per day with 0:100
treatment to 9.95 J/g DM fermented per day with 25:75 ratios
(Table 5). The results of this experiment suggested than it is
possible to reduce the methane emissions in low quality forage
diets with the use of strategic supplementation
Experiment 5 Dry matter intake (DMI) and organic matter
intake (OMI) were higher (P<0.01) when Leucaena foliage
was included in the grass forage (DMI: 3.32% vs 2.79% live
weight, with and without Leucaena, respectively) (Table 6).
Similar results were reported by Delgado et al. (1996) in
sheep.
There were no differences between treatments for the
apparent digestibility of DM, OM, and the NDF (Table 6).
The apparent digestibilities (in percent) were 45 and 47 for
DM, and 39 and 40 for NDF, for the treatments without and
with Leucaena, respectively.
Studies by Abdulrazak et al. (2006) with tanniferous
forages indicated increases in the intake without changes
in the digestibility and Myint et al. (2010) observed similar
DM intake and digestibility in goat supplemented with
Leucaena. However, Longo et al. (2008) found a significant
reduction in the apparent digestibility of nutrients in sheep
supplemented with this foliage and attributed it to a number
of factors, mainly among them to the contents of tannins and
lignin present in Leucaena.
When foliages of trees and shrubs are used in a diet, the
apparent digestibility can change depending on factors such
as supplementation level, animal species, type of foliage and
the basal pasture, the quantity and activity of condensed
tannins, and other secondary compounds present in the
plants, among others.
The inclusion of Leucaena in the diet did not significantly
reduce the total volume of methane produced by the animals
(6.55 vs 5.93 L/day with and without foliage, respectively).
However, it is interesting to note that when methane produc-
tion was calculated on DMI, the treatment with L. leucoce-
phala had lower methane production by 15.6% with respect to
the control, although the difference was not significant.
Methane emissions (in grams per kilograms DMI) from
animals fed forage legumes is usually but not always (Van
Dorland et al. 2007) lower than emissions from animals fed
predominantly with grasses. Possenti et al. (2008) observed
that inclusion of 20% and 50% of Leucaena hay in a ration
of C. dactylon increased methane production, but when
yeast Saccharomyces cerevisiae was added, the production
of methane diminished.
1103
Lower emissions from animals fed legumes are often
explained by the presence of condensed tannins, lower fiber
content, higher DMI, and faster rate of passage from the
rumen (Beauchemin et al. 2008). It is possible that the
inclusion levels of Leucaena or the experimental conditions
used in the experiment, among other factors, did not allow
reaching significant reductions in the methane production.
Further work is needed on optimizing the level of Leucaena
foliage to be used in in vivo conditions.
Conclusions
The present work revealed the potential of the plant foliages
tested as strategic supplements to ruminants fed with low
quality forages for reduction of methane production. S.
saman, A. lebbeck, T. diversifolia, L. leucocephala, T.
gigantea, S. saponaria, and M. alba produced lower meth-
ane as compared to grass forage (P. purpureum, clone Cuba
CT-115 or C. nlemfuensis). These plant foliages had mod-
erate amounts of tannins and none contained high amounts
of saponins. The results suggest that the supplementation
strategies based on feeding tropical foliages to animals fed
with low quality forage is an attractive and practical option
to reduce methane production and improve the efficiency of
rumen in the tropical areas. The study also demonstrates that
the tunnel method is a useful method to measure methane
production from animals fed with different diets.
Acknowledgments The Instituto de Ciencia Animal is grateful to the
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agricul-
ture for the financial support provided through Coordinated Research
Projects no. 12667.
References
Abdulrazak S. A, Kahindi, R. K. and Muinga, R. W., 2006. Effects of
Madras thorn, Leucaena and Gliricidia supplementation on feed
intake, digestibility and growth of goats fed Panicum hay. Live-
stock Research for Rural Development, 18, Article #124. http://
www.lrrd.org/lrrd18/9/abdu18124.htm. Reviewed: August 31,
2009
Anderson, M.A. and Horn, G.M., 1987. Effect of Lasalocic in weight
gain, ruminal fermentation and forage intake of stocker cattle
grasing winter wheat pasture, Journal of Animal Science, 65,
865-874
A.O.A.C., 1995. Official methods of analysis (15th Edition) Associa-
tion of Official Analytical Chemists. Arlington
Attwood, G. and MC Sweeney C., 2008. Methanogen genomics to
discover targets for methane mitigation technologies and options
for alternative H2 utilization in the rumen. Australian Journal of
Experimental. Agricultural, 48, 28-37
Beauchemin, K.A., Kreuzer M. O'Mara, F. and Mcallister, A., 2008.
Nutritional management for enteric methane abatement: A review.
Australian Journal of Experimental Agriculture, 48, 21-27
1104
Caldwell, D. R. and Bryant, M.P., 1966. Medium without fluid for non
selective enumeration and isolation of rumen bacteria. Applied
Microbiology, 14, 794-801
Carmona J.C., Bolívar D.M. and Girald, L.A., 2005. El gas metano en
la producción ganadera y alternativas para medir sus emisiones y
aminorar su impacto a nivel ambiental y productivo. Revista.
Colombiana de Ciencias Pecuarias, 18, 49-63
Colman, D. and Beever, D. E., 2009. Gaining from Improved Dairy Cow
Nutrition: Economic, environmental and animal health benefits. The
83 rd Annual Conference of the Agricultural Economics Society,
Dublin, 30th March to 1st April 2009. http://ageconsearch.umn.edu/
bitstream/51424/2/Colman_beever.pdf. Reviewed: Sept. 20/2011,
10:14 am.
Delgado, D.C., Galindo, J., Chongo B. and Curbelo, T., 1996. Efecto
del nivel de inclusión de la Leucaena leucocephala en el consumo
y la digestibilidad de la fibra en carneros. Revista cubana de
Ciencia Agrícola, 30, 283-287
Delgado, D.C., Galindo, J., González, R., Savón, L., Scull, I., González,
N. and Marrero, Y. 2010. In E. Odongo, M. García & G.J.Viljoen
(eds), Sustainable Improve of animal Production and health. Food
and Agriculture Organization of the United Nations, Rome, 2010,
49-54
Duncan, D.E., 1955. Multiple ranges and multiple F test. Biometrics,
11, 1-42
Elías, A., 1971. The rumen bacteria of animals fed on a high-molasses
urea diet. Dissertation. Aberdeen
Galindo, J.L; Delgado, D.C and Chongo, B. 2005. Papel de los árboles,
arbustos y leguminosas en la población microbiana ruminal.
Memorias II Curso Internacional Silvopastoril Cuba- Colombia.
Neiva-Bogotá- Valledupar. CORPOICA, Bogotá, ICA, Cuba. ,
130
Goel, G., Makkar, H.P.S. and Becker, K., 2008. Changes in microbial
community structure, methanogenesis and rumen fermentation in
response to saponin-rich fractions from different plant materials.
Journal Applied Microbiology, 105, 770–777
Goering, H.K and Van Soest, P.J., 1970. Forage fiber Analysis. Agri-
cultural Handbook, US. Department of agriculture, No 379.
Washington
Guo, Y. Q., Liu, J. X., Lu, Y., Zhu, W. Y., Denman, S. E. and Mc
Sweeney, C. S., 2009. Effect of tea saponin on methanogenesis,
microbial community structure and expression of mcrA gene, in
cultures of rumen microorganisms. Letters in Applied Microbiol-
ogy, 47, 421–426
Hess, H.D., Kreuzer, M., Diaz, T.E., Lascano, C.E., Carulla, J.E., Soliva,
C.R. and Machmuller, A., 2003. Saponin rich tropical fruits affect
fermentation and methanogensesis in faunated and defaunated ru-
men fluid. Animal Feed Science and Technology, 109, 79–94.
Holtshausen, L., Chaves, A. V., Beauchemin K. A., McGinn S.M.,
McAllister, T.A., Odongo, N.E., Cheeke, P.R. and Benchaa, C.,
2009. Feeding saponin-containing Yucca schidigera and Quillaja
saponaria to decrease enteric methane production in dairy cows.
Journal of Dairy Science, 92:2809–2821,
Hook, S.E., Wright, A-D.G. and McBride, B.W., 2010. Methanogens:
Methane producers of the rumen and mitigation strategies. Archaea,
http://downloads.hindawi.com/journals/arch/2010/945785.pdf.
Reviewed: Sept. 23/2011, 11:15 am
Hungate, P.E. 1970. A roll tube method for cultivation in microbiology
In: J.B. Morris and D.B. Ribbons (eds), New York, Academic 117.
Jayanegara, A., Togtokhbayar, N., Makkar, H.P.S. and Becker, K.,
2009. Tannins determined by various methods as predictors of
methane production reduction potential of plants by an in vitro
rumen fermentation system. Animal Feed Science and Technolo-
gy, 150, 230–237
Trop Anim Health Prod (2012) 44:1097–1104
Jayanegara, A., Wina, E., Soliva, C.R., Marquardt, S., Kreuzer M., and
Leiber, F., 2011. Dependence of forage quality and methanogenic
potential of tropical plants on their phenolic fractions as deter-
mined by principal component analysis, 163, 231-243
Kamra, D. N., PatraA, A. K., Chatterjee, P. N., Kumar, R., Agarwal N. and
Chaudhary, L.C., 2008. Effect of plant extracts on methanogenesis
and microbial profile of the rumen of buffalo: A brief overview.
Australian Journal of Experimental Agriculture, 48, 175–178
Lascano C.E. and Cárdenas, E., 2010. Alternatives for methane emis-
sion mitigation in livestock systems. Revista. Brasilera de Zoo-
tecnia, Special Supplement, 39, 175-182
Longo, C., Nozella, E. F., Cabral Filho, S. L. S., Lavorenti, N., Vitti, D.
M. S. S. and Abdalla, A. L., 2008. Voluntary intake, apparent
digestibility and nitrogen balance by sheep supplemented with
Leucaena leucocephala. Livestock Resources for Rural Develo-
ment. 20 (11)2008. http://www.lrrd.org/lrrd20/11/cont2011.htm.
reviewed: August 2/ 2009, 12:40 am
Lockyer, D.R. and Jarvi, S.C., 1995. The measurement of methane
losses from grazing animals. Enviromental Pollution, 90, 383-390
Martin, D. P., Morgavi, and Doreau, M., 2010. Methane mitigation in
ruminants: from microbe to the farm scale DOI:dx.doi.org . 2010.
Animal, 4, 351–365
Myint, K.H., Mu, K.S., Soe, T.M., Maw, N.M., Ming Gawng, L.T. and
NGwe, T., 2010. Evaluation of Leucaena leucocephala and Zizi-
phus Mauritania as sources of tannins and their interference with
nitrogen utilization in goats. In: E. Odongo, M. García and G.J.
Viljoen (eds), Sustainable Improvement of animal Production and
health. Food and Agriculture organization of the United Nations,
Rome, 2010, 159-166
Navas-Camacho, A., Laredo, M.A., Cuesta, A., Ortega, O. and
Romero, M., 1994. Evaluation of tropical trees with high or
medium saponins content as dietary alternative to eliminate ciliate
protozoa from the rumen. Proceeding of the of Nutrition Physiol-
ogy. 3, 204-210
Paterson, R.T., Quiroga, L., Sauma, G. and Samur, C. 1983. Creci-
miento de novillas Cebú-Criollo en la época de seca con acceso
limitado a la leucaena. Producción Animal Tropical, 8, 150-155
Possenti, R.A., Franzolin, R., Schammas, E. A. Demarchi, J.J.A.A,
Frighetto, R.T.S. and Lima, M.A., 2008. Efeitos de dietas con-
tendo Leucaena leucocephala e Saccharomyces cerevisiae sobre a
fermentação ruminal e a emissão de gás metano em bovinos.
Revista Brasileira de Zootechnia, 37, 1509-1516
Rochfort, S., Parker, A,J. and Dunshea, F,R., 2008. Plant bioactives for
ruminant health and productivity. Phytochemistry, 69, 299–322
Rondina, R.D.V. and Coussio. J.D., 1969. Estudios fitoquímicos de
plantas medicinales. Revista de Investigaciones Agropecuarias, 6,
351-36
Soliva C.R., Zeleke A.B., Clement, C., Hess, H.D., Fievez V., and
Kreuzer, M., 2008. In vitro screening of various tropical foliages,
seeds, fruits and medicinal plants for low methane and high
ammonia generating potentials in the rumen. Animal Feed Sci-
ence and Technology, 147, 53–71
Theodorou, M.K, Williams, B.A., Dhanoa, M.S., McAllan, A.B. and
France, J., 1994. A simple gas production method using a pres-
sure transducer to determine the fermentation kinetics of rumi-
nants feed. Animal Feed Science and Technology, 48, 185-197
Van Dorland, H.A, Wettstein., H.R, Leuenberger, H. and Kreuzer, M.,
2007. Effect of supplementation of fresh and ensiled clovers to
ryegrass on nitrogen loss and methane emissions in dairy cows.
Livestock Science, 111, 57–69.
Visauta, B., 1998. Análisis estadístico con SPSS para Windows. Esta-
dística multivariada. Vol II. Mc Graw-Hill/ Interamericana de
España, S.A.V, .358.
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