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

increase efficiency of production by up to fivefold with proper essential nutrients in the basal grass, according to the principle
dietary supplementation. This has been done without changing of strategic supplementation where efficiency of feed utiliza-
the basal feed resources and by identifying and providing tion from low quality forages would be achieved with
critical nutrients that are otherwise deficient in the diet. In this amounts of supplements not exceeding 30% in the diet.
way nutrient availability is balanced with animal requirements The in vitro gas production of Theodorou et al. (1994)
and forage intake is stimulated at the same time. Consequently, was used. The samples (500 mg of ground feed) were put in
substantial increase in animal productivity can be achieved 100-mL bottles with 30 mL of a mixture of rumen/buffer
(Colman and Beever 2009). (10 mL of rumen/20 mL of buffer as in Menke's syringe
Tree and shrub foliages can be used as catalytic supple- method). The bottles were sealed in anaerobic conditions
ments to provide rumen soluble protein and minerals. In addi- and incubated in water bath at 39°C. For the in vitro gas
tion, plant secondary metabolites such as tannins and saponins method, the ruminal inoculum was obtained before morning
present in some tree leaves and shrubs appear to have anti- feeding, from two rumen cannulated crossed zebu steers
methanogenic properties (Goel et al. 2008; Jayanegara et al. grazing star grass. One kilogram per day of commercial
2009). The use of leaves of trees and shrubs and plant extracts concentrate was also provided every day to these animals.
as rumen manipulators, with the aim to reduce methane pro- Four replicates were conducted.
duction, has also been reported (Kamra et al. 2008; Delgado et The mixture of gases from the fermentative vessel was
al. 2010). This communication presents and discusses main collected after 4, 8, 12, and 24 h of incubation by displacing
results obtained in Cuba from different experiments directed to the volume in 50-mL syringes. At each time, the gaseous
manipulate rumen fermentation and reduce rumen methano- content in the syringes was injected in a gas chromatograph
genesis by the strategic supplementation of tropical trees and to determine methane production. Total methane emitted
shrubs foliages to ruminants fed with fibrous diets of low after 24 h of incubation was obtained by summing methane
quality. 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.
Materials and methods The foliages used were screened for secondary metabolites
(Table 1).
The studies were conducted at the Institute of Animal Sci-
ence located at 22º 53′ latitude N, 82º 02′ longitude W, 92 m Experiment 2 Other 13 plants were supplemented with grass
above mean sea level. and defauning capacity, in vitro rumen methanogenesis and
populations of methanogens, cellulolytic bacteria, total viable
Assessment of protein-rich plants with respect to reduction bacteria, and protozoa were evaluated. The ratio of grass for-
in methane production and methanogen population age/foliages mixtures were 70:30. The plants selected were
Leucaena leucocephala, Gliricidia sepium, Samanea saman,
Experiment 1 The potential of three tropical plants (Tri- Albizia lebbeck, Azadirachta indica, Moringa olifera, Pithece-
chantera gigantea, Sapindus saponaria, and Morus alba) lobium dulce, Cordia alba, Guazuma ulmifolia, Enterolobium
to reduce methane production was evaluated. The plants cyclocarpum, Tithonia diversifolia accession 10, and T. diver-
were sown manually using stalks of 40 cm at a density of sifolia accession 23. The selection of these plants was based on
0.40 cm and 1-m apart. For preparation of the plant foliages, a previous study that showed the presence of secondary metab-
leaves with petioles and young stems were collected, simu- olites, particularly tannins and saponin in these plants. The
lating animal selection; stem diameter was not greater than procedure for collection of the plant foliages was similar to
0.5 cm, according to Paterson et al. (1983). The foliage was that mentioned in experiment 1. The screening for some plant
mixed to obtain a representative sample of approximately secondary metabolites was also realized (Table 2). Star grass
10 kg for each type of the plants. Pennisetum purpureum cv (Cynodon nlemfuensis) was the basal pasture. It was obtained
Cuba CT-115 was used as a basal diet in mixtures with the from a non-grazed area of the Institute of Animal Science. The
foliages. It was cut manually in grazing areas, dried in an sample was dried in an oven at 60°C for 48 h and ground to
oven at 60°C for 48 h and ground to pass through a sieve of pass through a sieve of 1.0 mm. The rumen inoculum was
1.0 mm for further analysis. obtained (using a vacuum pump) from two rumen cannulated
Seven treatment were studied: T1, Pennisetum; T2, Tri- buffalo grazing star grass supplemented with 1 kg day−1 of
chantera; T3, Sapindus; T4, Morus; T5, T6, and T7 were commercial concentrate. It was filtered through a Muslin cloth.
mixtures of foliage and grass in the ratio 25:75 of Trichan- The rumen liquor obtained from the two animals was pooled.
tera/Pennisetum; Sapindus/Pennisetum, and Morus/Pennise- The amount of sample incubated was 500 mg. Four replicates
tum, respectively. The foliage/grass ratio of 25:75 was taken were conducted. The gas and methane production were deter-
assuming as the appropriated level of foliage to provide 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 Trop Anim Health Prod (2012) 44:1097–1104

Experiment 5 Four male rumen-fistulated Pelibuey sheep, Phytochemical screening of plant materials to qualitative
aged 8 months (live weight 25 kg±3.5), individually kept in assess the presence of secondary metabolites was realized
metabolism cages, were used in a crossover design with two according to Rondina and Coussio (1969). The assays used
treatments and two replicates by treatment (two animals per to detect the secondary metabolites were: tannins by means
treatment). The treatments consisted of a control diet: P. of the Jello test, triterpenoides and steroids by means of the
purpureum clone CT-169, 63% and concentrate, 37% (on Lieberman–Bourchard reaction, alkaloids by the Dragen-
DM basis); and an experimental diet: P. purpureum 63%, dorff tests, flavonoids by the Shinoda reaction, and saponins
Leucaena 27%, and concentrate 10% (on DM basis). Pen- by the foam test.
nisetum was harvested in dry season (November–December
of 2008), after 4 months of establishment. Feeds were of- Statistical analysis Data were subject to ANOVA using
fered twice a day in equal portions. The feed intake and SPSS system for Windows (Visauta 1998). Differences be-
refusal were daily measured. Chemical composition of con- tween means were detected by Duncan test (1955). For the
centrate (percent on DM basis) was OM 95.1%, crude studies on effects of foliages on microbial populations,
protein 19.8%, and ash 4.9%. factorial design was used to evaluate treatments. The count-
ing of microorganisms was transformed to Log N, to guar-
Methane determination It was determined using a gas chro- antee the normality conditions in the growth curve. The
matograph Philips PU-4400 fitted with a flame ionization formula was (K + N). Applied for the analysis was 10×,
detector. One-milliliter gas mixture obtained in syringes where K is the constant representing the logarithm of the
from the tunnel was injected in the chromatograph. Pure dilution in which the microorganisms were inoculated; N is
methane (99.9%) was used for preparation of the calibration the logarithm of the colony forming units as colony forming
curve. Helium gas was used as a carrier gas (1 m/min), oven units per milliliters, tfu × mL-1, or cells per milliliters; 10 is
temperature was 60°C, attenuation to 200°C, and detector the base of the logarithms and X is the dilution
temperature was 200°C. In experiment 5, general lineal model, SSPS system, was
used to control the effect of treatment, animals, and periods.
Chamber method for in vivo methane determination Four In the necessary cases, the differences between means were
metabolism cages (200×82×147 cm) were adapted to obtain tested according Duncan (1955).
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 Results and discussion
(5-cm diameter) in which an exhaust pump was connected to
remove the inside air. A variable speed domestic fan was used Experiment 1 Table 1 shows the presence of a moderate
inside the chamber to keep the air circulated. During the level (++) of tannins in all plants while M. alba contained
adaptation periods, the polyethylene sheets on the walls of a high level (+++) of saponins. Pennisetum produced
the chamber were kept open to maintain free air flow. After 26.2 mL CH4/g DM fermented and when it was mixed with
adaptation for 17 days, the experimental chambers were foliages of Sapindus, Morus, or Trichantera at 25%, it not
sealed with the animals inside. Gas was manually taken every only improved the nutritional value of the ration by increas-
hour using a 100-mL capacity syringe for two consecutive ing the protein value (Table 1), but also reduced (P<0.01)
24-h periods. Two air samples were taken, one from inside and the amount of methane produced: 17.0, 19.1 and 18.0 mL/g
another from outside the chamber. The mean temperature of DM incubated, respectively (Table 2).
inside the chamber was 22°C. The airflow was determined These results are attributed, mainly, to the presence of
using a hand anemometer (Extech Instruments Corporation secondary compounds that seem to have antimethanogenic
series 451126, MA, USA) at the same time when the sample properties, specifically tannins and saponins. Both tannins
was taken for methane measurement. Samples were stored in and saponins have received attention for their ability to reduce
evacuated flasks of 60-mL capacity for later analysis. Before methane formation (Hess et al. 2003; Rochfort et al. 2008, and
keeping the animals inside the chambers, a known amount of Soliva et al. 2008) found that S. saponaria fruit decreased
methane was released (a balloon containing known amount of methane production and ruminal protozoa. Reduced CH4 pro-
methane was released) and recovery of this methane was 95%. duction from rumen fermentation by saponins has also been
demonstrated in various studies (Guo et al. 2009; Holtshausen
Chemical analysis DM, CP, and ash were determined by et al. 2009; Martin et al. 2010). Tropical plants rich in con-
the AOAC method (1995). Neutral detergent fiber (NDF) densed have been shown to reduce rumen methanogenesis
was determined by the method of Goering and Van Soest (Beauchemin et al. 2008; Soliva et al. 2008) probably due to
(1970). 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 1103

potential gas production. However, the lag phase decreased Lower emissions from animals fed legumes are often
(P < 0.01) with the increase in the level of supplement explained by the presence of condensed tannins, lower fiber
(Table 5). Inclusion levels of 15% and 25% reduced the content, higher DMI, and faster rate of passage from the
methane (in milliliters per gram DM fermented) by 37% rumen (Beauchemin et al. 2008). It is possible that the
and 42% with respect to the treatment without supplemen- inclusion levels of Leucaena or the experimental conditions
tation. The energy losses by means of the methane de- used in the experiment, among other factors, did not allow
creased from 17.45 J/g DM fermented per day with 0:100 reaching significant reductions in the methane production.
treatment to 9.95 J/g DM fermented per day with 25:75 ratios Further work is needed on optimizing the level of Leucaena
(Table 5). The results of this experiment suggested than it is foliage to be used in in vivo conditions.
possible to reduce the methane emissions in low quality forage
diets with the use of strategic supplementation
Conclusions
Experiment 5 Dry matter intake (DMI) and organic matter
intake (OMI) were higher (P<0.01) when Leucaena foliage The present work revealed the potential of the plant foliages
was included in the grass forage (DMI: 3.32% vs 2.79% live tested as strategic supplements to ruminants fed with low
weight, with and without Leucaena, respectively) (Table 6). quality forages for reduction of methane production. S.
Similar results were reported by Delgado et al. (1996) in saman, A. lebbeck, T. diversifolia, L. leucocephala, T.
sheep. gigantea, S. saponaria, and M. alba produced lower meth-
There were no differences between treatments for the ane as compared to grass forage (P. purpureum, clone Cuba
apparent digestibility of DM, OM, and the NDF (Table 6). CT-115 or C. nlemfuensis). These plant foliages had mod-
The apparent digestibilities (in percent) were 45 and 47 for erate amounts of tannins and none contained high amounts
DM, and 39 and 40 for NDF, for the treatments without and of saponins. The results suggest that the supplementation
with Leucaena, respectively. strategies based on feeding tropical foliages to animals fed
Studies by Abdulrazak et al. (2006) with tanniferous with low quality forage is an attractive and practical option
forages indicated increases in the intake without changes to reduce methane production and improve the efficiency of
in the digestibility and Myint et al. (2010) observed similar rumen in the tropical areas. The study also demonstrates that
DM intake and digestibility in goat supplemented with the tunnel method is a useful method to measure methane
Leucaena. However, Longo et al. (2008) found a significant production from animals fed with different diets.
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 Acknowledgments The Instituto de Ciencia Animal is grateful to the
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agricul-
lignin present in Leucaena. ture for the financial support provided through Coordinated Research
When foliages of trees and shrubs are used in a diet, the Projects no. 12667.
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
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