Effect of Albizia myriophylla bark and
its secondary metabolites on rumen
fermentation parameters and
microbial population
Research Project Department of Farm Animal Health
Faculty of Veterinary Medicine, University Utrecht
Iris Daamen (3894045)
Supervised by:
Dr. Goh Yong-Meng
Dr. J.T. (Thomas) Schonewille
Table of contents:

Abstract

Introduction

Aim of the study

Rumen physiology

Albizia myriophylla

Plant secondary metabolites

Materials and Methods

Statistical Analysis

Results and Discussion

Conclusion

References
Abstract
The objective of this study was to evaluate the in vitro effects of Albizia myriophylla bark and its
secondary metabolites on rumen fermentation parameters and microbial population, in particular its
effectiveness for mitigating ruminal methanogenesis and its potential as a feed additive to enhance
performance of ruminants. Control (50% concentrate + 50% Alfalfa), treatment 1 (10% Albizia
replaced Alfalfa) and treatment 2 (20% Albizia replacement) were added to the rumen fluid-buffer
mixture for 24h incubation. Total gas production, volatile fatty acids, pH, NH3-N, methane gas
production and ruminal microorganisms were measured. Estimates of rumen microbial population
using real-time PCR assay showed that supplementing different levels of Albizia did not affect the
relative abundance of total bacteria, Fibrobacter succinogenes, Butyrivibrio fibrisolvens,
Ruminococcus flavefaciens, Methanobacteriales, total protozoa and fungi but had a decreasing effect
on Ruminococcus albus. There were no significant changes (p > 0.05) on any of the measured
fermentation parameters (pH, NH3-N, VFA). Cumulative gas production and methane production at
different inclusion levels of Albizia myriophylla were not affected by the experimental treatments. It
was concluded that Albizia can probably be included in the diet of goats up to 20% without any
adverse effects on ruminal fermentation, however its potential as alternative performance enhancer
and methane mitigation agent was not confirmed.
Introduction
Ruminants are dependent on microbial fermentative digestion to obtain the necessary nutrients
from the ingested feed materials.1 Since the rumen is the main fermentation site, it plays a key role
in ruminant nutrition and the overall health of the animal. It is therefore an obvious target when one
seeks to improve feed digestion in ruminants.
The rumen contains a dense and diverse population of bacteria, protozoa and fungi.2 These
microorganisms ferment the feed material which leads to the production of volatile fatty acids
(VFA’s), the major energy source for the animal, and other products such as methane, carbon dioxide
and heat (which are considered energy losses).2 Methane also has a negative impact on the
environment due to its greenhouse effect, making the reduction of methane production one of the
most important aims.3 Feed transformation is performed by microbes and inevitably results in the
production of methane.1 Therefore nutritionists and rumen microbiologists have long been seeking
to manipulate the rumen microbial ecosystem to increase the efficiency of rumen metabolism and
ultimately the productivity of animals.4 Several chemical feed additives such as antibiotics,
ionophores, methane inhibitors and defaunating agents have been introduced into ruminant
nutrition in order to improve growth, enhance feed utilization and decrease methane production.4
However, increasing awareness of hazards associated with chemical feed additives, such as the
presence of chemical residues in animal products and the development of bacterial resistance to
antibiotics, have resulted in the search for safer natural alternatives.4 Plants or plant extracts
containing essential oils, tannins, saponins, flavonoids and many other plant secondary metabolites
(PSM) have been shown to improve rumen metabolism by decreasing methanogenesis and protein
degradation in the rumen, increasing microbial protein production, protein flow to the duodenum
and targeting specific groups of rumen microbes.4
Aim of the study
The aim of this study is to investigate the effects of Albizia on fermentation parameters (pH, volatile
fatty acids and NH3-N) and microbial population. After analyzing these factors, the ultimate goal of
this study is to understand the value of Albizia as a diet supplement that improves the efficiency of
rumen metabolism, and which can be used as an alternative performance enhancer and as a
potential methanogenesis suppressor.
Figure 1. Experimental lab work at the Faculty of Veterinary Medicine, Universiti Putra
Malaysia.

Rumen physiology
The major end products of the fermentative digestion in ruminants are volatile fatty acids (VFAs).2
The primary VFAs are acetic acid, propionic acid and butyric acid.2 There is a direct relationship
between acetic acid production and methane production.2 Likewise a reciprocal relationship exists
between methane production and propionic acid production.2 The more propionic acid is produced,
less need there is for methane synthesis.2
In the rumen, methane production is facilitated by methanogenic bacteria, such as
Methanobasterium ruminantium.2 When conditions are unfavorable for the survival of these
bacteria, methane production is reduced, shifting the metabolic pathways toward propionic acid
production.2 Some conditions that suppress methanogenic species are high levels of feed intake, use
of finely ground or pelleted feeds, and high-grain or high-starch diets.2 Under these circumstances
the rate of methane production is reduced , resulting in a lower rate of acetic acid production with a
concomitant increase in the propionic acid production rate.2
In the rumen, the ingested feed containing plant structural carbohydrates and proteins among other
organic material is degraded by anaerobic fermenters.5 This product is then further converted into
volatile fatty acids, CO2 and H2 by the ruminal microbes.5 Methanogens use the end products of
fermentation as substrates.5 The synthesis of methane contributes to the efficiency of the system by
keeping the H2 levels stable and therefore allowing the microbial enzymes to function normally.5
Fibrolytic microorganisms play a key role at the first level of feed transformation into volatile fatty
acids, CO2 and H2.5 Most fibrolytic microbes produce H2 as a main end product of fermentation,
which under normal conditions, is then rapidly used by methanogens.5 If this pathway is disturbed,
fermentation can be inhibited, resulting in less feed digestion.5 This is the reason why
methanogenesis is so closely linked to plant fibre degradation in the rumen.5 Anaerobic rumen
fibrolytic bacteria, protozoa and fungi degrade fibrous material, allowing ruminants to utilize plant
fiber for nutrition.6 Bacteria are the most numerous of these microorganisms and play a major role in
the biological degradation of dietary fiber.6 Fibrobacter succinogenes, Ruminococcus albus and
Ruminococcus flavefaciens are presently recognized as the major cellulolytic bacterial species found
in the rumen.6
 Albizia myriophylla
Albizia
belongs
to
the
subfamily
Mimosoideae
and
genus
Albizia.7,8
This genus consists of about 145 species, distributed in tropical and subtropical regions in the world
which includes Thailand, Malaysia, Vietnam and other countries.7,8 It is used in traditional
medicine as mouth wash in Thailand.7 The plant portion of Albizia myriophylla including roots,
fruits, and wood are used for several therapeutic purposes such as antitussive, expectorant, and
tonic.8 Previous biological activity study revealed that the ethanolic wood extract of this species
exhibited pronounced antibacterial activity against Streptococcus mutans.8 Furthermore, biological
screenings worldwide showed that many plants of the Albizia possess anticancer, antimalarial,
immunomodulatory, antioxidant, antimicrobial, anthelmintic and anti-inflammatory properties.8
As reported by Kim et al.9 various approaches, such as selection of rumen micro-organisms through
the elimination of protozoa, the inoculation of exogenous bacterial strains and vaccination against
methanogenic micro-organisms, have been studied with the aim of reducing methane emission of
digestive origin.9 Plant extracts as new feed additives have led to the interest in new, safe and
inexpensive ways to reduce methane emission from ruminants.9 Many plant extracts included tannin
and saponin have been studied as possible modifiers of rumen fermentation in order to decrease
methanogenesis.9
An overview of the different mitigation options and strategies that have been explored so far is
presented in Figure 2 (adapted from Patra, 2016)10, which involve intervention at the animal level,
dietary composition of animals, modulation of rumen fermentation, and inhibition of methanogenic
archaea.10 This review reveals the potential of methanogen-specific inhibitors, such as plant
metabolites, as effective mitigation agents.10
Figure 2: A schematic presentation of the potential targets of decreasing CH4 emissions from ruminants. Boxes without dark could be
the targets for suppressing CH4 emissions and boxes with dark shade are the options that have been studied in vitro or in vivo to
10
decrease CH4 production.
(Source: Recent Advances in Measurement and Dietary Mitigation of Enteric Methane Emissions in Ruminants; Amlan K. Patra, 2016)
 Plant secondary metabolites
The term plant secondary metabolite is used to describe a group of chemicals present in plants that
are not involved in the primary biochemical processes of plant growth and reproduction.11 Several
thousand different plant secondary metabolites have been reported.11 Secondary metabolites in
plants have been suggested to have different roles, with the most prominent being the protection of
plants from insect predation.11,12 This specificity of plant secondary metabolites against microbial
groups can be used for selective inhibition of some undesirable microbes in the rumen.12 The
methanogens classified as Archaea have a distinctly different cell wall structure from true rumen
bacteria.12 Thus, there exists a possibility that some of the plant secondary metabolites might act as a
selective inhibitor of methanogens and can be used as a feed additive for the manipulation of rumen
fermentation to achieve inhibition in methane emission by the livestock.12
Materials and methods
The experiment was conducted at the research farm of the Faculty of Veterinary Medicine, Universiti
Putra Malaysia, Serdang, Selangor. Rumen fluid was collected from two rumen fistulated Kacang
crossbred male goats that had received roughage and concentrate twice daily at 8:00 and 17:00h.
Rumen fluid was collected before feeding the animals in the morning. This procedure was repeated
twice, on two separate days. Water and mineral-vitamin block were allowed ad libitum.
The control diet consisted of 125mg of concentrate and 125mg of Alfafa, thus 50% each. Treatment 1
consisted of 50% concentrate, 40% Alfafa and 10% Albizia. Treatment 2 was similar except 20% of
the Alfafa was replaced with Albizia. Chemical composition of the feed is shown in Table 1.
Table 1. Nutrient composition (% as fed) of the experimental diets
% Nutrient
1
Basal substrate
Substrate
10% Albizia
20% Albizia
Ash
8.1
9.3
1.9
Dry matter
95.9
94.3
90.1
Crude Protein
16.5
27.1
5.2
Crude Lipid
3.2
1.3
nd*
Crude Fiber
11.2
18.7
64.2
Basal substrate: 50% Concentrate + 50% Alfalfa
A1: 10% replacement of Alfafa by Albizia
3
A2: 20% replacement by Albizia
*not detected
2
Before onset of the experiment, Albizia myriophylla bark samples were oven dried and ground to
pass a 1 mm sieve. Then, they were preserved in tightly closed plastic bags and stored in – 20oC until
further analysis. These samples were later analyzed for the contents of ash, moisture, protein, lipid
and fiber using the standard methods of AOAC (2000).13,14
Rumen samples were collected in a container under anaerobic conditions, carried to the laboratory
and filtered through four layers of cheesecloth. After this, the rumen fluid, together with the
phosphate(Na2HPO4.12H2O + NaH2PO4.H2O + NH4Cl) and bicarbonate (NaHCO3) buffers and the
corresponding diets were added to the syringes, 300ml of each liquid in a ratio of 1:1:1. Syringes
were incubated at 39oC for 24h. Volumes of the gas produced were determined after 0, 2, 4, 6, 8, 10,
12 and 24h of incubation. Net gas production values were corrected by subtracting blank values from
the samples. After 24h incubation, methane production was measured by injecting 1mL of the
headspace gas from each of the syringes into a gas chromatograph according to Ebrahimi, (2012).15
One milliliter of syringe content was immediately collected and frozen (-20oC) for DNA extraction and
microbial population quantification.
The pH of the contents of the syringes were determined at different times of incubation using a pH
electrode (Mettler-Toledo Ltd., England). The samples were acidified with 25% metaphosphoric acid
in water and centrifuged (10 min, 4oC at 15,000xg), followed by filtering. The filtrate was used to
determine volatile fatty acids (VFA) and ammonium nitrogen (NH3-N). Volatile fatty acids were
determined using gas-liquid chromatography according to Ebrahimi (2012) with Quadrex 007 Series
(Quadrex Corporation, New Haven, CT 06525 USA) bonded phase fused silica capillary column
(15m, 0.32mm ID, 0.25 µm film thickness) in an Agilent 7890A gas-liquid chromatography
(Agilent Technologies, Palo Alto, CA, USA) equipping with a flame ionization detector (FID). 15 The
injector/detector temperature was programmed at 220/230oC respectively. The column
temperature was set in the range of 70oC - 150oC with temperature programming at the rate of
7oC/ min increment to facilitate optimal separation. Peaks were identified by comparison with
authentic standards of acetic, propionic, butyric, isobutyric, valeric, isovaleric and 4-methyl-n-valeric
acids.16 NH3-N concentrations were determined using the colorimetric method described by
Solorzano (1969) and according to Ebrahimi (2012).15
For the rumen microbial populations the DNA was extracted from the rumen fluid sample using the
QIAamp DNA Stool Mini Kit (Qiagen Inc., Valencia, CA) according to the protocol of the manufacturer.
DNA samples were used as templates for real-time polymerase chain reaction (PCR) assay. The
relative abundance of each of the analyzed microbes in extracted samples from the goat was
measured by real-time PCR and the SYBR Green PCR Master Mix Kit (Bio-Rad Laboratories, Hercules,
CA). Real-time PCR was performed with the Bio-Rad CFX96 Touch (Bio-Rad Laboratories, Hercules,
CA) using optical grade plates. PCR primers used for amplifying target bacteria in the rumen are
shown in Table 2.
Tabel 2. Polymerase chain reaction primers used for amplifying target bacteria in the rumen
Microorganism
Sequence 5’ – 3’
Fibrobacter succinogenes F1
GTTCGGAATTACTGGGCGTAAA
Fibrobacter succinogenes R2
CGCCTGCCCCTGAACTATC
Butyrivibrio fibrisolvens F1
TAACATGAGTTTGATCCTGGCTC
Butyrivibrio fibrisolvensR
2
Ruminococcus albus F1
Ruminococcus albus R
2
CGTTACTCACCCGTCCGC
CCCTAA AAGCAGTCTTAGTTCG
CCTCCTTGCGGTTAGAACA
Ruminococcus flavefaciens F1
TCTGGAAACGGATGGTA
Ruminococcus flavefaciens R2
CCTTTAAGACAGGAGTTTACAA
Methanobacteriales F1
CGW AGGGAAGCTGTTAAGT
Methanobacteriales R2
TACCGTCGTCCACTCCTT
Total bacteria F
1
CGGCAACGAGCGCAACCC
Total bacteria R2
CCATTGTAGCACGTGTGTAGCC
Total protozoa F1
CTTGCCCTCYAATCGTWCT
Total protozoa R2
GCTTTCGWTGGTAGTGTATT
Statistical Analysis
All the presented data were analyzed using the general linear model (GLM) procedure of the
Statistical Analysis System Institute, Inc. (SAS, 2002). Differences among means were tested for
significance using Duncan’s multiple range test of SAS (2002). The microbial data were normalized
using repeated the log10-transformation prior to analysis. Values of p < 0.05 were considered
significant.
Results and Discussion
Table 3 illustrates the effect of Albizia on ruminal fermentation parameters after 24h in vitro
incubation. Data were presented as means ± standard error (except the acetic:propionic ratio). The
pH values range from a minimum of 7.17 up to 7.23, which in vivo would be considered slightly on
the higher side (optimum pH ranges from 6.5 -7).2 Nevertheless there was no significant difference in
terms of pH between the diets (p > 0.05).
Table 3. Effect of Albizia on ruminal fermentation parameters after 24h of in vitro incubation
Parameters
Basal
Substrate
A12
A2
pH
7.2 ± 0.0
7.2 ± 0.0
7.2 ± 0.0
0.6
NH3-N (mg/dl)
13.0 ± 0.5
13.5 ± 0.5
13.1 ± 0.5
0.8
Total VFA (mM)
40.4 ± 1.4
39.2 ± 1.5
38.1 ± 1.4
0.5
Acetic acid (%)
42.9 ± 0.5
41.1 ± 0.6
41.4 ± 0.5
0.08
Proprionic acid(%)
24.2 ± 0.4
24.2 ±0.5
24.3 ± 0.4
0.9
Butyric acid (%)
19.7 ± 0.1
20.4 ± 0.1
20.3 ± 0.1
0.0
1.8
1.7
1.7
1
Acetic:Propionic ratio
Pr > F
3
1
Basal: 50% Concentrate + 50% Alfalfa
A1: 10% replacement of Alfafa by Albizia
3
A2: 20% replacement by Albizia
2
In Figure 3 the effect of Albizia on total gas production over the course of 24h of incubation is
presented. Total gas production for control, 10% Albizia and 20% Albizia treatment is 63.2, 60.8 and
61.8 (ml/g DM), respectively. Regarding methane production the results are fairly similar in the
different groups, with the lowest values belonging to the 10% Albizia treatment and the highest to
the control group. Lack of significance in total gas production by increasing levels of Albizia means
this plant has no major negative effects on rumen fermentation characteristics. Remarkably, the gas
production curve (Figure 3) suggests that Albizia ferments to the same extent as Alfalfa and that they
have similar digestibility, however this seems unlikely and goes in disagreement with the results of
the chemical analysis (Table 1). Therefore this data should be treated with caution, when interpreting
results. For the remaining parameters we see similar results in the three groups, with little
fluctuation between the values (Table 3). There were also no significant changes (p > 0.05) on any of
the measured fermentation parameters (pH, NH3-N, VFA). The chemical composition of the feed
presented in Table 1 shows some odd results. For example, the increase in crude protein with the
addition of 10% Albizia followed by a steep decrease when adding an extra 10% Albizia is unfitting.
Also the very high fiber content of 20% Albizia substrate does not go in line with the control and 10%
Albizia composition. For practical reasons, it was not possible to redo the chemical composition
analysis, and therefore one should take caution when interpreting these results. We suggest that
adding Albizia to the diet did not reduce the methane production and none of the measured
parameters seem to be affected by the change in diet. Thus, so far we can conclude that this
concentration of Albizia has no apparent side effects on the microbial fermentation and feed
digestion, but also shows no benefits.
Figure 3. Effect of Albizia on total gas production over the course of 24h of incubation
Gas Production (ml/g DM)
70
60
50
40
Control
30
A1
20
A2
10
0
0
2
4
6
8
10
12
24
Time of incubation (h)
In an experiment by Wagner et al. 201417 on the effect of different acetic:proprionic (A:P) ratios on
the methanogenic community, significant differences on gas production, gas composition, and
volatile fatty acid (VFA) concentration, were observed between acetic:propionic ratios ≤1 and ≥2.17
Wagner et al. 2014 reported that generally ratios ≥2 resulted in a faster methane production
compared to ratios ≤1.17 In our current study the A:P ratio was around 1.7 for all the three groups
(Table 3), which goes in agreement with the results by Wagner et al. 2014, since there was no
significant decrease nor increase in the gas production or VFA concentration.
Table 4. Effect of Albizia on the microbial population after 24 h of in vitro incubation.
Log10 copy No/ml
Basal1
Substrate
A12
A23
Pr > F
Fibrobacter succinogenes
7.8
7.5
7.3
0.2
Butyrivibrio fibrisolvens
3.9
3.9
3.7
0.4
Ruminococcus albus
6.9
6.6
6.4
0.02
Ruminococcus flavefaciens
5.4
5.3
5.1
0.6
Methanobacteriales
4.8
4.8
4.6
0.6
Total bacteria
10.8
10.8
10.6
0.5
Total protozoa
5.2
5.1
4.9
0.5
Fungi
6.1
5.8
5.7
0.4
1
Basal: 50% Concentrate + 50% Alfalfa
A1: 10% replacement of Alfafa by Albizia
3
A2: 20% replacement by Albizia
2
Table 4 shows that the addition of Albizia to the diet had no effects (p > 0.05) on any of the
microorganisms, except R. albus (p = 0.02). Since none of the other microbial groups showed any
significant changes, it seems that the addition of Albizia in these concentrations does not have severe
adverse effects on feed digestion, although it could potentially cause some reduction in fiber
digestion (decrease in R. albus population). Nonetheless, our results show the highest fiber
composition with 20% Albizia substrate (as mentioned before, data that should be interpreted with
prudence).
Figure 4 shows a steady decrease in Ruminococcus albus population when increasing percentage of
Albizia is added to the diet of the goats. Studies have shown that Albizia has an antibacterial effect,
especially against gram positive bacteria, like R. albus.18 This would account for the decrease seen
after adding the two different treatments.
Conclusion
Our results suggest that Albizia can be added to a goat´s diet without adversely affecting ruminal
fermentation or having a negative effect on total gas production in vitro. Supplementing different
levels of this plant did not affect most ruminal microbes but had a decreasing effect on R. albus.
Unfortunately the obtained data does not provide a clue for some of the discrepancies we find in our
study, and therefore caution is wanted when generalizing current results.
The measured parameters in this study revealed no effect on methanogenesis or any reliable results
that prove the potential of Albizia as a methane mitigation agent for ruminants or as a useful diet
supplement.
Acknowledgements
I would like to thank the Faculty of Veterinary Medicine at the Universiti Putra Malaysia, where I was
allowed to participate in the research of Dr. Goh Yong-Meng. I am most grateful for the guidance and
help provided by Dr. Goh Yong-Meng and Dr. Mahdi Ebrahimi during my internship in Malaysia, who
made my stay abroad a wonderful and learning experience.
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