Trop Anim Health Prod
DOI 10.1007/s11250-016-1142-2
REGULAR ARTICLES
Supplementation of banana flower powder pellet and plant oil
sources on in vitro ruminal fermentation, digestibility,
and methane production
Sungchhang Kang 1,2 & Metha Wanapat 2 & Bounnaxay Viennasay 2
Received: 29 April 2016 / Accepted: 6 September 2016
# Springer Science+Business Media Dordrecht 2016
Abstract The objective of this study was to evaluate the ef-
fects of banana flower power pellet (BAFLOP-pellet) and
plant oil source on in vitro gas production, fermentation effi-
ciency, and methane (CH4) production. Rumen fluid was col-
lected from two rumen-fistulated dairy steers fed on rice
straw-based diet with concentrate supplement to maintain nor-
mal rumen ecology. All supplemented feed were added to
respective treatments in the 30:70 roughage to concentrate-
based substrate. The treatments were arranged according to a
3 × 3 factorial arrangement in a completely randomized de-
sign. First factor was different levels of BAFLOP-pellet sup-
plementation (0, 30, and 60 g/kg of dietary substrate) and
second factor was plant oil source supplementation [non-sup-
plemented, 20 g/kg krabok seed oil (KSO), and 20 g/kg coco-
nut oil (CO) of dietary substrate, respectively]. Under this
investigation, BAFLOP-pellet supplementation increased gas
production kinetics and in vitro digestibility (P < 0.05).
Ruminal pH was dropped post incubation time in the non-
supplemented group but was enhanced in BAFLOP-pellet-
supplemented treatments. On the other hand, supplementation
of KSO and CO depressed gas production and digestibility,
but did not influence ruminal pH. In addition, protozoal pop-
ulation and CH4 production were decreased by BAFLOP-
pellet and plant oil addition (P < 0.05). Based on this study,
it could be concluded that supplementation of BAFLOP-pellet
* Metha Wanapat
metha@kku.ac.th
1
Agricultural Unit, Department of Education, National Institute of
Education, Phnom Penh, Cambodia
2
Tropical Feed Resources Research and Development Center
(TROFREC), Department of Animal Science, Faculty of Agriculture,
Khon Kaen University, Khon Kaen 40002, Thailand
and plant oil source could enhance the in vitro fermentation
efficiency while reduced protozoal population and CH4 pro-
duction. It is suggested that BAFLOP-pellet (60 g/kg of die-
tary substrate) and KSO/CO (20 g/kg of dietary substrate)
could be used to manipulate rumen fermentation characteris-
tics fed on high-concentrate diet.
Keywords Banana flower . Plant oil . Rumen ecology .
Methane . Ruminant
Introduction
Medium chain fatty acid (MCFA; C8–C16) supplementation
in ruminant diets could improve rumen fermentation and re-
duce methane (CH4) production as it provides an alternative
hydrogen sink and inhabit rumen protozoa population.
Coconut oil (CO) and krabok seed oil (KSO) are rich in
MCFA namely lauric (C12:0; 458 and 420 g/kg, respectively)
and myristic acids (C14:0; 205 and 464 g/kg, respectively)
(Panyakaew et al. 2013). Supplementation of CO and KSO
could decrease CH4 and improve fermentation efficiency
(Yuangklang et al. 2010; Kang et al. 2016). In addition, feed-
ing tropical foliages or legumes containing saponins and tan-
nins reduced ruminal protozoal population and CH4 produc-
tion (Grainger et al. 2009; Lüscher et al. 2014; Calabrò et al.
2011; Guglielmelli et al. 2011). Kang and Wanapat (2013) and
Kang et al. (2014) reported that banana flower contains con-
densed tannins (CT) at 11.0 %, and supplementation of ba-
nana flower powder (BAFLOP) decreases CH4 production
and improves fermentation efficiency. Moreover, BAFLOP
has been investigated to use as a rumen buffering agent which
was promising in replacing NaHCO3 due to its high mineral
elements (Kang and Wanapat 2013; Kang et al. 2014, 2015).
Currently, most researchers are interested in making local feed
 Trop Anim Health Prod

resources and agricultural crop residues in the form of pellet Table 1 Feed ingredients and chemical compositions of experimental
diets
feed and used as high protein sources in ruminant feeding
(Wanapat et al. 2013). Furthermore, pellet feed could improve Items Concentrate BAFLOP- BAFLOP Rice
acceptability, density, and quality of feedstuffs. Therefore, this pellet straw
experiment was to determine the effect of banana flower pow-
Ingredients, g/kg
der pellet (BAFLOP-pellet) and plant oil source on gas
Cassava chip 600 –
production, fermentation efficiency, digestibility, and CH4
BAFLOP – 875
production using in vitro gas techniques.
Cassava starch – 5
Rice bran 120 –
Coconut meal 120 –
Materials and methods Palm meal 100 –
Urea 25 50
Dietary substrate, treatments, and experimental design Molasses 20 50
Mineral mixture 5 –
Fresh banana flowers were collected in Khon Kaen province, Salt 5 10
Thailand and were chopped and sun-dried for 3 or 4 days until Sulfur 5 10
air-dried. BAFLOP-pellet was formulated according to the Chemical composition, g/kg
respective ingredients shown in Table 1. All pellet ingredients Dry matter 864 865 883 934
were ground to pass a 1-mm screen using the Cyclotech Mill Organic matter 909 786 824 874
(Tecator, Höganäs, Sweden) and mixed well with water at Crude protein 159 258 141 23
ratio of 0.5:1 (water: pellet meal). Neutral detergent fiber 208 617 664 741
All treatments were arranged according to a 3 × 3 factorial
Acid detergent fiber 122 459 449 585
arrangement in a completely randomized design (CRD).
Ash 91 214 176 126
Factor A was different levels of BAFLOP-pellet supplemen-
Condensed tannins 96 116
tation (0, 30, and 60 g/kg of dietary substrate), while factor B
Macro minerals, g/kg
was plant oil source supplementation (non-supplement,
Nitrogen 19.8 22.6
20 g/kg KSO, and 20 g/kg CO of dietary substrate).
Phosphorus 3.2 3.7
Roughage to concentrate ratio (R:C) at 30:70 was used as a
Potassium 70.3 80.3
dietary substrate.
Sodium 0.4 0.5
Dietary samples were dried at 60 °C, ground to pass a 1-
Calcium 0.3 0.3
mm sieve (Cyclotech Mill, Tecator, Sweden), and kept for
Magnesium 4.0 4.6
chemical composition analysis and in vitro gas study. All
Sulfur 1.5 1.7
samples were analyzed for dry matter (DM), ash, crude pro-
Micro minerals, ppm
tein, acid detergent fiber (AOAC 2012), and neutral detergent
Iron 490 560
fiber (NDF; Van Soest et al. 1991). Mineral elements of
BAFLOP and BAFLOP-pellet were analyzed using wet di- Manganese 180 210
gestion (nitric-perchloric digestion), spectrophotometry (total Copper 20 20
P), atomic absorption spectrophotometry (total Ca, Mg, Na, Zinc 50 60
K, Fe, Mn, Zn, Cu), and tubidity (total S) (model: analytic jena BAFLOP banana flower powder
nova 350). Condensed tannin was analyzed by means of
vanillin-HCl method as modified by Wanapat and
warmed in a water bath at 39 °C for 1 h before filling with
Poungchompu (2001).
30 ml of the rumen inocula mixture.

Rumen and substrate inocula Medium solution preparation

Two rumen-fistulated dairy steers (180 ± 15 kg BW) were
used as source of rumen inocula, and 1000 ml of rumen fluid
was collected before morning feeding. The method in this
study was based on the technique described by Menke et al.
(1979). Two hundred milligrams of dietary substrate (R:C)
were weighed into 60-ml glass bottles which were then sealed
with rubber stoppers and aluminum caps. All bottles were pre-
Medium solution of 3000 ml was prepared for 88 bottles per
each run (Makkar et al. 1995). Reducing medium (2000 ml)
consists of 950 ml distilled water, 480 ml rumen buffer solu-
tion (35.0 g NaHCO3 and 4 g NH4HCO3 made up to 1 l with
distilled water), 480 ml macro-mineral solution (6.2 g
KH2PO4, 5.7 g Na2HPO4, 2.22 g NaCl, and 0.6 g MgSO4.
7H2O made up to 1 l with distilled water), 0.24 ml micro-
Trop Anim Health Prod

mineral solution (10.0 g MnCl2. 4H2O, 13.2 g CaC12. 2H2O,
1 g CoCl2 .6H2O, 8.0 g FeC13. 6H2O and made up to 100 ml
with distilled water), 2.44 ml resazurine (0.1 g made up to
100 ml with distilled water), and 99 ml freshly prepared re-
duction solution (95 ml distilled water, 672 mg Na2S. 9H2O,
and 4 ml 1 M NaOH). Rumen fluid was mixed with the re-
ducing medium at ratio of 2:1 (reducing medium: rumen flu-
id). The mixture was kept stirred under CO2 pumping at 39 °C
using a magnetic stirrer fitted with a hot plate. The portion of
30 ml medium solution was transferred into each bottle and
incubated in the water bath at 39 °C.
Samples collection and analysis
Gas production kinetics: During the incubation, gas pro-
duction was recorded at 1, 2, 4, 6, 8, 12, 24, 48, 72, and
96 h by extraction using glass syringes. Cumulative gas
production data were fitted to the model of Orskov and
McDonal (1979): y = a + b (1-e(−ct)); where a = gas pro-
duction from immediately soluble fraction, b = gas pro-
duction from the insoluble fraction, c = gas production
rate constant for the insoluble fraction (b), t = incubation
time, (a + b) = the potential extent of gas production. y =
gas produced at the time Bt.^
Fermentation parameters: The pH was measured at 6, 12,
24, and 96 h incubation time using a portable pH temperature
meter (HANNA Instruments HI 8424 microcomputer,
Singapore). At 24 h post inoculations, the portion of 1 ml fluid
was collected and kept in a plastic bottle to which 9 ml of 10 %
formalin solution (1:9 v/v, rumen fluid: 10 % formalin) were
added and stored at 4 °C for the direct count of bacteria,
protozoa, and fungal zoospores (Galyen 1989) using a
haemacytometer (Boeco, Hamburg, Germany).
In vitro digestibility: At 12 and 24 h post inoculation,
in vitro digestibility was measured based on the following
equation according to Van Soest and Robertson (1985): True
digestibility (TD) = ((DM of feed taken for incubation-NDF
residue) × 100)/DM of feed taken for incubation.
Methane production: Samples of gas (3 ml) were extracted
at 4, 8, and 12 h post incubation for analysis of CH4 concen-
tration using gas chromatography (Instruments by GC-17A
System, Shimadzu; TCD detector; column Shin carbon;

Table 2 Effect of BAFLOP-pellet and plant oil source supplement on gas production and in vitro digestibility

Trts BAFLOP-pelleta Oil sourcesb Gas production kineticsc Gasd In vitro digestibility (%)

a b c a+b True NDF

12 h 24 h 12 h 24 h

T1 0 Non 1.31 85.0 0.06 86.3 86.0 35.4 67.2 10.8 37.4
T2 30 Non −3.17 93.3 0.07 90.1 90.0 42.0 72.2 22.3 51.1
T3 60 Non −1.14 99.1 0.07 97.9 97.8 49.1 81.5 27.7 57.3
T4 0 KSO 0.25 65.1 0.08 65.3 65.3 25.1 46.2 10.1 30.4
T5 30 KSO 0.28 69.9 0.08 70.1 70.1 29.1 57.1 23.2 40.4
T6 60 KSO −3.34 86.9 0.08 83.5 83.5 32.5 63.3 27.7 46.8
T7 0 CO −1.65 58.1 0.09 56.5 56.5 22.6 45.0 8.9 18.3
T8 30 CO −1.50 63.6 0.10 62.1 62.1 31.2 51.1 20.4 31.6
T9 60 CO −0.56 69.2 0.09 68.7 68.6 32.1 55.6 22.6 39.4
SEM 1.05 3.59 0.004 3.54 3.53 2.82 2.13 2.62 1.41
Interactions
BAFLOP-pellet 0.2194 0.0008 0.4719 0.0018 0.0018 0.0348 0.0010 0.0012 0.0001
Oil sources 0.9508 0.0001 0.0001 0.0001 0.0001 0.0036 0.0001 0.5403 0.0001
BAFLOP-pellet*oil sources 0.0798 0.5745 0.4101 0.8898 0.8919 0.8906 0.7417 0.9808 0.7629
Comparisons
Non vs KSO, CO 0.9176 0.0001 0.0001 0.0001 0.0001 0.0013 0.0001 0.5986 0.0001
KSO vs CO 0.7665 0.0046 0.0241 0.0049 0.0049 0.9438 0.0852 0.3488 0.0006
a
BAFLOP-pellet: banana flower powder pellet supplement at 0, 30, and 60 g/kg of total substrate
b
Oil sources: non-supplement (non), krabok seed oil (KSO; 20 g/kg of total substrate), and coconut oil (CO; 20 g/kg of total substrate)
c
a, the gas production from the immediately soluble fraction; b, the gas production from the insoluble fraction; c, the gas production rate constant for the
insoluble fraction; a + b, the gas potential extent of gas production
d
Cumulative gas production at 96 h (ml/0.2 g DM substrate)
NDF neutral detergent fiber
 Trop Anim Health Prod

column size 3 m × 3 mm, activated charcoal 60/80 mesh) Table 3 Effect of BAFLOP-pellet and plant oil sources supplement on
ruminal pH
adapted from Sittijunda et al. (2010).
Trts BAFLOP- Oil Ruminal pH
Statistical analysis pelleta sourcesb
6h 12 h 24 h 96 h Mean
All the obtained data were subjected to General Linear Model T1 0 Non 6.00 5.94 5.93 5.94 5.95
(GLM) procedures of SAS (2013) according to a 3 × 3 facto-
T2 30 Non 6.29 6.18 6.21 6.18 6.21
rial arrangement in a completely randomized design.
T3 60 Non 6.44 6.34 6.42 6.34 6.38
Differences among treatment means were contrasted by the
T4 0 KSO 5.96 5.91 5.94 6.01 5.95
Tukey’s multiple comparison test (Crichton 1999).
T5 30 KSO 6.30 6.29 6.26 6.20 6.26
Comparison between plant oil source supplementation was
T6 60 KSO 6.47 6.38 6.44 6.38 6.42
tested by orthogonal contrast.
T7 0 CO 6.00 5.90 5.95 5.94 5.95
T8 30 CO 6.26 6.24 6.28 6.24 6.26
T9 60 CO 6.56 6.50 6.55 6.51 6.53
Results and discussion
SEM 0.06 0.07 0.07 0.07 0.06
Interactions
Gas production from the immediately soluble fraction (a) was
BAFLOP-pellet 0.0001 0.0013 0.0003 0.0011 0.0002
not affected by the dietary supplementation while gas produc-
Oil sources 0.8746 0.7807 0.6267 0.6179 0.6821
tion from the insoluble fraction (b), gas potential extent of gas
BAFLOP-pellet* oil 0.8440 0.8806 0.9740 0.8298 0.9110
production (a+b), and cumulative gas production at 96 h were
sources
increased by BAFLOP-pellet supplementation (Table 2; Comparisons
P < 0.05). However, supplementation of plant oil sources re- Non vs KSO, CO 0.8050 0.5018 0.4511 0.4013 0.4569
duced gas production kinetics (P < 0.05) and this could be due KSO vs CO 0.6702 0.7902 0.5649 0.6891 0.6638
to the most probably negative effect of CO and KSO had on
a
the extent of NDF digestion by coating cell skin of microbes BAFLOP-pellet: banana flower powder pellet supplement at 0, 30, and
(Kongmun et al. 2010). In addition, supplementation of KSO 60 g/kg of total substrate
b
reduced fiber digestion, but increased propionate in goat meat Oil sources: non-supplement (non), krabok seed oil (KSO; 20 g/kg of
total substrate), and coconut oil (CO; 20 g/kg of total substrate)
(Yuangklang et al. 2010). The increasing of gas production
kinetics in treatments with BAFLOP-pellet supplementation
could be due to the enhancement of ruminal pH in these Plant oil supplementation did not affect ruminal pH
groups, and this was also supported in the study of Kang (P > 0.05). The decline of ruminal pH from 6.8 to 5.5 or lower
and Wanapat (2013). On the other hand, plant oil source sup- would occur when readily fermentable carbohydrates are sud-
plementation decreased True and NDF digestibility while denly fed to the ruminants without prior adaptation (Dijkstra
BAFLOP-pellet supplementation could enhance the in vitro et al. 2012). Therefore, the dietary buffering agent would pre-
digestibility. It could be explained that mineral elements in vent the depression of rumen pH associated with high concen-
BAFLOP could regulate ruminal pH and increase microbe trate feeding. In the present study, supplementation of
activity (Kang and Wanapat 2013; Kang et al. 2014, 2015). BAFLOP-pellet could enhance the pH which was also found
In addition, CT in BAFLOP-pellet did not affect gas produc- in the study of Kang and Wanapat (2013). In addition, Kang
tion and digestibility in the present study. Frutos et al. (2004); et al. (2014) reported that ruminal pH was decreased when
Calabrò et al. (2011), and Guglielmelli et al. (2011) reported cattle were fed diet of R:C at 30:70 and BAFLOP supplemen-
that if CT >50 g/kg DM in the feed, there will be a reduction of tation at 20 and 30 g/kg DMI could maintain the pH above
feed intake and nutrient digestibility. If CT level was <50 g/kg 6.0. Moreover, Kang et al. (2015) stated that supplementation
DM, it would help to protect protein from rumen digestion, of BAFLOP had similar buffering capacity to NaHCO3 in
thereby increasing bypass protein or rumen undegradable pro- lactation dairy cows. Supplementation of KSO and CO did
tein. This suggested that selective suppression of cellulolytic not affect ruminal pH in this study (P > 0.05) which was in
bacteria by saponins and tannins did not occur in the present consistency to the finding of Kongmun et al. (2010).
study. The population of protozoa was reduced by BAFLOP-
pellet and plant oil supplementation, while bacterial popula-
Ruminal pH, microorganisms, methane production tion was increased by BAFLOP-pellet (Table 4). Medium-
chain saturated fatty acid, specifically lauric, is known toxic
In table 3, the pH was dropped below 6.0 in non- to ruminal protozoa (Hristov et al. 2004). Furthermore, the
supplemented groups. However, supplementation of reduction of protozoal population could be due to the presence
BAFLOP-pellet could buffer the pH in the present study. of CT in BAFLOP-pellet. The sensitivity of microbes toward
Trop Anim Health Prod

Table 4 Effect of BAFLOP-pellet and plant oil source supplement on microorganism growth and methane production

Trts BAFLOP-pelleta Oil sourcesb Microorganisms (cell/ml; 24 h post incubation) Methane production (ml/L)

Protozoa (×105) Fungi (×106) Bacteria (×109) 4h 8h 12 h Mean

T1 0 Non 15.0 3.0 31.0 60.6 88.5 94.7 81.3

T2 30 Non 12.0 3.5 41.8 55.2 73.7 88.8 72.6
T3 60 Non 10.0 3.5 30.8 47.2 67.7 82.9 65.9
T4 0 KSO 11.5 5.5 19.3 48.7 67.1 88.2 68.0
T5 30 KSO 8.5 2.5 21.8 44.6 67.8 72.8 61.7
T6 60 KSO 5.5 4.0 26.8 39.2 57.4 62.8 53.1
T7 0 CO 10.5 5.0 22.5 67.8 77.1 84.0 76.3
T8 30 CO 9.8 4.3 22.3 46.6 58.8 67.4 57.6
T9 60 CO 6.3 2.3 28.3 43.0 59.4 63.5 55.3
SEM 1.41 0.50 1.43 3.35 3.66 3.78 2.08
Interactions
BAFLOP-pellet 0.0016 0.0995 0.0548 0.0079 0.0121 0.0052 0.0002
Oil sources 0.0051 0.4838 0.0002 0.0603 0.0283 0.0075 0.0014
BAFLOP-pellet* oil sources 0.8201 0.0659 0.0203 0.3805 0.4414 0.7132 0.3545
Comparisons
Non vs KSO, CO 0.0023 0.2777 0.0001 0.1280 0.0112 0.0030 0.0005
KSO vs CO 0.7381 0.7805 0.3216 0.0789 0.8174 0.5122 0.3937
a
BAFLOP-pellet: banana flower powder pellet supplement at 0, 30, and 60 g/kg of total substrate
b
Oil sources: non-supplement (non), krabok seed oil (KSO; 20 g/kg of total substrate), and coconut oil (CO; 20 g/kg of total substrate)

plant secondary compounds may be explained by the presence
of sterols in cell membranes, but not bacterial membranes
(Goel and Makkar 2012). Supplementation of BAFLOP-
pellet and plant oil reduce CH4 production in the present study
(Table 4; P > 0.05). Dohme et al. (2001) and Panyakaew et al.
(2013) found that supplementation of CO and KSO could
reduce CH 4 production due to its highly inhibitory to
methanogenesis. Lauric and myristic fatty acids in plant oil
could be primarily accountable in reducing methanogenesis in
the present study. It was reported that fiber digestion was
reduced by dietary fats, and it diminishes the availability of
hydrogen for methanogenesis, lowers protozoa-associated
methanogenesis, and directly inhibits the methanogenic activ-
ity. In addition, CT in BAFLOP-pellet reduced CH4 produc-
tion as it shifted hydrogen from CH4 pathway to produce
propionate (Carulla et al. 2005). Tannins at low level in feed
have potential to modulate the rumen fermentation toward
maximizing microbial protein synthesis (Zhou et al. 2011).
A reduction rate of the feed digestion by tannins could help
synchronize a release of various nutrients, which is responsi-
ble for microbial efficiency enhancement.
Conclusions and recommendations
Based on this experiment, it could be concluded that supple-
mentation of BAFLOP-pellet at 60 g/kg and plant oil source at
20 g/kg of total dietary substrate could improve the in vitro
fermentation and remarkably reduce rumen CH4 production.
Acknowledgments The most sincere thanks are extended to the
Tropical Feed Resources Research and Development Centre
(TROFREC), Khon Kaen University (KKU), and Thailand Research
Fund (TRF) through the International Research Network (IRN) program
for their kind support on research fund and facility.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Ethical guideline All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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