Combination
Effect of Clove and Orange Peel Oils on In vitro Digestion of Dairy Total Mixed
Ration Using ANKOM DAISYII Incubator
Muhamad N. Rofiq1
LAPTIAB 612 Bld.PUSPIPTEK, Centre for the Agriculture
Production Technology-BPPT,Tangerang Selatan 15310, Indonesia
Email: nasir_rofiq@yahoo.co.id
Murat Gorgulu
Animal Science Departmen
Cukurova University, Balcali Campuss, Adana, 01330, Turkey
Email: gorgulu@cu.edu.tr
Abstract—Clove
and orange peel oils were used for rumen manipulation in ruminant animal production.
However there is limited study with true
in vitro rumen digestibility. The objective of this study was to
evaluate combination effect of clove and orange peel oils on in vitro digestion
of Dairy Total Mixed Ration (TMR) using ANKOM DAISYII Incubator.
Ruminal fluid for in vitro digestion technique
was prepared as in vitro digestibility
ANKOM method. The results indicated that in vitro true DM disappearance
(IVTDMD) and in vitro neutral detergent fiber disappearance (IVNDFD) of dairy TMR
were significant (P <0.01) affected by clove, orange peel oils and their
combination. Clove increased IVTDMD and IVNDFD and energy estimate (TDN, ME and
NEl) of TMR, while orange peel oils decreased. Therefore, there was antagonistic
effects between CO and OP 300 ppm when they were used together in combination
treatment for decreasing in vitro digestion of
dairy TMR.
Index Terms—Clove oil, Orange Peel oil, In vitro Digestion, ANKOM DaisyII
Incubator, TMR.
I.
Introduction
Some essential oils which have been used for animal feed additive may substitute the use
of growth promoters such as antibiotics and hormones. Clove and orange peel
essential oils were used for rumen manipulation in ruminant animal
production. Major component of clove oil were eugenol and
b-caryophylene which is a phenolic non-nutrient component which reported that
they had effect on all in vitro rumen fermentation products, VFA, N-NH3
and rumen microbe [1,2]. Limonene as major component of essential oils in
orange peel essential oil was less used as rumen modifier, but it was reported
that it could increase dry mater and NDF digestibility doses 50, 100 and 150
ppm with wheat straw as substrate [3]. The combination between essential oils
may result in additive and/or synergic effects that may enhance efficiency of
rumen microbial fermentation and nutrient utilization in ruminants. The
combination of clove and orange peel oils at optimum doses might effect on
rumen microbe balance for ruminal digestion.
The digestibility of feeds could be estimated by
some methods known as in vitro techniques. In vitro digestibility method was
developed by Tilley and Terry[4] has long been regarded as an accurate in vitro
method for predicting diet digestibility. It had been modified by the method
and in vitro buffer composition [5] and result in modified equipment and
reagent that use are in the method [6,7]. In vitro digestion method was
developed for multiple analysis of feedstuff, reducing labour demands and
improving the precision of the assay. DaisyII incubator (ANKOM
Techology Corp., fairport, NY. USA) makes in vitro dry matter disappearance
study easy and efficient because it use an equipment which was designed with
four rotating digestion jar and maintains constant, uniform heat and agitation
within a controlled chamber (preset at 39.5 oC ). The filter bag
(ANKOM F57) in DaisyII incubator method was reported gave a more
precise prediction of conventionally measured digestibility estimates than the
alternative bags[8]. The bags ensure a more standardized, repeatable
alternative to the Tilley and Terry method. Because the in vitro rotating jar technique is
a simple apparatus, further improvement would probably be obtained by reducing
the laboratory differences in rumen collection procedures and type of animal
donors, which, however, reflect practical conditions[9].
The objective of this study were to determine
the effect of clove oil ppm (CO 300), orange peel oil 300 ppm (OP 300) and
their combination (OP 300/CO 300) at 30 hour incubation on rumen in vitro true
DM disappearance (IVTDMD) and neutral detergent fiber diaspperance (IVNDFD) of
dairy total mixed ration (TMR) using
ANKOM DAISYII incubator.
II.
Material and Methods
A. Ruminal
Fluid Donor Animals
Three ruminally cannulated cows were
used and individually penned indoors at
Research and Application Farm, Faculty of Agriculture, University of Cukurova,
Turkey. Animal was adapted with total mixed ration (TMR) contaning 60%
concentrate and 40% alfalfa hay sized 1-2 cm for 2 weeks before taking ruminal fluid for inoculums
medium at in vitro digestion assay. Ruminal fluids were collected from
different sites within the rumen before morning feeding at 08.00 am.
B. Experimental
Diets
Experimental diet was
total mixed ration containing 60% concentrated feed and 40% alfalfa hay.
Alfalfa hay was chopped to be 1.5 – 2 cm size. Nutrients contain in alfalfa
hay, concentrated fed and TMR were described in table 1. TMR was used for
feeding to cannulated cows and used for
substrate fermentation in in vitro digestion assay. Alfalfa hay, concentrate,
TMR and other feed ingredients which were used for this experiment were
analysed for proximate[10], Fiber analysis[7]. Fiber analysis for neutral
detergent fiber and acid detergen fiber (ADF) were analysed using a heat stable
α-amylase at 0,2 ml/g DM. Neutral detergent insoluble N (NDIN) or crude protein
CP (NDICP) represents the protein associated with cell wall and insoluble in
neutral detergent solution. After NDF data recorded, the filter bag was placed
in Kjehdahl destilation tube for CP analysis as described above (Residual CP).
Residual CP was corrected by CP values from blank filter bag.
TABLE I.
Total Mixed ration as experimental diet :
ingredients and its chemical composition
|
|
Concentrate (%)
|
Alfalfa hay (%)
|
TMR (%)
|
|
Ingredients :
|
60
|
40
|
100
|
|
Barley grain
|
9.59
|
|
|
|
Corn grain
|
3.00
|
|
|
|
Wheat middlings
|
3.59
|
|
|
|
Wheat brand
|
4.19
|
|
|
|
DGGS
|
15.32
|
|
|
|
Sun flower meal
|
11.98
|
|
|
|
Soybean meal
|
6.25
|
|
|
|
Mineral
|
1.88
|
|
|
|
Salt
|
0.34
|
|
|
|
Molasses
|
3.59
|
|
|
|
Vitamin
|
0.09
|
|
|
|
DCP
|
0.17
|
|
|
|
Chemical composition :
|
|
|
|
|
Dry Matter (%)
|
89.95
|
92.80
|
91.95
|
|
OM (% DM)
|
91.91
|
91.79
|
91.70
|
|
CP (% DM)
|
23.56
|
14.73
|
20.29
|
|
EE (% DM)
|
2.15
|
1.15
|
1.39
|
|
CF (% DM)
|
11.21
|
36.51
|
21.33
|
|
NFE (% DM)
|
54.98
|
39.41
|
48.69
|
|
Ash (% DM)
|
8.09
|
8.21
|
8.30
|
|
NDF (% DM)
|
28.53
|
60.30
|
41.23
|
|
ADF (%DM)
|
19.84
|
49.27
|
31.61
|
|
ADICP (% CP)
|
10.28
|
8.54
|
9.22
|
|
NDICP (% CP)
|
7.20
|
4.91
|
8.09
|
|
ME (MCal/kg)1
|
|
|
2.41
|
DM = dry matter, OM = organic matter, CP =
crude protein, EE = extract ether, CF = crude fiber, NFE = nitrogen free
extract, NDF = neutral detergent fiber, ADF = Acid detergent fiber, ADICP =
Acid detergent insoluble crude protein, NDICP = Neutral Detergent insoluble
crude protein, 1)metabolizable energy
estimated by equation 5.
C. In
vitro Digestion Methods
In vitro
digestion method using ANKOM DAISYII incubator had several steps :
0.25 g substrat TMR preparing in filter
bag (F57); buffer solutions, rumen inoculum and essential oils mixing; 30 hours
incubation and NDF analysis. The
final bag weight after NDF analysis was recorded as final weight (W3)
which its values of samples was used for energy and digestibility estimation.
Estimation energy of feed was approached by UC Davis (UCD) factorial[11] using
equation 3. In vitro true DM disappearance (IVTDMD) and in vitro NDF
disappearance (IVNDFD) were calculated with equation (1 and 2)
IVNDFD (% DM) = 100 x [(W2 x %NDFFeed)
– (W3 – (W1
x C1))]/(W2 x %DMFeed ) (1)
IVTDMD
(%DM) = 100 – [(W3 – (W1 x C1)) x 100 ]
(W2 x % DMFeed) (2)
Where W1 is weight of filter bag, W2 is weight of sample, W3 is final weight (Filter bag +
sample), NDFFeed is % of NDF
contain in Feed (%DM), DMFeed is % of dry matter contain in feed and
C1 is correction of factor (blank filter bag NDF value).
IVNDF or digestible NDF (dNDF) is used for energy estimation
approached by energy equation of UC Davis (equation 3, 4, 5,6) result in TDN,
DE, ME and NEL values.
TDN(1xM) = ((CP-ADICP)*(FT/5)*0.98)
+((CP–ADICP)*(1-(FT/5))*0.80)
+((EE-1)*0.98*2.25) +(NDF*dNDF)
+ (0.98*(100-ASH-EE-NDF-CP))) (3)
DE(1xM) = (0.04409 x TDN(1xM) (4)
ME(1xM)
= ((DE(1xM))*1.01) – 0.45
(5)
NEL (1xM) = ((TDN(1xM))*0.0266) – 0.12 (6)
TDN(1xM) is the total digestible nutrient value of a feed or
diet at maintenance intake. Because net energy for lactation (NEL) at
maintenance (NEL)(1xM) is not representative of the energy value of
feed or diet at production level, a discount factor was developed to correct
for decrease net energy level at production (NEL, 3xM)). In UCD
factorial approach to estimate feed energy levels. A discount energy factor was
formulated based on NDF and non structural carbohydrate (NSC) content of
feedstuff and NEL(1xM) values as % per unit of energy intake (M)
which it is calculated with equation 7.
Discount = ((0.033 + (0.132*NDF(%DM)))
-
(0.033*NEL(1xM),MCal/Kg)))
+(NSC(%DM)*0.05) (7)
D. Statistic
Analysis
Treatments of this experiment were 1) control, 2) Orange
peel oil 300 ppm (OP 300), 3) Clove oil 300 ppm (CO 300) and 4) combination
between clove oil 300 ppm and orange peel oils 300 ppm (OPCO 300) in ruminal
fluid, which were assigned and analyzed in two by two factorial arrangement in
a completely randomized design. The data was analyzed by using GLM procedure of
SAS 9.1.3 for windows statistical package [12].
Yijk = µ + αi + βj + (αβ)ij + eijk (8)
Where Yijk is observed value, µ is
general mean, αi is clove essential oils 300 ppm effect, βj is orange peel oil 300 ppm effect, (αβ)ij
is their combination effect and eijk
= standards error.
III.
Result and Discussion
Clove oil
300 ppm (CO 300) was significantly increased (P <0.01) in vitro true DM
disappearance (IVTDMD), in vitro NDF
disappearance (IVNDFD), and estimated energy contents (TDN, ME and NEL) values
compared to control using DaisyII Incubator, while orange peel oils
300 ppm (OP 300) decreased all of parameters values after 30 hour incubation
(Table 2). CO significantly (P<0.01) increased IVNDFD values compare to control
after 30 hour incubation (48,27% from 35.67%), while PO was decreased. The
value of IVNDFD of CO 300 ppm or OP 300 ppm linearly resulted to same effect of
some energy estimated values (TDN, ME and NEL). it was caused by the equation
of energy estimated which used IVNDFD 30 hours as a variable. CO 300 ppm
increased TDN value 7.73%, ME value 6.72, NEL value 8.44% from control treatmen
of TMR. OP 300 decreased TDN value 9.15%, ME value 13.03 NEl value 9.74% from
control TMR. However, CO 300 ppm was reported had no effect to increase in
vitro rumen gas production using Hohenheim gas technique (HGT) after 96 hour
fermentation[13] indicated no negative effect on digestibility of insoluble
fraction of TMR, because CO at 0.25 ml and 0.50 ml level of extract could
inhibit enzyme CMCase, xylanase and acetylesterase [14]. Other in vitro
fermentation culture also was reported that eugenol oil from doses 3 to 5000
mg/L had effect on all rumen
fermentation products while the eugenol in this research is containing 97,26% or close to 300 mg/L [15]. An
unpublished experiment reported that OP 300 ppm increased gas production after
48 fermentation using HGT which is containing more in vitro methane gas
production refer to uneffective rumen fermentation.
Combination
of CO 300 and OP 300 was significant (P<0.01) affected on IVTDMD, IVNDF and
some energy estimated values of TMR. The combination result was affected by OP
300 which was decreased IVNDF, IVTDMD and some energy values significantly
(P<0.05). There was no synergistic effect of combination but antagonistic
effect when CO 300 ppm and OP 300 ppm were used together in in vitro rumen fermentation.
This result
might have been a reflection of high doses of orange peel oil 300 ppm which its
high doses of main component (Limonene) having highly antibacterial
characteristics especially gram-positive bacteria[16], such as Methanobrevibacter ruminantium[17] which
is living synergic with rumen cilia protozoa in rumen. Protozoa in rumen was
reported have improvement in digestion of lignocellulose due to bacterial and
cilia protozoa synergistic effect[18]. High doses of CO 300 ppm had no negative
effect on IVNDFD and IVTDMD when it was used alone.
The
combination effect of CO 300 and OP 300 showed a negative effect to IVTDMD,
IVNDFD and some energy estimated values because of stronger microbial activity
of CO 300 and OP 300 while they were used together for 30 hour rumen
incubation. Generally, main
action mechanism of plant essential oils as rumen manipulator is antimicrobial
effects of main component of the essential oils. It was reported that cell
membrane disruption would be happen when plant EO’s active with microorganism
cell membrane including electron transport, ion gradients, protein
translocation, phosphorilation and other enzyme-dependent reactions [19,20].
The combination effect was not only affected by doses but also with type of
essential oil. It was reported that combination of clove oil (800 mg/L) with
other essential oil (cinnamaldehyde) had no effects on deaminative activity of
rumen bacteria and ammonia N Concentration in vitro rumen fermentation [21]
(Benchaar et al 2008), while other combination using less doses of clove oil
(90 mg/day and 300 mg/day) affected N metabolism in the rumen by increasing the
concentration of small peptide plus amino acid N and decreasing ammonia N
concentration [22]. Combination effect of OP 300 with CO 300 ppm increased gas
production after 96 fermentation HGT compare to control and single addition of
CO and OP. Combination between CO and OP had no negative effect on gas
production after 24 hour incubation, which led to an increase in ME of TMR.
Menke et al (1988) suggested that GP after 24 hour incubation has a positive
correlation with ME in feedstuff. CO 300 ppm had similar effect with the
control but did not decrease the ME value of TMR [23].
All of combination between CO and OP had no
advantage according to in vitro digestibility because they could reduce methane
production but could not keep the in vitro digestibility to be similar with without
essential oil. The result could be explained because of activity of limonene in
PO and clove in CO which have methane reducing compounds. There was a
synergistic effect of combination for methane reducing but also antagonistic
effect of combination for decreasing in vitro digestibility. The Advantage of
CO 300 in combination is covering of negative effect of 300 ppm OP result in
similar effect between combination and control (without essential oil).
TABLE
II. IVTDMD,
IVNDFD and energy estimated values of 30
hours Fermented TMR with CO, OP and their combination
|
Parameter
|
CO-0
|
CO300
|
SE
|
P Value
|
||||
|
OP-0
|
OP-300
|
OP-0
|
OP-300
|
CO
|
OP
|
CO*OP
|
||
|
IVNDFD (% DM)
|
35.67b
|
20.74a
|
48.27c
|
19.36a
|
3.99
|
0.18
|
0.01
|
0.10
|
|
TDN1xM
(%)
|
62.38b
|
56.67a
|
67.20c
|
56.15a
|
1.53
|
0.18
|
0.01
|
0.10
|
|
ME1xM(Mcal/Kg)
|
2.33b
|
2.07a
|
2.54c
|
2.05a
|
0.07
|
0.18
|
0.01
|
0.10
|
|
NEl3xM(Mcal/kg)
|
1.54b
|
1.39a
|
1.67c
|
1.37a
|
0.04
|
0.18
|
0.01
|
0.10
|
|
IVTDMD (%)
|
75.41b
|
69.70a
|
80.23c
|
69.18a
|
1.53
|
0.18
|
0.01
|
0.10
|
Where : CO= Clove oils, OP =
orange peel Oils, Same letter at the
same row indicate to no difference between treatment (P > 0.05)
The
chemical composition of clove and orange peel essential oil samples indicated
that eugenol contained in clove oil is 97.26% and limonede contained in orange
peel oil is 98.08%. Mostly pure active component of essential oils containing
in CO and OP were used in this experiment.
In vitro rumen fermentation method using Daisy incubatorII
(ANKOM) proved that the in vitro true digestibility of DM and NDF could explain
estimation of energy values more accurate than other in vitro rumen
fermentation method.
IV.
Conclussion
Clove and
orange peel essential oils 300 ppm affected in vitro digestion of dairy TMR. However,
there was antagonistic effect on in vitro digestion value while they were used
together in in vitro rumen fermentation due to stronger microbial activity of
CO 300 ppm and OP 300 ppm.
Acknowledgements
This research was funded by Cukurova University Research Project Unit
with Grant no ZF2011D10
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1988.
Muhamad N. Rofiq
was born in Jakarta 15th January 1974. Started to study in primary
school, junior, and high school in
Jakarta. In 1996, accepted as bachelor student in animal science in Bogor
Agriculture University (IPB) in Bogor Indonesia. In 2003, graduated from IPB as
master science of animal science and continue to study in PhD of Animal science
in Cukurova University, Turkey in 2009 to 2013.
Professional worker as young scientist at Directorate
of Primary Industry System Analysis , The Agency of The Assessment and
Application of (BPPT) from 1997 to 2007 and in middle level of researcher at Centre
of Agriculture Production Pechnology, BPPT from 2007 until now. He gained
hand-on experience in UMR-SAS INRA for gas analysis from animal husbandry.
Dr. Muhamad Nasir Rofiq coordinate is a member of Indonesian
Association of Nutrition and Feed (AINI) and lead some project included the project of
Dissemination Technology for animal science – in South Borneo (Research and
Technology Department of Indonesia) in
2004 and Animal Protein Project
in BPPT in 2008.
Murat. Gorgulu
was born in Ermenek-Karaman 15th Novembery 1963. Started to study in
primary school, junior, and high school
in Ermenek-Karaman. In 1986, accepted as bachelor student in animal science in Cukurova
University in Adana Turkey and get PhD in 1990 at same university. Some experience of Animal science education
were gained from some country : Poland and Netherland.
He was honoured to be a Professor of Animal Science in
Cukurova University in 2003.
Professor Murat Gorgulu lead some projects of
experiment in Animal Science in Turkey. He wrote a lot of journal about
ruminant animal science.
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