Sunday, March 8, 2015

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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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.


 Manuscript received January 15, 2014; revised February 25, 2014; accepted February 1, 2014.
1) corresponding author .

Combination Effect of Clove and Orange Peel Oils on In vitro Digestion of Dairy Total Mixed Ration Using ANKOM DAISY II Incubator ...