Combination Effect of Clove and Cinnamon Oil on in Vitro
Rumen Gas and Methane Production
M.N. Rofiq1,*, S. Martono1, M. Görgülü2 & M. Boga³
1Centre for the Agriculture Production Technology,Agency for the Assessment and
Application of Technology (BPPT), MH.Thamrin, Jakarta,10340, Indonesia, *E-mail: nasir_rofiq@yahoo.com
2Animal Science Department, Cukurova University, balcali, Adana, 01330, Turkey, 3Bor Vocational School, Nigde University, Nigde, Turkey
Abstract
Clove and cinnamon oils were used for rumen manipulation in ruminant animal production. Their major component, eugenol and cinnamadehyde were proved to optimize rumen metabolism and microbial composition. The objective of this study was to evaluate combination effect of clove and cinnamon oil on rumen gas and methane production by using an in vitro rumen fermentation system. Three ruminal cannulated cows were used as donors of ruminal fluid and were individually penned indoors. The cattle were fed daily total mix ratio (tmr – 60% concentrate 40% alfalfa hay) and had free access to water at all time. Ruminal fluid for in vitro rumen fermentation system was prepared as in vitro hohenheim gas test method. The treatments were 1) control, 2) cinnamon oil 300 ppm, 3) clove oil 300 ppm and 4) combination of clove and cinnamon oil which were assigned in order to factorial design. The results indicated that insoluble gas fraction (b; 40.8, 46.91, 40.36 and 47.46 ml respectively) and potential of extent of gas production (׀a׀+ b; 43.01, 51.03, 42.96 and 50.45 ml respectively) were significantly different (p < 0.01) between treatments. Soluble gas fraction (a) and rate of gas production (c) were not different between treatments. Clove oil, cinnamon oil and their combinations affected methane production and both essential oil decreased methane production (18.15 ml/ g dm vs 11.80 ml/ g dm and 11.55 ml/g dm, p<0.05). However there were no additive or synergistic effect when they were used together (11.80 ml/g dm vs 11.55 ml/g dm). These results explained that some essential oils were used together as blend oil may not give additional beneficial effect.
Introducton
Publc awareness of the potental health rsk and envronmental problem caused by the excessve use of n-feed antbotcs, growth hormones and some pharmaceutcal food producton lead to prohbton of some antbotcs snce 1998 n EU member state. Some aromatc herbs and essental ols whch have been used for anmal health management may substtute the usng of growth promoters such as antbotcs and hormones. Current nterest n rumnant anmal producton ndustry s focused on tryng to mprove producton effcency and reduce envronment effect (Methane emsson and ammona excreton) wth usng essental ols. Clove and cnnamon essental ols have been evaluated at some doses. Ther major component, eugenol and cnnamaldehyde were proved to optmze rumen metabolsm and mcrobal composton.
Some in vitro research of clove essental ols and euogenol ol from doses 3 to 5000 mg/L in vitro fermentaton culture was reported had effect on all of rumen fermentaton products (Busquets, 2005a) decreasng VFA concentraton (Busquet, 2005b; Castllejos et al., 2006). In vivo experments usng used blend ols contan eugenol (Agoln rumnant) 0.5 g/day/cow whch had no effected on dry matter n-take and mlk yeld, but ncreased fat contan n mlk (Santos, 2010). Cnnamon leaf essental ol (500 mg/l) contanng 76% eugenol decreased N-ammona con-centraton, molar of branched-chan VFA (Fraser et al., 2007), acetate but ncreased proponate and butyrate at doses 312 mg/l (Busquet et al., 2005b). Cnnamon leaf ol (250 mg/l) also reduced methanogeness actvty of rumen bactera and methane concentraton n the fermentaton gases, wthout alterng total VFA (Chaves et al., 2008). In vivo studes reported that supplementaton of cnnamaldehyde (200 mg/kg of dry matter) had no effect on dry matter ntake, gan, feed effcency, carcass char-acterstcs, meat qualty and rumen N-ammona concentraton n growng lamb fed barley or corn-based dets (Chaves, 2008).
The combnaton between essental ols or ther components may result n addtve and/or synergstc effects that may enhance effcency of rumen mcrobal fermentaton and nutrent utlzaton n rumnants. Thus, ths research was evaluated the effects of clove (eugenol), cnnamon(cnnamaldehyde) and ther combnatons n the in vitro fermentaton system.
Materals and Methods
Three rumnal cannulated cows were used as donors of rumnal flud and were ndvdually penned ndoors. The cow was fed daly total mx rato (TMR – 60% concentrate 40% alfalfa hay) twce at 08.00 and 14.00 h and had free access to water at all tme.
from clove bud and cnnamon bark. The chemcal composton of clove and cnnamon essental ol samples ndcated that eugenol contan n clove ol s 97.26% and cnnamaldehyde contan n cnnamon ol s 17.79%. The TMR that was used for rumnal anmal donor, was also used as a substrate for in vitro rumen fermentaton system. The TMR contaned proten 18.81%, ADICP 9.22 %CP, NFE 49.87%, crude fbre 19.77%, NDF 38.22 and ADF 29.24%
The treatments were 1) control, 2) Cnnamon ol (CIN) 300 ppm, 3) Clove ol (CO) 300 ppm and 4) Clove ol (CO) 300 ppm + Cnnamon Ol (CIN) 300 ppm. Addton of CO, CIN and ther combnaton were evaluated by in vitro Hohenhem Gas Test (HGT). The method of the HGT system s descrbed n detal by Menke and Stengass (1979).
Anaerobc technques were used n all procedures durng the rumen flud trans-fer and ncubaton perod. Rumen flud was collected from rumen-fstulated cows before mornng feedng after 2 weeks feed adaptaton. Fltered Rumen flud was added to buffer medum n proporton 1 : 2 v/v, keep wth carbon doxde flushng for 10 -15 mnute before startng to fll up the syrnges. CO and CIN ols 300 ppm added to mxed rumen flud and buffer medum as a treatments of ths experment. Through the nlet of the HGT glass syrnges whch substrate s placed nsde t , 30 ml ncubaton mxed medum (rumen flud, buffer medum and CO,CIN or comb-naton CO-CIN) then dspensed nto the pre-heated HGT glass syrnge (39 oC) wth
the help of a sem-automatc ppette. The ncubaton was conducted for 96 hours nsde the modfed water bath (39 oC). Buffer medum contanng some soluton
whch s descrbed by Menke and Stengass (1979).
Gas producton data was collected at 3, 6 , 9, 12, 24, 48, 72, 96 hours. After 6 h of ncubaton methane gas was measured usng a catharometer methane sensor OLC20 (Oldham ®, USA). The methane sensor was calbrated at 0–100% methane at gas produce at the tme, and was calbrated agan usng pure methane gas (99,7%). Cumulatve gas producton data were ftted to the model of Orskov and Mcdonald (1979):
y = a + b(1-exp-ct)
where : a - gas producton from mmedately soluble fracton (ml). b- gas producton from the nsoluble fracton (ml). [a + b] – potental gas producton (ml). c – gas producton rate constant for the nsoluble fracton (ml/h). t – ncubaton tme (h). y – gas produce at tme t (ml)
Estmaton of metabolc energy of TMR was calculated by equatons from Alderman (1985) usng proxmate values of TMR and from Boguhn et al (2003) usng gas producton value. The predcted value of ME of TMR for ths experment s 2.70 Mcal/Kg.
produc-ton knetcs and methane producproduc-ton usng the General Lnear model (GLM) of the SAS. The sgnfcance of dfferences between ndvdual means wre determned us-ng Duncan’s multple comparaton test.
Results and Dscusson
Soluble gas fracton (a) and rate of gas producton (c) were not dfferent between treatments. These data suggested that a lag phase due to delay n mcrobal colonzaton of the TMR may occur n the same tme of ncubaton. The fermentaton TMR by usng CO, CIN and ther combnaton had sgnfcant effect on nsoluble gas fracton (b), potental of extent of gas producton (|a|+b), gas producton after 96 h ncubaton and methane gas producton after 6 h ncubaton (Table 1). The value for a ntercept for all treatments was same .
The addton of CIN 300 ppm and combnaton between CO and CIN resulted n hgher value of gas volume at asymptote (b) than control treatment but athe addton
Table 1. Methane producton at 6 hour ncubaton, characterstc and cumulatve gas volume producton throughout 96 hour of rumen ncubaton wth TMR, CO, CIN, and combnaton between CO-CIN at 300 ppm
Gas Parameter CO-0 CO300 SE P < 0.05
CIN-0 CIN300 CIN-0 CIN300 CO CIN CO*CIN
Gas characters :
a -2.22 -4.12 -2.60 -2.99 0.66 0.59 0.11 0.28
b 40.80b 46.91a 40.36b 47.46a 1.17 0.97 0.00 0.68
c 0.08 0.08 0.09 0.07 0.01 0.36 0.36 0.11
[a] + b 43.01b 51.03a 42.96b 50.45a 1.97 0.27 0.03 0.28
Gas Producton :
6 h 12.50b 14.21a 12.86ab 12.53b 0.44 0.16 0.15 0.04
24 h 31.28b 36.17a 30.75b 34.59a 0.59 0.1 0.00 0.39
48 h 37.31b 41.69a 37.27b 42.13a 0.97 0.84 0.00 0.81
96 h 38.74b 42.87a 37.70b 44.56a 1.76 0.85 0.01 0.45
pH 6.78 6.76 6.76 6.74 0.01 0.19 0.14 1.00
ME (MCal/ KgDM)*
2.25b 2.30a 2.25b 2.29a 0.01 0.10 0.00 0.39
Methane % 26.55a 21.11b 23.18b 18.86c 0.79 0.01 0.06 0.09
(ml/g DM) 18.15a 11.80c 14.90b 11.55c 0.01 0.02 0.07 0.08
MR (%) 34.99 17.91 36.36
of CO 300 ppm had same value wth control. The gas volume at asymptote (b) represented the fermentaton of the nsoluble fracton. Ths result mght ndcated that the addton of CO at 300 ppm had no negatve effect on dgestblty of nsoluble fracton of TMR, because CO at level 0.25 ml and 0.50 ml could nhbt CMCase, xylanase and acetylesterase actvty (Patra, 2010). The addton of CIN 300 ppm and combnaton between CO and CIN could ncreased dgestblty nsoluble fracton of TMR due to hgh value of gas producton at asymptote (b) and total gas producton at 96 h after ncubaton. There s no nteracton effect between CO and CIN on gas producton at asymptote (b) value (P >0.05).
The Addton of CIN 300 ppm and combnaton between CO 300 ppm and CIN 300 ppm had potental extent of gas producton value hgher than control, but the addton of CO had same value wth control. These results ndcated that the addton CIN and combnaton CO and CIN affect dgestblty of nsoluble fracton of TMR. Gas producton 24 h after ncubaton ncreased wth the addton of CIN and combnaton CO-CIN whch t mght effcency of energy usng from TMR. Addton of CIN 300 ppm and combnaton CO and CIN had predcted metabolsm energy after 24 h of ncubaton hgher than control. Menke et al (1988) suggested that gas volume at 24 h after ncubaton has a relatonshp wth metabolzable energy n feedstuff. .3320@ 1642?@6/686@E ;3 6:?;8A/82 3>.0@6;: ;3 *$( .? <>;1A0@6;: 5 .3@2> 6:0A/.@6;: 6:0>2.?21 C6@5@52 .116@6;:;3!%.:10;9/6:.@6;:&!%C56056@9645@2336062:0E;32:2>4EA?6:43>;9 *$( 116@6;:;3!%<<9.:10;9/6:.@6;:&.:1!%5.1<>2160@2192@./;86?92:2>4E .3@2>5;36:0A/.@6;:56452>@5.:0;:@>;8 $2:722@.8?A442?@21@5.@4.?B;8A92.@5 .3@2>6:0A/.@6;:5.?.>28.@6;:?56<C6@592@./;86F./822:2>4E6:3221?@A33
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Fgure 1. Cummulatve gas volume estmated by Y = a + b [1-exp( - ct)] throughout 96 hour. substrat s TMR added wth CO, CIN and ts combnaton at 300 ppm
CIN 300 ppm. It ndcated that there was no synergy effect between CO 300 ppm and CIN 300 ppm at n vtro gas producton test.
Conclusons
The addton of cnnamon 300 ppm, clove 300 ppm and ther combnaton affected on methane reducton. Methane reducton value of addton cnnamon was twce hgher than addton of clove, but there had no synergy effect of methane re-ducton value of addton combnaton between cnnamon and clove. Methane gas reducton of addton clove, cnnamon and ther combnaton due to effcency of energy avalable n TMR from nsoluble fracton of TMR.
Acknowledgement
Ths research was funded by Cukurova Unversty Research Project Unt wth Grant no ZF2011D10.
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