Methane Mitigation from Goat in Indonesia

Combination Effect of Clove and Orange Peel Oils on In Vitro and In Vivo Rumen Methane Production in Goat

M. N. Rofiq¹, M. Görgülü²

¹Centre for the Agriculture Production Technology - BPPT, LAPTIAB 612 Bld., Kawasan PUSPIPTEK, Serpong,15314, Indonesia

²Animal Science Department, Cukurova University, Balcali, Adana, 01330, Turkey

nasir.rofiq@bppt.go.id

ABSTRACT

Feed additive which favourable affect the environment had been an interesting focus since the GHG’s emission from livestock was reported increasing. Essential oils had been selected since the garlic oil has a good effect for rumen methane gas reduction. There is no more information for other essential oils which was tested for rumen methane reduction. Clove and orange peel oils were used for rumen manipulation in ruminant animal production. Therefore, this study  was carried out to evaluate the effect of combination of clove and orange peel oil on in vitro and in vivo rumen methane production in goat. The treatments were 1) control, 2) orange peel oil 300 ppm or 1.8g/d, 3) clove oil 300 ppm or 1.8 g/d and 4) combination between clove oil 300 ppm or 1.8 g/d and orange peel oils 300 ppm or 1.8 g/d in ruminal fluid, which were assigned and analyzed in a two by two factorial arrangement in a completely randomized design and latin square design. Ruminal fluid for in vitro rumen methane production was prepared using the in vitro Hohenheim gas test method (HGT). 4 Jawarandu goats were used for in vivo rumen methane production using open respiration chamber method. The results indicated that in vitro rumen methane production was not different with in vivo. CO, OP and COOP decreased in vitro and in vivo rumen methane production (32.16%, 17.89%, 18.77% and 39.53%, 44.23%, 28.24%, respectively). There was antagonistic effect of combination on in vivo and in vitro rumen methane production (ml/gDM and g/DMI). In conclusion, clove and orange peel oil at 300 ppm or 1.8 g/day could be used as feed additive for rumen methane reduction but not for combination.

Keywords:  Clove, Orange Peel, Essential oils, Goat, Methane.  

INTRODUCTION

Essential oils as rumen manipulator. Plant essential oils were assessed for categorized to zoo-technical additives group by the European parliament and the Council (EC) regulation number 1831/2003. The following functional zoo-technical additives group are included digestion enhancer, gut flora stabilisers, substance which favourable affect the environment and others (EC, 2003). Some essential oils have been evaluated for in vitro rumen methane reducer and associated rumen fermentation effect. In vivo experiments using plant essential oils was mostly conducted in cattle and a few of them were conducted for goats. The conversion of feed substances to CH₄ emission in the rumen involves the integrated activities of different microbial species, with the final step carried out by methanogenic bacteria. Methane production mitigation from rumen was assessed by some experiments using ration manipulation, feed additives or biotechnological intervention. For all the reasons, the experiments still has not enough yet for establish the using of essential oils as feed additive. The objective of this study  was carried out to evaluate the effect of combination of clove (CO) and orange peel oil (OP) 300 ppm or 1.8 g/d on in vitro and in vivo rumen methane production in goat especially as ruminants feed additive.

MATERIALS AND METHODS

Animal and Experimental Diet. Three ruminal cannulated cows were used as donors of ruminal fluid for in vitro gas and methane production technique. The cows were fed daily with a total mixed ration (TMR–60% concentrated feed and 40% alfalfa hay) twice a day. The TMR was also used as a substrate in the in vitro rumen gas production technique. The TMR has 18.81% crude protein (CP), 9.22 % CP acid-digestible insoluble crude protein (ADICP), 49.87% nitrogen-free extracts (NFE), 19.77% crude fiber (CF), 38.22% neutral detergent fiber (NDF) and 29.24% acid detergent fiber (ADF). The estimation of metabolic energy (ME) of TMR was calculated by an equation from Boguhn et al (2003). The animal which was used for in vivo methane gas production is Jawa randu goat, the crossbred goat from Peranakan etawah and Kacang goats (Indigenous Indonesian goats). 4 goats were adapted with in vivo experimental diet for 14 weeks before data collection in animal laboratory, LABTIAP-BPPT, Indonesia. The goat had 21.65±4.11 kg of average live weight.  

In Vitro Rumen Gas Methane Production Technique: Addition of CO 300 ppm, OP 300 ppm and their combination (COOP 300 ppm) into rumen fluid were evaluated by the in vitro Hohenheim Gas Test (HGT). The operation of the HGT system is described in detail by Menke and Steingass (1979). CO and OP oils 300 ppm added to mixed rumen fluid and the buffer medium as a treatment of this experiment. The incubation of GHT was conducted for 96 hours inside a modified water bath (39 ^OC). Gas production is observed from calibration scale at HGT glass syringe and methane was measured at 6^thhour of incubation with methan gas sensor OLCT 20 (Industrial Scientific Corp.).

In Vivo Rumen Methane Production Technique. Rumen methane production from goats were measured by open respiration system. After treatments with clove (CO) and oral peel oils (OP) in adaptation pen, goats were located in open circuit respiratory chamber for 5 days (3 days for chamber adaptation and 2 days respiration measurements). Goats were daily fed ad libitum using TMR and injected with 1.8 g/d clove and orange peel oils by oral infusion once a day. Respiration measurements using MARS Sable System USA^(R), an open circuit respiratory system for measuring gas containing in gas respiration, pressure and flow. Chamber with close head animal keep gas respiration near animals head that would took by pump flow meter via plumbing. The gas from flow meter is filtered into the scrubber and gas dryer before entre to gas analyser.

Statistical Analysis.  A two (CO0-CO300 ppm) by two (OP0-OP300) factorial arrangement in a completely randomized designed was used to compare gas production kinetics and methane production using the General Linear model (GLM) of the SAS. In Vivo experimental used the same treatment as in vitro which was designed as latine square design.   

RESULTS AND DISCUSSION

In Vitro Rumen Methane Production.  The addition of CO, OP and their combination reduced rumen methane production after 6 h incubation from the control (32.16%, 17.89% and 18.77%, respectively). The methane reduction value of addition of CO 300 ppm had nearly twice the value as addition of OP 300 ppm, but the combination of CO and OP had the same value of methane reduction with OP 300 ppm. This indicated that there was negative effect with addition of the combination due to eugenol activity or limonene activity in the rumen. However, there was no effect  decreasing digestibility of the insoluble fraction of TMR which was explained from in vitro total gas production. treatment OP and combination (COOP) 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 gas production after 24 hour incubation has a positive correlation with ME in feedstuff. CO had similar effect with the control but did not decrease the ME value of TMR (Table 1). Methane gas reduction of the clove oils , orange peel oil and their combination due to efficiency of energy available in TMR from insoluble digestibility fraction of TMR.

Table 1. Characteristic and cumulative gas volume production throughout 96 hours of rumen incubation with TMR, CO, OP and combination between CO-OP at 300 ppm, CH4 production at 6 hours incubation and Estimated ME.

Parameter		Control		OP300		CO300		CO-OP300
		Avg	SE	Avg	SE	Avg	SE	Avg	SE
Gas Product	6 h, ml	12.50b	0.12	17.65a	1.45	12.86b	0.53	19.63a	0.49
	24 h, ml	31.28c	0.20	38.60b	1.85	30.75c	0.69	43.43a	0.44
	48 h, ml	37.31c	0.36	45.14b	2.02	37.27c	1.11	49.97a	0.34
	96 h, ml	38.74c	0.61	46.58b	1.53	37.70c	2.42	51.65a	0.40
ME(MCal/KgDM)*		2.25c	0.00	2.33b	0.02	2.25c	0.01	2.37a	0.00
Methane  %		26.55a	0.85	23.18b	0.64	14.71c	0.54	14.99c	0.64
CH4 (ml/g DM)		18.15a	0.52	14.90b	0.54	12.31c	0.69	14.74b	0.88
MR (%)				17.89		32.16		18.77

 Where : MR = methane reduction, ME* = predicted by equation from Boguhn J et al (2003),

Table 2. Effect of CO 1.8 g/d, OP 1.8 g/d and their Combination (CO 1.8 g/d +OP 1.8 g/d) on in vivo CH₄ production and Gas respiration volume

	CO-0		CO300			P<0.05
	PO-0	PO-300	PO-0	PO-300	SE	CO	PO	CO*PO
- CH4(l/d)	16.88b	11.41a	11.54a	12.07a	0.91	0.03	0.03	0.27
- CH4(g/day)	10.82b	7.31a	7.40a	7.74a	0.59	0.03	0.03	0.26
- CH4(g/DMI	15.33b	8.55a	9.27a	11.00a	1.23	0.04	0.04	0.12
- CH4(g/Kg W0.75)	0.93b	0.65a	0.64a	0.72a	0.03	0.04	0.04	0.17
VCO2 (l/d)	267.74ab	253.58a	303.09b	265.33a	29.41	0.51	0.55	0.76
VO2 (l/d)	73.71	69.17	83.55	72.64	7.96	0.49	0.55	0.77
Composition of Gas(%) :
	1	2	3	4
O2	20.57	20.70	20.98	20.75
CO2	74.72	75.89	76.12	75.80
CH4	4.71	3.41	2.90	3.45
Total gas  l/d	358.33	334.16	398.18	350.04

  Where : VCO₂ = CO₂ production from respiration, VO₂ = O₂ consumption from respiration,

In Vivo Rumen Methane Production. Oral infusion of CO 1.8g/d, OP 1.8 g/d and combination between CO 1.8 g/d and OP 1.8 g/d (COOP) decreased in vivo CH₄ production but there were no interaction between CO and PO (P>0.05). CO, OP and COOP decreased in vivo CH₄ production according to control (9.27; 8.55; 11.00; 15.33 g/Kg DMI, respectively), although there had no combination effect of COPO according to individual of oral infusion CO and PO (P >0.05). Limonene as major component of orange peel oils (PO) was reported had low recovery rate after 24 hour incubation with ruminal fluid due to the interaction with the rumen flora as well as acidity (Haider, 2005). Daily oral infusion of PO, probably increased the limonene recovery inside the rumen fluid. Therefore, the daily in vivo CH₄ production was decreasing. These results were similar with their effect on in vitro methane gas production. Good nutritional status of TMR is one factor that oral infusion of essential oils not effect to DMI and weight gain because it may prevent or hide essential oils effects . Oral infusion CO, OP and their combination (COOP) decreased in vivo methane production and increased weigh gain if the animals were given enough of nutrient above  their requirements. In similar, the commercial blend oil, CRINA (thymol, eugenol, vanillin and limonene) 1.2g/d had no benefit in feed with low concentrate for cows in early lactation and preprartum (Tassaoul et al., 2008). Other blend oils commercial, Agolin Ruminant (eugenol, geranyl acetate and coriander oil) 0.5 g/d did not increase milk yield of early lactation of Holstein cows but increased daily milk fat production (Santos et al., 2010).

              

Acknowledgements. This research was funded by Cukurova University Research Project Unit with Grant no ZF2011D10

CONCLUSSIONS

Addition of clove oil (CO) 300 ppm or 1.8 g/d, orange peel oil (OP) 300 ppm or 1.8 g/d and their combination (COOP, 300 ppm + 300 ppm or 1.8 g/d + 1.8 g/d) affected methane production compared to the control. There was antagonistic effect of combination on in vivo and in vitro rumen methane production (ml/gDM and g/DMI). Methane gas reduction of the addition CO, OP and COOP due to efficiency of energy available in TMR from insoluble digestibility fraction of TMR. The results indicated that in vitro rumen methane production was not different with in vivo. Clove and orange peel oil at 300 ppm or 1.8 g/day could be used as feed additive for rumen methane reduction but not for combination.

REFERENCES

Boguhn J., Kluth H., Steinhofel O., Peterhansel M., Rodehutscord M., 2003 Nutrient digestibility and prediction of metabolizable energy in total mixed rations for ruminants. Arch. Anim. Nutr., Vol.57(4) : 253-266

European Commission (EC). 2003. Regulation (EC) no. 1831.2003 of the European and the council of 22 September 2003 on additives for use in animal nutrition. Offcial journal of The European Comission.

Haider V. 2005. Dissertation, University of Veterinery Medicine, Vienna.

Menke K. H. and Steingass H. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development. 28: 7-55.

NRC. 2007. Nutrients requirements of small ruminants.( sheep, goat, cervids and new word camelide). The National academic press. Washington DC.

Sable System International CO. 2007. Respirometry System Measurements. http://www.sablesys.com/index.php. Las Vegas.

Santos, M. B. P.H. Robinson., P. Williams and R. Losa. 2010. Effects of addition of an essential oil complex to the diet of lactating dairy cows on whole tract digestion of nutrients and productive performance. J. Of Anim. Feed Sci. Technol. 157(2010) : 64-71

Tassoul, M. D. and R. D. Shaver. 2008. Effect of an essential oil blend on performance of perparturient and early lactation dairy cows. J. dairy sci. 91 (E-Suppl. 1) : 265.