Efficacy of different essential oils in modulating rumen fermentation in vitro using buffalo rumen liquor

Aim: Present study was conducted to examine the modulatory effect of different essential oils on rumen fermentation pattern in vitro using wheat straw based diet (concentrate: wheat straw 50:50). Materials and Methods: Four essential oils i.e. cinnamon, garlic, oregano and rosemary oils were tested at concentration of 0, 30, 300 and 600 mg/litre (ppm) of total culture fluid using in vitro gas production technique. Total gas production, methane production, nutrient degradability, volatile fatty acid (VFA) production and ammonia nitrogen concentration were studied in vitro using buffalo rumen liquor. Results: Results indicated that all four essential oils decreased gas production significantly (P<0.05) at 600ppm concentration. However, in case of garlic oil, 300 ppm concentration was also found to be effective in decreasing total gas production. Reduction in methane production was found maximum (P<0.05) at higher doses in most of the oils. Maximum reduction in methane was noticed with garlic oil at 600ppm dose. Ammonia-N concentration was also decreased significantly (P<0.05) with essential oils and was found minimum with oregano oil at 600 ppm dose. Partition factor was found to be significantly (P<0.05) higher in 600 ppm concentration of garlic and oregano oil. The degradability of dry matter decreased significantly with higher concentration of essential oil in most of treatment combinations. Conclusion: Supplementation with different essential oils on wheat straw based diet modulates rumen fermentation and reduced methane and ammoniaN production and improved utilization of nutrients.


Introduction
condition, present study was conducted to see the effect of different EO on rumen fermentation in vitro in The use of antibiotics as animal feed additive is buffaloes on wheat straw based diet.facing reduced social acceptance due to the appearance of residues and resistant strains of bacteria.As

alternatives, plants and their extracts are attractive with
Ethical approval: Rumen liquor was collected from consumer opinion that most things 'natural' are good.
two donor fistulated Buffalo bull maintained by the Essential oils (EO) are plant secondary metabolites and herd of National Dairy Research Institute (NDRI), the word "Essential oils" has come from "essence" Karnal, Haryana, India.Fistulation of Buffalo bull was which means sweet fragrance.The odour of EOs is due performed by surgeon as per regulation of Institutional to the presence of active compounds (thymol, carvacol, Animal Ethics committee constituted as per the article eugenol, limonene, allicin, diallyldisulphide etc.).Some no. 13 of the CPCSEA rules laid down by Government EOs have antimicrobial activities and are currently of India.considered safe for human and animal consumption, Source of essential oils: Cinnamon, garlic, oregano, and are categorized as Generally Recognized as Safe rosemary oils were supplied by Sigma-Aldrich (GRAS) [1].Potential use in the diet of ruminants has chemicals Pvt.Limited (USA).Each EO was diluted to been reviewed recently [2,3].A number of researchers prepare three dilutions i.e. 30, 300 and 600 ppm in have conducted studies using some patented EO 30ml of incubation medium from standard supplied by combinations got promising result both in vitro [4][5][6][7] Sigma-Aldrich chemicals .Use of essential oil as antimicrobial substances has also been studied [16].
Animal feeding and sample analysis: Rumen liquor Due to paucity of studies on the effect of was collected from donor animals fitted with individual EO on rumen fermentation pattern in Indian permanent rumen fistula, before morning feeding into a pre-warmed thermo-flask and brought to the laboratory.Donor animals were fed on wheat straw and concentrate based diet (3.0 kg concentrate mixture and wheat straw ad libitum).Degradability of feed: For the determination of true DM and OM degradability, the content of each syringe Proximate analyses of substrate: Dry matter (DM) was transferred quantitatively into centrifuge tube and (ID number 930.15), Organic matter (OM) and ash (ID centrifuged at 5000 rpm for 15 min.True degradability number 942.05) and Crude protein (CP) (N×6.25,ID was estimated after 24 h incubation as per the standard number 954.01), Ether extract (EE) (ID number 920.39), procedure [20].Truly degradable organic matter in Neutral detergent fibre (NDF) and Acid detergent rumen (TDOMR) was calculated as the amount of fibre (ADF) of concentrate mixture and wheat straw substrate OM incubated minus the amount of substrate were determined by standard procedures [17,18].recovered as residue after ND solution treatment, and the partitioning factor (PF) was calculated as the ratio In vitro gas production test: The incubations were of TDOMR (mg) to gas volume (ml) produced from it carried out in 100 ml graduated glass syringes [19].200 during 24 h of incubation.mg of concentrate mixture and wheat straw (Triticum aestivum) in 50:50 ratio were used as substrates and Ammonia-N concentration (NH -N): 5 ml of with CO for about 2 minutes and filtered through 4 2 calculated as per standard procedure [21].layers of muslin cloth.After medium became colorless, Volatile fatty acid (VFA) estimation: For total VFA the required amount of strained rumen liquor (SRL) estimation [22], one ml of supernatant was taken into was added.The ratio of medium to rumen liquor was Markham's distillation apparatus and steam distilled 2:1.Corresponding dose of EO were injected in with 1 ml of oxalate buffer containing 10% potassium specified syringes before injecting inoculums.Each oxalate and 5 percent oxalic acid.About 100 ml of treatment was incubated in triplicate.30 ml of distillate was collected and then titrated against incubation medium was injected to each syringe using standard 0.01N sodium hydroxide solution.auto dispenser.The syringes were shaken gently and Phenolphthalein was used as indicator, which gave residual air or air bubble, if any, was removed and the light pink colour as an end point.For the estimation of outlet was closed.The level of piston was recorded and o individual VFAs, a 5 ml of supernatant was treated with the syringes were placed in an incubator (39 ± 0.5 C).
1 ml meta-phosphoric acid (25%) and kept overnight at The syringes were shaken every 30 minutes for first 2 h o 4 C [23].Thereafter, it was centrifuged at 3000 rpm for from the start of the incubation and thereafter every 2 h 10 min and used for the VFA estimation using gas up to 24 h of incubation.
chromatograph (Nucon 5700, India) equipped with Estimation of total gas and methane production: Gas flame ionization detector (FID) and stainless steel production in blank (containing inoculum) and test column (length 4'; o.d ¼ "; i.d 3 mm) packed with syringe (containing inoculum and substrate) was Chromosorb -101.Temperature of injection port, measured after 24 h.The gas produced in standard o column and detector was set at 200, 180 and 210 C, syringe (containing mulberry leaves) was used to respectively.The flow rate of carrier gas (nitrogen) examine day to day variation in the quality of inoculum.
through the column was 40 ml/min; and the flow rate of Estimation of methane production: Methane (CH ) hydrogen and air through FID was 30 and 300ml/ min, 4 respectively.Sample (2 l) was injected through the was estimated using Nucon-5700 gas chromatograph injection port using Hamilton syringe (10 ).Different equipped with flame ionization detector (FID) and VFA's of the samples were identified on the basis of stainless steel column packed with Porapak-Q.For this their retention time and their concentration (mmol) was 3 ml gas was sampled from the headspace of syringe in calculated by comparing the retention time as well as an airtight syringe and injected into Nucon-5700 gas the peak area of standards after deducting the correschromatograph.The standard gas used for methane ponding blank values.estimation (Spantech Calibration gas, Surrey, England) composed of 50% methane and 50% CO .The peak of 2 Statistical analysis: The generated data were methane gas was identified on the basis of retention statistically analyzed by one way ANOVA considering time of standard methane gas and the response factor dose of essential oil as factor using the general linear obtained was used to calculate methane percentage in model procedure (univariate) according to Yij = µ + th the gas sample.The methane produced from the Di+ e with Yij as the studied parameter (j observation ij substrate during 24 h incubation was corrected for the th on the i treatment), µ as population mean, Di as effect blank values.The volume of CH (ml) produced was th 4 of the essential oil (effect of the i group) and e as ij calculated as follows; th residual error associated with the ij observation.Means Methane production (ml) = Total gas produced (ml) × were compared using Tukey's test [24].Significant % methane in the sample.
reduced in all the concentration of garlic oil compared to control (Table -3).

Results and Discussion
With increase in dose rate, oregano oil decreased Chemical composition: Chemical compositions of methane production significantly (P<0.01);whereas, concentrate mixture and wheat straw are given in rosemary oil reduced it significantly (P<0.01) at Table-1.The DM, OM, CP, EE, CF, NFE and total ash 30ppm dose rate, but no further decrease was found at contents are similar to the findings of earlier workers higher doses ( diopenten, and β-caryophyllene whereas cinnamon production: Gas production remains unaffected in contains cinnamaldehyde [7,28,29].Among those, cinnamon oil supplemented groups (Table -2

), whereas
Carvacrolis is the major active compounds of oregano it reduced significantly (P <0.05) in 600 ppm garlic and oil (average of 660 mg/g) [29].Decrease in gas and oregano oil treated groups (Table -3 and 4).In case of methane production by higher dose level of oregano oil rosemary oil supplementation, gas production was in the present study might be due to depressing effect found similar in control and treated groups (Table -2 on microbial fermentation [30].Previous reports [4, 5] and Table-5).Methane production per unit of DM and   suggested that the antimethanogenic effect of garlic significantly (P<0.05) at higher doses of garlic and oregano oil (Table -3 and Table-4); whereas, degradability and its active components was the result of direct was not affected (P>0.05) by supplementation of inhibition of Archaea microorganisms in the rumen.rosemary oil.Similarly, partitioning factor was found Archaea have unique membrane lipids that contain significantly higher (P<0.05) in 600 ppm oregano and glycerol linked to long chain isoprenoid alcohols garlic oil treated groups (Table -3 and Table-4).essential for the stability of the cell membrane.The Busquet et al. [31] showed that the effect of oregano oil synthesis of the isoprenoid units in methanogenic was most apparent at 500 mg/l and similar to those by Archaea is catalyzed by hydroxymethylglutaryl coenzyme oregano oil and carvacrol at 300 mg/l in combination.A (HMGCoA) reductase, an enzyme that has also been Decrease in degradability at higher dose rate of garlic described in the liver and that participates in the and oregano oil was reflected in total VFA production synthesis of cholesterol.Garlic oil and some derived which was decreased significantly (P<0.05),but molar organosulfur compounds are strong inhibitors of HMG proportion of acetate was reduced significantly -CoA reductase, and as a result, the synthesis of the (P<0.05)(Table -3 and Table-4).On the contrary, total isoprenoid unit is inhibited, the membrane of Archaea VFA content and acetate proportion was increased at becomes unstable, and the cells die.In vitro studies higher dose levels of cinnamon oil (P<0.05).The active demonstrated that garlic reduced CH (µmol): VFA 4 compounds with phenolic structures, such as thymol (µmol) ratio from 0.20 to 0.05 [5].In the present study, and carvacrol, are more effective antimicrobials versus we also found significant (P<0.05)decrease in non-phenolic compounds, and the presence of hydroxyl methane production at all the dose level of garlic oil group in their phenolic structure enhances this activity.supplemented group.
Therefore, high doses of oregano oil (500 mg/l) seem too strong to show positive effects on rumen fermentation.
Effect of essential oils on feed degradability, partitioning factor and VFA concentration: In vitro However, the lowest doses of oregano oil (5 and 50 DM and OM degradability percent was reduced mg/l) may help improve efficiency of rumen fermen-    tation by increasing VFA concentrations [32].Similar revision of the manuscript.All authors read and trend was found in our study where higher dose levels approved the final manuscript. of oregano oil were found to improve rumen fermen-

Table - 1
: Chemical compositions (%DM) of concentrate mixture and wheat straw

Substrate Dry matter Organic matter Crude protein Ether extract Crude fiber NFE Total Ash NDF ADF
a,b,c

Table - 2
: Effect of different levels of cinnamon oil on in vitro fermentation in wheat straw based (Concentrate: Roughage= 50:50) diet

Table - 4
: Effect of different levels of oregano oil on in vitro fermentation in wheat straw based (Concentrate: Roughage= 50:50) diet

Table - 5
: Effect of different levels of rosemary oil on in vitro fermentation in wheat straw based (Concentrate: Roughage= 50:50) diet