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EFFECT OF SOME AUSTRALIAN NATIVE SHRUBS ESSENTIAL OILS ON IN VITRO RUMEN MICROBIAL FERMENTATION OF A HIGH-CONCENTRATE DIET.

Byline: H. Jahani-Azizabadi, Z. Durmic, J. Vadhanabhuti and P. E Vercoe

Keywords: essential oils, methane, ammonia-N, rumen fermentation, in vitro.

INTRODUCTION

Rumen fermentation of feedstuff consumed by the animal provides energy for the animal, but includes some disadvantages, such as methane emission and ammonia production and excretion. Methane represents 8% to 12% loss of gross energy intake in ruminants (Johnson and Johnson, 1995), and it is a potent greenhouse gas that has a global warming potential 21 times higher than that of CO2 (Crutzen, 1995). Further, valuable protein consumed by ruminant is metabolized by rumen microbes into nitrogen (N) and over 80% of N consumed is excreted in feces and urine (Tamminga, 1992). Ruminant nutritionists have been interested in enhancing energy and protein efficiency, by decreasing methane and ammonia-N excretion. The use of feed additives such as ionophores that can modify rumen fermentation has been found useful in this respect and widely applied (Van Nevel, and Demeyer, 1988; McGuffey et al., 2001).

However, increased public concerns about the risk of the use of these synthetic, subtherapeutic levels of antimicrobials resulted in a ban on their use as animal feed additives in EU (European Commission, 2003). Plant essential oils (EOs) have been studied in recent years as a natural alternative for these in-feed antibiotics, in particular for a decrease of rumen methane production and ammonia nitrogen (N) excretion (Busquet et al., 2005; Busquet et al., 2006; Castillejos et al., 2007; Jahani-Azizabadi et al., 2011). Our recent preliminary findings have implied that selected EOs from Australian plants may also affect rumen fermentation including methane production in vitro (Durmic et al., 2014).The study focused on one relatively high dose of EOs that, while being very potent at reducing on methane, also had an effect on other fermentation parameters.

Several authors have found that it is possible to dilute out negative effects of EOs, while maintaining desirable bioactivity (Castillejos et al., 2007; Castillejos et al., 2008; Hungate, 1966). Therefore, the aim of the present study was to investigate the effect of selected Australian EOs on in vitro rumen fermentation characteristics at various doses to find out if there is an optimal dose that affects methane and ammonia N without affecting other fermentation parameters.

MATERIALS AND METHODS

The effects of eight commercial Australian EOs were examined in an in vitro batch fermentation system (Durmic et al., 2014). This experiment was conducted from Jun to Agust 2012 on anaerobic lab of department of animal science of university of Western Australia. The experimental substrate used was a concentrate-based diet (Ellenbank pellet, Debenham Australia, Pty Ltd) which was ground to pass 1-mm screen. Concentrate-based diet was selected because the targeted animals for this feed manipulation are likely to be in intensive systems and fed this type of diet. The ingredients and chemical composition of experimental diet are shown in Table 1. Rumen fluid for incubations was obtained from three adults ruminally-fistulated sheep (49.5 +- 2.5 kg, body weight), that were acclimatized for two weeks on a diet of 1kg of oaten chaff, 250 g of lupin grain and 25g mineral mix (per head/day). This fibrous diet for donor sheep was selected in order to promote methanogen population in their rumen (McDougall, 1948).

The ruminal content was collected on experimental day, 2 h after the morning feeding to obtain an inoculum with the most active microbial population. It was immediately transferred to the laboratory in a pre-warmed thermos flask, then strained through one layer of cheesecloth to eliminate large feed particles and placed in an anaerobic chamber (Coy Vinyl Anaerobic Chamber; Coy Laboratory Products Inc., USA). Buffer (Durmic et al., 2014) was added to strained rumen fluid until pH reached to 7.1, and then 10 ml of the mix was dispensed into a 30-ml anaerobic tube (Bellco tube, Bellco Glass Inc., Vineland, NJ, USA) containing 0.1 g dry matter (DM) of the experimental diet and the appropriate dose of EO.

Treatments were control (substrate+ 0.20 ml of 70% ethanol), and EO from Agonis fragrans, Eucalyptus plenissima, Leptospermum pettersoni, Melaleuca alternifolia, Melaleuca ericifolia, Melaleuca teretifolia, Santalum spicatum and Lavandula angustifolia, obtained commercially from Paperbark Co., Harvey, Weste rn Australia. Four different doses of EO were tested: 62.5, 125, 250 and 500 ul/g DM incubated (DMI), with three replicates for each treatment and control, run in a single batch. All EO were individually dissolved (v/v) in 70% ethanol prior to inclusion in the assay. Control included tubes containing substrate only and an equivalent amount of 70% ethanol. Three Bellco tubes that contained buffered rumen fluid only provided a blank to correct gas produced from nutrient content of initial rumen fluid Each tube was sealed with a butyl rubber stopper and aluminum cap and placed in a shaking incubator (at 50 rpm) for 24 h at 39 AdegC.

At the end of the incubation, the gas pressure in the tubes was recorded using pressure transducer (Greisinger electronic GmbH, Regenstauf, Germany) and a sample of the gas (5 ml) was collected into a 10 ml evacuated Exetainer vial (Labco, UK). The tubes were then transferred to an ice bath to stop fermentation. Samples for ammonia-N (0.2 ml of 2MHCl was added to 1 ml of culture liquid) and volatile fatty acid (VFA, 0.2 ml of 1M NaOH was added to 1ml of culture liquid) were collected and frozen for analysis. Finally, each bottle content was filtered (42 um pore size), the residual was oven dried (60 AdegC for 48 h) and used to calculate in vitro dry matter disappearance (IDMD).

Chemical analysis: Samples for VFA analysis were prepared as described by Durmic et al. (2010). Briefly, samples were centrifuged and the supernatant was analyzed by gas chromatography (Aligent, 6890 Series, Technologies Inc., Santa Clara, CA, USA) with HP 6890 injector, capillary column HP-FFAP, 30 m x 0.53 mm x1.0 m, FID detector and HP Chemstation software. Temperatures were 240AdegC, 260AdegC and 265AdegC in the oven, injector and detector, respectively. The carrier gas (hydrogen) flow was adjusted to 6.6 ml/min. Total VFA were calculated as the sum of acetate, propionate, butyrate, isobutyrate, valerate and isovalerate, while individual main VFA were analyzed as the proportion in total VFA. Branched-chained VFA (BCVFAs) were calculated as the sum of isobutyrate and isovalerate.

The ammonia N concentration in the fermentation uid was determined by spectrometry with a Boehringer Mannheim Test kit 1112732 (R-Biopharm AG, D-64293 Darmstadt, Germany) on Cobas Mira S auto-analyzer (Roche Diagnostics, F Hoffman-La Roche Ltd., CH-4002 Basel, Switzerland), using principles of enzymatic assay described by Bergmeyer and Beutler (1985). Methane content of the produced gas was determined using gas chromatography (Micro gas Chromatograph, Varian 3600; Varian Inc., Palo Alto, CA, USA). The instrument had 60 m HP-1capillary column using helium as the carrier gas. The injector temperature was 50AdegC, and the column was held constantly at 60AdegC during analysis, while the ame ionization detector temperature was 60AdegC. Carrier gas flow was adjusted to 24 ml/min. Methane content was calculated by external standard regression curve which provided by standard gas mixture (contains methane, hydrogen and carbon dioxide; 20, 30 and 30 ml/100 ml, respectively).

Calculations and statistical analysis: Total gas production was reported as ml/24 h of incubation (cumulative gas) and methane production was reported as ml/24h and ml/mg IDMD. Substrate fermentation efficiency (SFE) was calculated as mg IDMD/ml cumulative gas [18]. In vitro DM disappearance (IDMD) was calculated using flowing formula:

IDMD (g/g) = (IDM-remained DM after 24h of incubation)/IDM

Gas pressure was converted into volume using a formula total volume of gas (TVM) = [(Gas pressurekPa + 101.3kPa) x 20mL]/101.3kPa.

Data were statistically analyzer using GLM procedure of SAS (1999) with following statistically model; y=u+Ti+ eij, where y= depended variable, u= overall mean, Ti= effect of EO and eij= residual error. The standard error of the mean (SEM) determined with MEAN procedure. Dunnett's test was used to compare the means with those of the control (P< 0.05). Linear and quadratic orthogonal contrasts were tested using the CONTRAST statement of SAS (1999).

Table 1. Chemical and ingredients composition of experimental diet.

###Amount

Ingredients(g kg-1 DM)

Lucerne Hay###125

Wheat grain###406

Powdered Molasses###50

Minerals###30

Cold pressed Canola###388

Raspberry Flavour###1

Chemical composition(% of DM)

Crude protein(CP)###23.0

Soluble protein(% CP)###44.0

Neutral detergent fiber###20.7

Acid detergent fiber###9.8

ME(Mcal/kg DM)###3.3

Table 2. Effect of essential oils from selected Australian shrubs on fermentation variables measured in gas and solid phase after 24h in vitro rumen microbial fermentation of a concentrate-based diet.

Treatment###Dose( ul/g DMI)###Effects

###0###62.5###125###250###500###SEM###Linear###Quadratic

A. fragrans

IDMD###0.77###0.79###0.79###0.68*###0.67*###0.010###*###NS

SFE###5.53###6.35###6.30###6.15###8.46###0.070###NS###*

Gas(ml/24h)###14.0###12.5###12.4###11.1*###8.0*###0.212###NS###*

Methane(ml/24h)###1.26###1.33###1.24###1.03###0.27*###0.027###*###NS

Methane(ml/mg IDMD)###0.016###0.017###0.016###0.015###0.004*###<0.001###*###NS

E. plenissima

IDMD###0.77###0.80###0.76###0.68*###0.68*###0.010###*###NS

SFE###5.53###5.40###5.43###5.88###5.90###0.120###NS###*

Gas(ml/24h)###14.0###14.89###14.07###11.62*###7.62*###0.182###*###NS

Methane(ml/24h)###1.26###1.87*###1.78*###1.12###0.34*###0.029###*###NS

Methane(ml/mg IDMD)###0.016###0.023*###0.023*###0.016###0.005*###0.002###*###NS

L. pettersoni

IDMD###0.77###0.70###0.67*###0.57*###0.52*###0.010###NS###*

SFE###5.53###6.07###6.91###6.54###10.50*###0.420###NS###NS

Gas(ml/24h)###14.0###11.58*###9.75*###9.63*###5.10*###0.769###NS###*

Methane(ml/24h)###1.26###0.95*###0.31*###0.31*###0.14*###0.036###*###NS

Methane(ml/mg IDMD)###0.016###0.014###0.005*###0.006*###0.003*###0.004###NS###*

M. alternifolia

IDMD###0.77###0.78###0.68*###0.63*###0.62*###0.010###NS###*

SFE###5.53###5.80###6.31###7.87###9.05###0.240###NS###*

Gas(ml/24h)###14.0###13.50###10.96*###8.00*###6.91*###0.343###NS###*

Methane(ml/24h)###1.26###1.51*###0.87*###0.41*###0.34*###0.088###*###*

Methane(ml/mg IDMD)###0.016###0.019###0.013###0.007*###0.005*###0.007###*###*

M. ericifolia

IDMD###0.77###0.70###0.72###0.67*###0.63*###0.010###NS###*

SFE###5.53###5.70###7.38###7.56###31.70*###1.120###NS###*

Gas(ml/24h)###14.0###12.25###9.83*###8.88*###2.05*###0.206###NS###*

Methane(ml/24h)###1.26###1.30###0.85*###0.59*###0.08*###0.022###*###*

Methane(ml/mg IDMD)###0.016###0.019###0.011*###0.009*###0.001*###<0.001###*###*

M. teretifolia

IDMD###0.77###0.71###0.65*###0.61*###0.49*###0.010###NS###*

SFE###5.53###5.64###6.71###8.20###33.50*###0.490###NS###*

Gas(ml/24h)###14.0###12.62###9.77*###7.47*###1.47*###0.131###*###*

Methane(ml/24h)###1.26###1.41###0.32*###0.24*###0.05*###0.011###*###*

Methane(ml/mg IDMD)###0.016###0.020###0.005*###0.004*###0.001*###<0.001###*###*

S. spicatum

IDMD###0.77###0.74###0.73###0.69###0.67*###0.010###NS###*

SFE###5.53###5.68###5.77###5.60###5.38###0.060###NS###NS

Gas(ml/24h)###14.0###13.05###12.68###12.41###12.44###0.223###NS###*

Methane(ml/24h)###1.26###0.86*###0.69*###0.66*###0.63*###0.028###*###*

Methane(ml/mg IDMD)###0.016###0.012*###0.009*###0.009*###0.009*###<0.001###*###*

L. angustifolia

IDMD###0.77###0.73###0.63###0.58*###0.52*###0.020###NS###*

SFE###5.53###5.52###6.13###11.60*###35.90*###0.550###NS###*

Gas(ml/24h)###14.0###13.28###11.31*###4.52*###1.47*###0.295###*###*

Methane(ml/24h)###1.26###1.50*###1.09###0.14*###0.04*###0.010###*###*

Methane(ml/mg IDMD)###0.016###0.021*###0.015###0.003*###0.001*###<0.001###*###*

Table 3. Effect of some of Australian native shrub essential oils on ammonia N and total and individual volatile fatty acids (VFA) concentration compared with control after 24h in vitro rumen microbial fermentation of a concentrate-based fermentation substrate.

Treatment###Dose(ul/g DMI)###Effects

###0###62.5###125###250###500###SEM###Linear###Quadratic

A. fragrans

Ammonia N(mg/l)###173###105*###114*###105*###70.0*###2.40###NS###*

Total VFA(mmol/l)###110.2###92.6*###80.7*###68.2*###49.9*###2.50###*###*

Individual, mol/100 mol

Acetate###55.2###54.3###53.4###64.2*###65.3*###0.93###*###*

Propionate###26.5###29.8*###24.7###18.0*###18.1*###0.71###*###*

Butyrate###12.3###10.4###17.1*###12.1###12.3###0.23###NS###*

BCVFA###2.63###2.27###1.77*###1.67*###1.93*###0.06###NS###*

C2:C3###2.08###1.82###2.16###3.14*###3.61*###0.06###*###*

E. plenissima

Ammonia N(mg/l)###173###136*###102*###109*###60*###2.22###NS###*

Total VFA(mmol/l)###110.2###98.2*###90.5*###68.2*###47.2*###1.62###*###*

Individual, mol/100 mol

Acetate###55.2###54.5###54.4###58.7###63.1*###0.67###*###*

Propionate###26.5###28.3###26.8###14.7*###18.8*###0.53###*###*

Butyrate###12.3###11.6###13.1###-###13.1###0.44###NS###*

BCVFA###2.63###2.50###2.27###1.77*###2.10*###0.04###NS###*

C2:C3###2.08###1.92###2.03###4.02*###3.35*###0.058###*###*

L. pettersoni

Ammonia N(mg/l)###173.0###87.5*###61.0*###42.0*###50.3*###2.65###NS###*

Total VFA(mmol/l)###110.2###61.5*###49.7*###44.6*###40.6*###1.95###*###*

Individual, mol/100 mol

Acetate###55.2###55.8###67.4*###66.1*###63.1*###1.66###NS###*

Propionate###26.5###14.5*###16.8*###18.2*###18.5*###0.33###*###*

Butyrate###12.3###25.4*###12.1###11.6###13.9###1.18###*###*

BCVFA###2.63###1.67*###1.70*###1.83*###2.30###0.22###*###NS

C2:C3###2.08###3.83*###4.03*###3.63*###3.43*###0.15###*###*

M. alternifolia

Ammonia N(mg/l)###173###129*###134*###106*###81.0*###3.24###*###*

Total VFA(mmol/l)###110.2###84.6*###77.5*###53.8*###47.1*###1.95###NS###*

Individual, mol/100 mol

Acetate###55.2###52.8###51.6###57.2###63.4*###0.612###*###*

Propionate###26.5###30.2*###27.8###21.1*###18.7*###0.464###*###*

Butyrate###12.3###11.2###15.4###16.9*###13.3###0.686###NS###*

BCVFA###2.63###2.50###2.03*###1.83*###2.00*###0.10###NS###*

C2:C3###2.08###1.75###1.86###2.72*###3.38*###0.06###*###*

M. ericifolia

Ammonia N(mg/l)###173.0###162.0###110.0*###74.0*###70.3*###0.10###NS###*

Total VFA(mmol/l)###110.2###80.8*###82.4*###58.5*###42.7*###2.76###NS###*

Individual, mol/100 mol

Acetate###55.2###55.2###54.7###70.2*###58.9###0.48###*###*

Propionate###26.5###26.9###22.9*###15.2*###20.6*###0.55###NS###*

Butyrate###12.3###11.9###17.8*###10.6###15.6###0.63###NS###*

BCVFA###2.63###2.63###1.77*###1.60*###2.30###0.63###NS###*

C2:C3###2.08###2.05###2.41###2.61*###2.86*###0.07###*###*

M. teretifolia

Ammonia N(mg/l)###173.0###74.3*###50.3*###37.0*###55.7*###1.50###*###*

Total VFA(mmol/l)###110.2###76.1*###54.2*###49.2*###43.7*###1.05###NS###*

Individual, mol/100 mol

Acetate###55.2###53.8###68.4*###66.2*###56.4###0.42###*###*

Propionate###26.5###16.9*###16.9*###18.4*###20.1*###0.30###*###*

Butyrate###12.3###25.4*###11.2###11.2###18.6*###0.53###*###*

BCVFA###2.63###1.40*###1.60*###1.73*###2.10*###0.07###*###*

C2:C3###2.08###3.20*###4.04*###3.60*###2.80*###0.06###*###*

S. spicatum

Ammonia N(mg/l)###173.0###118.0*###94.7*###102.0*###71.0*###3.03###*###*

Total VFA(mmol/l)###110.2###87.4*###86.8*###92.6*###88.5*###1.51###NS###NS

Individual, mol/100 mol

Acetate###55.2###52.0###51.9###52.4###52.4###0.19###*###*

Propionate###26.5###31.6*###32.8*###32.5*###32.6*###0.23###*###*

Butyrate###12.3###11.0###10.9###10.4###10.5###0.24###*###*

BCVFA###2.63###2.30###1.77*###1.73*###1.70*###0.64###NS###*

C2:C3###2.08###1.65*###1.58*###1.61*###1.60*###0.02###*###*

L. angustifolia

Ammonia N(mg/l)###173.0###122.0*###105.0*###68.3*###71.3*###2.75###*###*

Total VFA(mmol/l)###110.2###90.7*###72.3*###48.2*###43.9*###1.78###NS###*

Individual, mol/100 mol

Acetate###55.2###54.8###56.1###63.3*###55.1###0.59###NS###*

Propionate###26.5###27.2###18.7*###18.8*###19.8*###0.31###NS###*

Butyrate###12.3###12.8###20.7*###12.3###17.2*###0.57###*###*

BCVFA###2.63###2.30###1.47*###1.83*###2.20*###0.05###*###*

C2:C3###2.08###2.02###3.01*###3.37*###2.77*###0.08###NS###*

RESULTS AND DISCUSSION

Microbial gas and methane production: Effects of EOs on gas production and methane are presented in Table 2. Relative to the control, the addition of A. fragrans and E. plenissima EOs (at 250 and 500 ul/g DMI), M. alternifolia, M. ericifolia, M. teretifolia and L. agustifolia EOs (at 125, 250 and 500 ul/g DMI) and L. pettersoni EOs at all doses resulted in a decrease (P<0.05) in gas production. The gas production was affected linearly in E. plenissima, M. ericifolia, M. teretifolia and L. angustifolia Eos and quadratically in A. fragrans, L. pettersoni and M. alternifolia EOs. The addition of S. spicatum EO did not have a significant effect on total gas production at any of tested levels.

Methane production (ml/24h) was lower (P<0.05) in A. fragrans and E. plenissima (at 500 ul/g DMI), M. ericifolia and M. teretifolia (at 125, 250 and 500 ul/g DMI), L. angustifolia (at 250 and 500 ul/g DMI) and in M. alternifolia EOs treatments (at 125, 250 and 500 ul/g DMI, except at 125 ul/g DMI for ml/mg DMI), compared to control. When expressed as ml/mg IDMD, methane was reduced at all levels in S. spicatum and L. pettersoni EOs, except at 62.5ul/g DMI in L. pettersoni EO. With increasing dose, methane decreased linearly in all EOs treatments, except quadratically in L. pettersoni. The results from the current study confirmed that EOs from selected Australian plants may have a noticeable effect on rumen microbial fermentation and the effect occurs in a dose-dependent manner. When EO were supplied at high levels (250 ul/g DMI and above), the majority of treatments resulted in significant reduction of methane concentration, but also in other fermentation parameters (gas production, IDMD and total VFA).

The inhibitory effect on microbial fermentation confirmed potent antimicrobial activity of EO (Reuter et al., 1996; Davidson and Naidu, 2000) and agree with previous in vitro batch culture reports where high doses of EO have been tested (Busquet et al., 2005; Busquet et al., 2006; Castillejos et al., 2007; Durmic et al., 2014; Jahani-Azizabadi et al., 2011).

The study provided further evidence on the bioactivity of these EOs, either aligning or contrasting other studies. For example, Castillejos et al. (2008) reported that addition of L. angustifolia could not modify rumen microbial fermentation, while we have found a significant effect of this EOs, in particular on methane production and VFA concentration. Conversely, a recent study found that methane was decreased and propionate increased in vivo when buffalo diet was supplemented with of eucalyptus leaves (Thao et al., 2015) and our study had confirmed similar effects and further revealed that it is likely to be associated with extractable compounds (EOs) in this plant (Zrira et al., 2004). These findings may reflect some differences or similarities in methodologies and approaches, but also imply the need to look into specific plant secondary compounds.

The most potent EOs in terms of reducing methane was L. angustifolia (reduction up 96%) and Melaleuca species were highly effective in this respect (reduction ranged in methane from 31 to 94%) and these EOs were highly effective in reducing in vitro methane even at lower doses. The high antimicrobial potential of Melaleuca EOs is not surprising (Hammer et al., 2003; Durmic et al., 2014) and while species of Melaluca have been reported to have anti-methanogenic effect (Castillejos et al., 2008; Durmic et al., 2014) to our knowledge this is the first report that L. angustifolia may have that property. However, S. spicatum was the only EO causing a significant reduction in methane (up to 50%) at all doses that were coupled with relatively low effect on overall fermentation.

IDMD and VFA and ammonia-N concentrations: When compared to the control, the IDMD was significantly reduced (P<0.05) with as little as 125ul/g DMI in L. pettersoni, M. alternifolia and M. teretifolia treatments, 250 ul/g DMI in A. fragrans, E. plenissima, M. ericifolia and L. angustifolia, but this decrease for S. spicatum treatment was observed only at high inclusion levels (at 500 ul/g DMI). When compared to control, the SFE was higher (P<0.05) in L. pettersoni, M. alternifolia, M. ericifolia and M. teretifolia EO treatments at 500ul/g DMI; and in L. angustifolia EO treatment (at 250 and 500 ul/g DMI), while, S. spicatum, A. fragrans and E. plenissima EOs had no effect on SFE (Table 2).

The addition of A. fragrans and E. plenissima EOs resulted in a linear decrease in IDMD and quadratic increase in SFE, whereas L. pettersoni (highest at 62.5 ul/g IDMD and lowest at 500 ul/g DMI), M. alternifolia, M. ericifolia, M. teretifolia, S. spicatum and L. angustifolia (highest at 62.5 ul/g DMI and lowest at 500 ul/g DMI) resulted in a quadratic effect. The SFE changed linearly in M. ericifolia and M. teretifolia. Effects of EOs on VFA concentration and proportion of individual VFA are reported in Table 3. The addition of all EO and at all levels tested resulted in a reduction (P<0.05) in total VFA concentrations, but the reduction was the least in S. spicatum (up to 20%). Total VFA concentrations relative to control were reduced linearly in A. fragrans, E. plenissima and L. pettersoni EOs, and quadratically in the others.

When Australian EOs were tested at higher doses, there was a decrease in IDMD and total VFA concentrations, especially in genus Melaluca, and this agrees with our previous in vitro studies when these EO were used at high doses (Durmic et al., 2014). This also confirmed findings of other researchers where EOs inhibited IDMD, gas production and VFA production (Kumara et al., 2009b; Jahani-Azizabadi et al., 2011, Jahani-Azizabadi et al., 2014).

Relative to the control, the molar proportion of acetate was higher (P<0.05) in all EO excluding S. spicatum. This effect was quadratic in all EOs that caused the effect. The molar proportion of propionate generally decreased with increasing concentration in all treatments except S. spicatum EO. The quadratical effect was observed in M. ericifolia and L. angustifolia and linear in the other EO treatments. In contrast, the molar proportion of propionate linearly increased (P<0.05) in S. spicatum EO compared with control. The molar proportion of butyrate increased (P<0.05) in all EO treatments, except S. spicatum where there was no effect. The molar proportion of BCVFAs decreased (P<0.05) with selected doses of EOs and with increasing dose, the effect was quadratic in all treatments. With the increased dose, the proportion of acetate to propionate (C2:C3) decreased quadratically in S. spicatum EO (highest at 62.5ul/g DMI and lowest at 125 ul/g DMI). In other treatments, the values increased quadratically.

The VFA are a major source of metabolizable energy for ruminants (Van Soest, 1982) and it important that these are maintained when developing rumen manipulation strategies. We have now demonstrated that at lower doses, the bioactive effects on rumen fermentation in at least one EO may dissociate and became more specific, targeting only selected microbes and pathways. The most promising candidate appears to be S. spicatum EO, as it caused a significant reduction in methane and ammonia-N, with a lesser effect on VFA. Another interesting finding is that S. spicatum was the only EO that was actually promoting propionate (up to 22% compared to control). Propionate is considered as a valuable energy fuel for the animal, and hence this is a highly desirable effect and comparable to that of some in-feed antibiotics (Kobayashi, 2010).

Propionate is often implied as an alternative hydrogen acceptor in methane mitigation, and it is likely that these two were coupled in S. spicatum treatments, but further studies are needed to confirm this. In contrast with ionophores or other feed additives such as tannins that decrease ruminal methane production by affecting overall microbial fermentation (Chen and Wolin, 1979; Newbold et al., 1988; MOSS et al., 2000) this particular EO appears to have a more specific antimicrobial nature and apparently targeting methane-producing microbes and/or pathways. Other EOs were also effective in reducing methane and ammonia-N, but in these, a significant reduction in VFA production occurred, usually greater than 20%. Interestingly, other fermentation parameters, such as microbial gas production or dry matter disappearance were not impaired with any of the low-dose treatments.

While later is considered a nutritionally-favorable effect in ruminants, these findings also imply that we can explore the doses further and potentially find a dose that is also more gentle towards VFA-producing microbes.

The addition of all EO and at levels, except M. ericifolia at 62.5 ul/g DMI, resulted in a decrease (P<0.05) in ammonia N concentration compared with control (Table 3). This decrease effect with increasing dose was quadratical for all EO treatments. All EOs, except for one, significantly reduced ammonia N at the lowest doses tested. Microbes involved in N metabolism in the rumen seem to be very sensitive to Eos (McIntosh et al., 2003), and Australian EOs seem not to be an exception in that respect. While this effect may have been the result of a desirable decrease in rumen degradation of substrate protein (Van Nevel and Demeyer, 1988) it is also possible that the bacteria involved in deamination process were altered (Allison et al., 1962), as the effect was readily associated with a reduction in VFA.

Nevertheless, results of the present study suggested that selected Australian EOs may have a potential to reduce ammonia-N losses in the rumen, but further studies are needed to confirm the exact mechanism of this effect.

Conclusions: In conclusion, this study revealed an interesting candidate amongst Australian EOs, i.e. S. spicatum EO that may have the potential to improve rumen microbial fermentation characteristics of a high concentrate diet by reducing methane, ammonia-N and promoting propionate production. Future studies should focus on progressing these findings toward optimizing doses and confirming effect in vivo. It is also necessary to examine and identify specific plant secondary compounds in this and other EOs directly responsible for the effect, as well as the mechanisms by which they act. The findings from this and future studies may warrant further investigation of these Eos (especially S. spicatum EO) into possible applications in animal diets.

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