INTRODUCTION

Ionophore antibiotics have been successfully used in rumens for many years to reduce energy (methane emission) and protein (ammonia nitrogen emission) losses (1). While ionophore antibiotics have been widely used in conventional production systems for the control of rumen fermentation, the European Commission (EC) decided to phase out, and ultimately ban (January 1st 2006), the use of antibiotics in feed (70/524/EEC Directive and EC Regulation No:1831/2003)(2). Hence, alternative substances were started to be explored for antibiotics. One of these possible alternatives are the plant extracts, which are also called as essence oils. The term essential oil is misleading as they are not -as their name suggests- ‘essential’ for nutrition or metabolism nor are they oils in the sense of being glycerol based lipids. Thus, it would be more appropriate to use the terms “plant extract” or “essence oil” instead of the term “essential oil”.

Hart et al (3) have been reported that plant extracts decreased methane production with a subsequent decrease in rumen methanogens. Methane is one of the most important greenhouse gases that contribute to global warming. The population of ruminants is an important source of methane, contributing approximately 15% of the total atmosferic methane flux (4). Despite the greenhouse effect of methane at a life-threatening rate, no chemicals with long-term effects that would prevent methane production could have been developed so far. The only method used for reducing methane production is suppressing the number of microorganisms consuming H2 – which is used in methane synthesis- by using antibiotics or ionophores. Nevertheless, this method has resulted only a 25% reduction in methane production (5). Takashi (6) reported that methane production from ruminants in the developing countries may be high since their diets are often lack of critical nutrients for efficient microbial growth in the rumen. Plant extracts have been reported to decrease methane production with a subsequent decrease in rumen methanogens (3). One of the most important goals of stock breeders is manipulation of rumen microbial ecosystem to improve ruminant performance and to reduce methane emissions generated by ruminants. Methane is one of the most important greenhouse gases that contribute to global warming. Besides constituting a significant loss of energy for the animal, methane is a very potent greenhouse gas since it is given off atmosphere (7). Methane is 23 times more potent as a greenhouse gas than carbon dioxide (8). The global population of ruminants in 2014 was reported as 3.875 billion (9). Global ruminant population is significantly growing accompanied with significant increase in atmospheric methane concentrations. There is a strong correlation between increasing atmospheric methane concentrations and growing global ruminant population (10).

Feeding strategies are required to reduce methane production generated by ruminants. One of the alternative feed supplements are plant extracts. In this study, Oreganum vulgare, Thymus vulgaris, Zingiber officinale, Syszygium aromaticum essence oils were supplemented to TMR, concentrate and hay to explorepotential capacities of these essence oils in reducing methane productionat 2, 8 and 24 h in in vitro conditions. The aim of the study is to determine the effect of different plant essence oil supplementation to TMR, concentrate and hay on methanogenesis and at 2, 8 and 24 hours (h) using in vitro gas production technique in cattle.

MATERIALS AND METHODS

Animals and feeds. Three fistulated Holstein dairy cows were used for rumen fluid collection for application of in vitro gas production technique. Fistulated diary cows were fed according to the caring level with a diet containing alfalfa hay (%60) and concentrate (%40) twice a day. For each feed (TMR, Concentrate and Hay), ruminal fluid samples were collected twice day on different days. Four essence oils (T. vulgaris, O. vulgare, S. aromaticum, Z. officinale) were used as plant extracts. T. vulgaris, S. aromaticum, and Z. officinale essence oils were obtained from Ege Lokman San. Tic. Company in Manisa Province (Turkey) and O. vulgare essence oil from Aksu Gıda San. Tic. Company in Mersin Province (Turkey). All plant extracts were extracted with distilated water. For each extract, different doses were tested to determine harmful and usable doses. The samples were incubated in vitro rumen fluid in calibrated glass syringes. The 200 mg samples were weighed into calibrated glass syringes of 100 ml. The syringes were pre-warmed at 390C before the injection of 30 ml rumen fluid-buffer mixture into each syringe followed by incubarion in a water bath at 390C. Incubation was run for each regulation in 2, 8 and 24 h time periods. Total mixed ration (TMR), concentrate and hay were used as substrates. In the 2, 8 and 24 h of the experiment methane levels were measured.

The group with only rumen liquor was assigned as the negative control group and the group with rumen fluid and feed samples was assigned as the positive control group. The groups with rumen fluid, feed samples and thymol, oregano, zingiber and syzygium essence oils were the treatment groups. The compositions of TMR, concentrate and hay used in the experiment are presented respectively in table 1. The effects of Syzygim aromaticum, Zingiber officinale, Thymus vulgaris, Oreganum vulagare supplementation to TMR, concentrate and hay on methanogenesis production were assessed in the present experiment by incubation of in buffered rumen fluid using an in vitro gas production technique (HFT).

Methane measurements in in vitro gas production study. Methane measurement depending on experimental and in vitro gas production of study has been carried out at Animal Science Department of Hohenheim University, Germany. Methane concentration was measured in the gas produced during 2, 8 and 24h incubation by methane gasmitter® (AGM30/CH4; Firma PRONOVA Analysentechnik GmbH & Co; measurement range 0 - 5 Vol.%).

Statistical analysis. The descriptive statistics for the examined parameters were expressed in terms of average and standard errors. Factorial Analysis of Variance (Factorial ANOVA) was conducted to determine any differences between the means of supplements and feed types with respect to the examined parameters. Additionally, Repeated Measures ANOVA was performed to determine any differences with respect to feeds and durations (hours). Following analyses of variance, Tukey test was conducted to determine varying means (11). Statistical significance level was set at 5% and calculations were performed by SPSS Ver13 statistical software package.

RESULTS

Descriptive statistics and comparative results on methane production (%) for feed groups according to time period and supplement groups are presented in table 2. Changes in methane production (%) at 2, 8 and 24h with essence oil supplementation for TMR, concentrate and hay are depicted in figures 1, 2 and 3, respectively.

Methane production values significantly decreased in time in all treatment groups for all feeds (p<0.05). Methane production values for TMR negative control and TMR positive control groups at 24 h were respectively 12.34 and 10.45 while the lowest methane production value (1.65) was recorded in the TMR group with Syzygium 200 supplementation (p<0.05). For concentrate group, methane production values at 24 h significantly decreased with Thymol 25, Oregano 25, Zingiber 200 and Syzygium 200 supplementations compared to negative control and positive control groups (p<0.05).

The lowest methane production value (3.28) for concentrate group was recorded with Syzygium 200 supplementation at 24 h (p<0.05) as similarly observed for the TMR. For hay group, methane production values at 24 h significantly decreased with Thymol 25, Oregano 25, Zingiber 200 and Syzygium 200 supplementations compared to negative control and positive control groups. The lowest methane production value (1.98) for hay group was recorded with Syzygium 200 supplementation at 24 h as observed for TMR and concentrate substrates. Syzygium 200 supplementation resulted 84.21%, 69.49% and 80.34% reduction in methane production at 24 h in TMR, concentrate and hay groups compared to the positive control group, respectively. As indicated by the findings of the study, essence oils have significant effect on reduction of methane production and this effect becomes more apparent at higher doses of essence oil supplementations.

DISCUSSION

Effects of essence oils on methanogenesis. Methanogenic (methane producing) bacteria convert H2 and CO2 into methane. In the rumen, formation of methane is the major way of hydrogen elimination through the following reaction (12):

CO2 + 4H2→ CH4 + 2H2O

Methanogens reduce hydrogen and carbondioxide to produce methane and meet their energy requirement. The main methanogens in rumen microbial ecosystem are Methanobrevibacter ruminantium, Methanomicrobium mobile and Metha-nosarcina. Using high starch concentrate feed instead of roughage reduces methane production, energy loss and global pollution (13). The loss of feed energy as methane in mature ruminants depending on ration composition ranges between 2-12% gross feed energy intake (14). Hydrogen in rumen is produced as a result of fermentation of nutrients in anaerobic conditions. This hydrogen is used in the synthesis of volatile fatty acids and microbial proteins (15).

Higher amount of hydrogen is released during microbial production of acetic and butyric acid. Hydrogen is utilized by methanogenic bacteria and converted into methane with CO2 (16). In contrary to the process in acetic and butyric acid, ambient hydrogen ions are utilized during the production of propionic acid. Acetic and butyric acid can not be used in gluconeogenesis like propionic acid. Hence, conversion of volatile fatty acid profile in favour of propionate will ensure more efficient use of feed energy (16).

Protozoa produce much hydrogen as with gram positive bactaria in rumen and increases methane production by creating a symboitic relationship with methanogenic bacteria. Although protozoa have useful effects, decreasing of them in number affects productivity in ruminants in a positive way (16). It has been reported that extract of Myristica fragrans significantly reduces the number of protozoa. Decreasing of protozoa number, increasing of bacteria, fungi number, increasing of propionate production and decreasing of methanogenes improve performance in ruminants (15). Energy loss in rumen fermentation (methane) can limit production performance and can also lead to environmental pollution (1).

As seen in table 2, methane production rates significantly decreased in time in all treatment groups for all feeds (p<0.05). Methane production rates, depending on time period, type of essence oil and dose amount, ranged between 13.85 and 1.65 for TMR, between 13.67 and 3.28 for concentrate, and between 15.93 and 1.98 for hay. The lowest methane production rates at 24 h for TMR, concentrate and hay are recorded respectively as 1.65, 3.28 and 1.98 in the group with Syzygium 200 supplementation (p<0.05).

Syzygium 200 supplementation resulted 84.21%, 69.49% and 80.34% reduction in methane production at 24 h in TMR, concentrate and hay groups, respectively. Pen et al (17) reported that while Yucca schidigera extract, depending on dose, resulted upto 42% reduction in methane production, Quillaja saponaria extract had no effect on methane production. Bodas et al (18) demonstrated that Cardus pynocephalus, Populus tremula, Prunus avium, Quercus robur, Rheum nobile and Salix caprea supplementation to 50:40:10 alfalfa:hay:barley sheep rations significantly decreased methane production at 24 h of incubation in in vitro conditions. Bunglavan et al (19) reported that water-based extracts of Zingiber officinale rhizomes supplemented to 60:40 roughage:concentrate rations significantly decreased methane production. Ahari et al (10) observed that thyme supplementation (0.25 and 0.50 ml) to 100% roughage (alfalfa) rations significantly decreased methane production at 4, 12, 16, 24, 48, 72 and 96 h in in vito conditions. Canpolat et al (20) demonstrated that carvacrol supplementation significantly decreased methane production. Goel et al (14) added 1, 2 and 3 ml of Cinnamomum zeylanicum (Cinnamon) extracts to sorghum based rations including low, medium and high doses of cellulose. They observed that 3 ml of extract supplementation to the rations with high doses of cellulose resulted reduction in methane production at maximum level (45.37%).

Sallam et al (21) reported that essence oils extracted from Achillea santolina (75 µl), Artemisia judaica (75 µl) and Mentha microphylla (25, 50, 75 µl) significantly suppressed methane production at 24 h. Shabestari et al (7) demonstrated that clove methanol extract (CME) did not have any effect on reduction of methane production during early hours (2 and 4 h) of incubation, but CME significantly reduced methane production in later stages of incubation both in roughage and barley based rations. Patra and Yu (22) reported that increased doses of clove oil, eucalyptus oil, garlic oil, origanum oil and peppermint oil supplementations reduced methane production respectively by 34.3%, 17.6%, 42.3%, 87.0% and 25.7%. Santra et al (23) revealed that among 96 different extracts (water, methanol and ethanol) of 32 aromatic plants, 29 plant extracts significantly reduced methane production by 20% and over. Sirohi et al (24) showed that Myristica fragrans extract resulted upto 48% reduction in methane production compared to negative control group and they observed significant reduction in number of methanogens (p≤0.05). Tekippe et al (25) examined use of essence oils as natural products for suppression of methane production in cattle under in vitro conditions. According to their findings, lavender (Lavandula latifolia) essence oil reduced methane production by 20-30% and Oregano (Oreganum vulgare) essence oil reduced methane production by 31%.

Bhatta et al (8) reported that aromatic plants with tannin content such as Clerodendrum inerme, Gymnema sylvestre and Sapindus laurifolia have the potential to suppress in vitro methanogenesis. Chaudhary and Gupta (26) demonstrated that Melia azadiracta seed and S. aromaticum essence oils reduced methane production respectively by 27.7% and 29.34% compared to control. Schizium cumin essence oil was found to reduce methane production by 5.09%. The essence oils Delbergia sisso leave, Schizium cumin bark, Emblica officinalis fruit, Melia azadiracta seed, Allium cepa bulb, Psidium guajava, Azadiracta indica leave, Syzygium aromaticum were reported to reduce methane production by over 15%. Oskoueian et al (27) showed that flavonoids significantly reduce methane production in cattle without inducing any negative effect on rumen microbial fermentation. Kim et al (28) determined in in vitro study carried out in ruminants that medical plant exract of Arisaema ringens decreases methane production more than 43 percent for 24 hours. The findings of these researchers support the findings of the present study. As indicated by research findings, essence oils have significant effect on reduction of methane production and this effect becomes more apparent at higher doses of essence oil supplementations.

In contrary to the current study, Storlien et al (29) reported that supplementation of hop and steroidal saponin extracts in barley-based (47%) and barley silage-based (43%) rations did not reduce methane production under in vitroconditions. Moreover, combined use of these extracts was found to significantly increase methane production. Al-Hadeethi et al (30) reported that 20 and 30 g/kg DM doses levels of exrtracts of green tea (Camellia sinensis), turmeric (Curcuma longa) and garlic do not have a remerkable effect on formation of rumen methane. This case can be associated with the fact that avaliable additives do not create an important effect on rumen fermentation. Such differences in research findings can be attributed to the differences in ration composition, plant extracts used, extraction methods and dose amounts.

It can be concluded that essence oil supplementations to ruminant rations have noteworthy potential of reducing greenhouse gases that cause global warming. Reduced methane production resulted by essence oil supplementations can be attributed to:1) possible inhibition of methanogenic bacteria due to strong antimicrobial effects of essence oils against gram positive and gram negative bacteria; and 2) reduction of methane production by the shift of H2 protons to propionate production which will prevent reaction of CO2 with H2 and result in reduced methane production. Energy loss as methane not only worsens animal performance, but also increases environmental pollution.

One of the most important goals of stock breeders is manipulation of rumen microbial ecosystem to improve ruminant performance and to reduce methane emissions generated by ruminants. There are several methods to reduce methane emission in rumen. One of these methods is the use of essence oils extracted from aromatic plants. In the prsent study, among all plant extracts, Syzygium 200 ppm supplementation resulted the lowest methane production values. Also, Thymol 25, Oregano 25, Zingiber 200 and Syzygium 200 ppm supplementations significantly decreased methane production for TMR, concentrate and hay with increased durations.

In conclusion it has been concluded that increasing doses of essence oil highly decreases methane production in ruminants. Essence oils can be used as alternatives to chemical supplements (such as anti-biotic, ionophore) used for rumen regulators. Being natural, these substances are environmental friendly. Reduction of methane production is internationally considered as important for mitigating global warming, hence, this study produces highly valuable outputs in this regard.

Acknowledgements

The authors are grateful to Hohenheim University (Germany) for its financial and technical support.

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