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In vitro screening of the potential of numerous plant species as antimethanogenic feed additives for ruminants
Available online at www.sciencedirect.com
Animal Feed Science and Technology 145 (2008) 245–258
In vitro screening of the potential of numerous plant species as antimethanogenic feed additives for ruminants夽 R. Bodas a , S. L´opez a,∗ , M. Fern´andez a , R. Garc´ıa-Gonz´alez a , A.B. Rodr´ıguez a , R.J. Wallace b , J.S. Gonz´alez a a
Departamento de Producci´on Animal, Universidad de Le´on, E-24071 Le´on, Spain b Rowett Research Institute, Greenburn Road, Aberdeen AB21 9SB, Scotland, UK Accepted 30 April 2007
Abstract A screening experiment was conducted to evaluate the potential of 450 plant species as antimethanogenic additives in ruminant feeds. Effects of addition of these plants, which were incorporated to the fermentation substrate as a dry powder, on ruminal fermentation, fibre digestion and methane production were studied in vitro in batch cultures of mixed rumen microorganisms. Serum bottles containing 500 mg of substrate (500 g alfalfa hay/kg, 400 g grass hay/kg and 100 g barley grain), 50 mg of the plant additive tested and 50 ml of buffered rumen fluid (10 ml sheep rumen fluid + 40 ml culture medium) were incubated at 39 ◦ C for 24 h. After incubation, gas and methane production, pH and volatile fatty acid (VFA) concentration in the incubation medium and dry matter and neutral detergent fibre disappearance were recorded. Of the 450 samples tested, 35 decreased methane production by more than 15% versus those with corresponding control cultures and, with 6 of these plant additives, the depression in methane production was more than 25%, with no adverse effects on digestibility, total gas and VFA production. With these six samples, incubations were repeated to confirm their effects on methane production in vitro. Some candidates, in particular Rheum nobile Abbreviations: DM, dry matter; NDF, neutral-detergent fibre; VFA, volatile fatty acids This paper is part of a special issue entitled “Enzymes, Direct Fed Microbials and Plant Extracts in Ruminant Nutrition” guest edited by R. J. Wallace, D. Colombatto and P. H. Robinson. ∗ Corresponding author. Tel.: +34 987 291 291; fax: +34 987 291 311. E-mail address: [email protected] (S. L´opez). 夽
0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.04.015
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and Carduus pycnocephalus, consistently decreased methane production without adversely affecting other parameters of the rumen fermentation. © 2007 Elsevier B.V. All rights reserved. Keywords: Methane; Plant additive; Botanicals; Plant secondary compound; Rumen fermentation
1. Introduction Many plants produce secondary metabolites, a group of chemicals that are not involved in the primary biochemical processes of plant growth and reproduction but are important to protect the plants from insect predation or grazing by herbivores. Several thousand plant secondary metabolites have been reported (Kamra et al., 2006). Some of them, for example phenolic compounds, essential oils and sarsaponins (Chesson et al., 1982; Wallace et al., 1994; Kamel, 2001) have antimicrobial activity (Cowan, 1999). Accordingly, it has been suggested that secondary metabolites could be used as alternatives to antibiotics in ruminant feeds (Greathead, 2003), as they may modify ruminal fermentation thereby enhancing the efficiency of utilization of feed energy while decreasing methane emissions (Garcia-Gonzalez et al., 2006). Manipulating the rumen microbial ecosystem to enhance digestibility of fibrous feeds, and reduce methane emission and N excretion by ruminants to improve their performance are some of the most important goals for animal nutritionists. Methane is a greenhouse gas many times more potent than CO2 . Its concentration in the atmosphere has doubled over the last century and continues to increase (Gibbs et al., 1989; Crutzen, 1995). Ruminants are major contributors to biogenic methane formation, and it has been estimated that preventing methane formation from domesticated ruminants could contribute to stabilising atmospheric methane concentrations (Gibbs et al., 1989; Crutzen, 1995; Johnson and Johnson, 1995). However, researchers in this area have failed to find a chemical inhibitor of ruminal methane formation whose effectiveness persists for more than several days (Clapperton, 1977; Van Nevel and Demeyer, 1996). The only effective method in common use is ionophores which inhibit H2 formation by species that provide H2 to the methanogens (Nagaraja et al., 1997), and decrease methane emissions by up to 25% (Van Nevel and Demeyer, 1996). There is a need for feed additives with the potential to reduce ruminal methanogenesis. Plant extracts with high concentrations of secondary compounds are potential candidates to achieve this objective (Teferedegne, 2000), as they are available in large number, and on an industrial scale, while considered as natural products that are generally authorised for human consumption by national regulatory agencies. Some plant extracts have been examined for their effects on ruminal microbial fermentation, including sarsaponins (Ryan et al., 1997), phenolic compounds (Evans and Martin, 2000) and essential oils (Cardozo et al., 2004; Busquet et al., 2005). Some plant extracts having high content of flavonoids decrease methane production and stimulate microbial metabolism leading to an increase in degradability of crude protein (CP) and neutral detergent fibre (NDF), and in the efficiency and yield of microbial biomass production (Broudiscou et al., 2000, 2002).
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Tannins have also been shown to reduce ruminal methane production (Woodward et al., 2001). However, the effectiveness of plants and plant extracts with high levels of saponins, flavonoids and tannins varies depending on their source, type and concentration (Patra et al., 2006a). Screening of natural products is an early step in discovery and development of new compounds and feed additives. Research to identify new compounds or novel uses for existing natural products is expensive, but is essential to discover new active compounds given the high level of molecular diversity in these products (Borris, 1996). The aim of this study was to identify plants that decrease ruminal methanogenesis, as one of the objectives of the EU project ‘Rumen-Up’ (QLK5-CT-2001-00992) which was commissioned to explore and identify plant-based alternatives to antibiotic growth promoters for ruminants.
2. Materials and methods 2.1. Plant material Plant samples were derived from the ‘Rumen-Up’ collection of 450 samples of plant parts (mainly foliage) from species listed at http://www.rowett.ac.uk/rumen up/. After collection, plants were freeze-dried, ground to pass through a 1 mm sieve, and stored in tightly closed glass jars in a dry, dark and cool place. 2.2. In vitro batch cultures 2.2.1. Screening assay Effects of plant samples were examined in vitro using cultures of mixed ruminal microorganisms. Substrate used for the batch cultures was a mixture of alfalfa hay (500 g/kg), grass hay (400 g/kg) and barley grain (100 g/kg) which was ground to pass a 1 mm sieve. Contents of organic matter, NDF and CP were 921, 450 and 133 g/kg dry matter (DM), respectively. Three adult rumen cannulated sheep were used as donors for rumen fluid. Ewes were fed alfalfa hay once a day, and rumen fluid was collected before morning feeding, strained through four layers of cheesecloth and kept under flushing CO2 . Incubations were completed in 120 ml serum bottles in which 500 mg DM of substrate and 50 mg DM of each plant were weighed, and 10 ml strained rumen fluid and 40 ml medium (according to Menke and Steingass, 1988) were dispensed anaerobically. Bottles were sealed and placed in an incubator at 39 ◦ C, with three replicates/plant incubated, and blanks (i.e., no substrate no plant) and controls (i.e., with either 500 or 550 mg DM of substrate and without any plant additive) were used. In order to examine all plant samples, 16 incubation batches were completed. After 24 h of incubation, accumulated head space pressures and gas volumes were measured using a pressure transducer (Bailey & Mackey Ltd., Birmingham, UK) and by collecting the gas in a calibrated syringe as described by Theodorou et al. (1994). A sample of the gas collected was transferred to a 10 ml vacuum tube (Venoject® , Terumo Europe N.V., Leuven, Belgium) for methane analysis. Bottles were swirled in ice to stop fermentation,
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and then opened to measure pH in the incubation medium. A sample of supernatant (0.8 ml) was added to 0.5 ml of deproteinising solution (20 g metaphosphoric and 4 g crotonic acids/l of 0.5N HCl) for volatile fatty acids (VFA) analysis. All contents remaining in the bottle were filtered through sintered Pyrex® crucibles (pore number 1), with the residue dried (100 ◦ C for 48 h) and weighed. A sample of the dry residue was weighed in F57 ANKOM filter bags to be extracted with ND (100 ◦ C for 1 h) in an ANKOM fibre analyzer (ANKOM Technology, Macedon, NY, USA). 2.2.2. Confirmation assay Based on the analysis of the frequency distribution, the criteria used for selection of candidates for anti-methane effects were a decrease in methane production by more than 20%, and no adverse effects on DM digestibility, total gas and VFA production and consistent changes in the propionate to acetate ratio. After assessment of results, further in vitro experiments were completed to investigate the consistency of the response in the screening experiment. In vitro incubations were completed using the same experimental procedure described above to obtain 16 observations per plant additive. Thus, four vials for each plant sample selected and six vials for the control were incubated in each of four incubations. 2.3. Chemical analysis The NDF (expressed inclusive of residual ash) was determined according to Van Soest et al. (1991), using sodium sulphite, but not ␣-amylase, in boiling ND solution. Methane concentration was determined by gas chromatography (GC) using a Shimadzu GC-14 B GC (Shimadzu, Tokyo, Japan) equipped with a CarboxenTM 1000, 45/60, 2 m × 1/8 in. column (Supelco, St. Louis, MO, USA) and flame ionization detector (FID). Temperatures were 170, 200 and 200 ◦ C in column, injector and detector, respectively. Carrying gas (He) flow was adjusted to 24 ml/min. A sample of 0.5 ml of gas was manually injected using Pressure-Lok® syringes A-2 series of 500 l (Supelco). Methane content was calculated by external calibration, using a certified gases mixture with (l−1 ): 100 ml CH4 , 250 ml N2 , 50 ml H2 and 600 ml CO2 (Carburos Met´alicos, Valladolid, Spain). The VFA were determined by GC using a Perkin Elmer Autosystem XL GD (Perkin Elmer Inc., Waltham, MA, USA), equipped with a semi-capillary, TR-FFAP, 30 m × 0.53 mm × 1 m column (Supelco), FID and auto-sampler. Temperatures were 140, 250 and 250 ◦ C in the column, the injector and the detector, respectively. Carrier gas (He) flow was adjusted to 13 ml/min. Each sample was injected automatically with a split ratio of 1/3. Chromatograms were integrated using Star Chromatography Workstation 6.2 software (Varian Inc., Palo Alto, CA, USA). 2.4. Calculations and statistical analysis Parameters studied were total gas, methane and VFA production after 24 h of incubation (expressed in mmol/g DM incubated or mmol/g DM digested), methane proportion in gas as mmol methane/mol gas, propionate to acetate ratio, DM disappearance after 24 h of in vitro incubation (i.e., DM digestibility), pH and fermentation efficiency (i.e., mg DM digested/ml gas produced).
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Volumes of gas and methane (ml) were converted to mmol assuming 1 mol is equivalent to 25.4 l of gas under the atmospheric pressure and temperature conditions of gas measurement in our laboratory. Fermentation efficiency was calculated as described by Blummel et al. (1997). Comparisons for each parameter in the screening assay were established using Student’s t-test (Steel and Torrie, 1980) between the average value of the three bottles containing any plant species and the average value of the control bottles incubated in the same batch. Effects of each plant additive on any fermentation parameter (i.e., difference from the control) was the relative increase (positive) or decrease (negative) in relation to the average value of the controls for that incubation batch. In the confirmation assay, results were analysed using a one-way analysis of variance with plant sample as the treatment factor and incubation run as a blocking factor. Multiple comparisons among mean values used Duncan’s test (Steel and Torrie, 1980).
3. Results 3.1. Screening assay Plant samples were derived from the ‘Rumen-Up’ collection of 450 samples of plant species that were screened for their effects on ruminal fermentation, in particular methane production. The relative effect of each plant sample on methane production (i.e., mmol/g DM incubated) after 24 h incubation is in Fig. 1. Most points are spread within the interval between −10% to +10% of change relative to control values. Few plants caused an increase greater than 20% and not many points can be seen below −20% effect, indicating that not many plants had a large effect on methane production.
Fig. 1. Effect as percent of increase (positive) or decrease (negative) of each plant on methane production (mmol/g DM incubated).
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Fig. 2. Histogram of grouped frequency (a) and grouped cumulative frequency (b) of effects (increase or decrease of control values) on methane production (mmol/g dry matter incubated) on the horizontal axis (values grouped into class intervals) and frequency (number of plants) on the vertical axis.
Fig. 2a is the histogram of grouped frequency of all plant additives such that each bar represents the number of plants for each class interval that caused a range of change in methane production (increase or decrease in relation to the corresponding control value). The frequency distribution plot is “bell-shaped”, showing that data approached normal distribution. A similar distribution occurred for total gas and VFA production, propionate to acetate ratio and DM digestibility. Effects on methane production, expressed either per mol of gas produced or per unit DM digested, did not differ. When a histogram of grouped cumulative frequency distribution was plotted (Fig. 2b), 35 plant samples (8% of the total examined) decreased methane production (mmol/g DM incubated) by more than 15%. Only a few samples (12, or 3% of the total) reduced methane production by more than 20% (values lower than −20%) and had no effect on VFA production (Fig. 3), VFA molar proportions or DM digestibility (data not shown). Effects on DM digestibility and VFA
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Fig. 3. Relative changes (increase or decrease of control values) in methane production (mmol/g dry matter incubated) and total volatile fatty acids production (VFA, mmol/g dry matter incubated) after 24 h incubation in response to the addition of plant samples.
production, of those samples that reduced methane production by more than 20%, are in Fig. 4. Based on this information, plant species represented by the points enclosed within a circle in Fig. 4 were selected as the most promising candidates and their effects were validated in subsequent confirmation assays. These plant species were Carduus pycnocephalus L., Populus tremula L., Prunus avium (L.) L., Quercus robur L., Rheum nobile Hook. f. & Thoms. and Salix caprea L. 3.2. Confirmation assays Effects of selected candidates on methane and gas production, DM digestibility, fermentation efficiency, VFA production, propionate proportion, propionate to acetate ratio and pH after 24 h of incubation are in Table 1. All plants depressed methane produc-
Fig. 4. Relative changes (increase or decrease of control values) in total volatile fatty acids production (VFA, mmol/g DM incubated) and dry matter (DM) digestibility in response to the addition of plant samples for those plants which showed a decrease in methane production lower than 20%.
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Methane (mmol/g DM incubated) Methane (mmol/g DM digested) Methane (mmol/mol gas) DM digestibility Gas (mmol/g DM incubated) Fermentation efficiency (mg DM digested/ml gas) Propionate to acetate ratio VFA (mmol/g DM incubated) Propionate (mmol/mol VFA) pH
Control
Carduus
Populus
Prunus
Quercus
Rheum
Salix
S.E.D.
P
1.094 a 1.664 a 174 a 0.662 b 6.31 ab 4.14
0.999 b 1.500 b 163 bc 0.666 b 6.14 b 4.29
1.001 b 1.495 b 160 c 0.671 ab 6.23 ab 4.28
1.043 b 1.544 b 163 bc 0.673 ab 6.40 a 4.15
0.991 b 1.502 b 161 bc 0.664 b 6.13 b 4.29
0.941 c 1.388 c 151 d 0.678 a 6.22 b 4.32
1.041 b 1.566 b 168 ab 0.668 ab 6.20 b 4.26
0.0247 0.0390 3.3 0.0054 0.084 0.071
<0.001 <0.001 <0.001 0.036 0.034 0.130
0.295 b 4.95 b 206 b 6.68
0.295 b 4.99 b 206 b 6.60
0.290 bc 4.90 b 204 bc 6.64
0.283 cd 5.01 b 201 cd 6.65
0.278 d 4.92 b 198 d 6.60
0.316 a 5.16 a 216 a 6.59
0.288 bc 4.90 b 202 c 6.65
0.036 0.069 1.7 0.020
<0.001 <0.001 <0.001 0.466
Means with different letters differ P<0.05.
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Table 1 Effects of Carduus pycnocephalus, Populus tremula, Prunus avium, Quercus robur, Rheum nobile and Salix caprea on rumen fermentation parameters in vitro after 24 h incubation
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tion (P<0.001). R. nobile caused the greatest, and most consistent, decrease in methane production (P<0.001), with positive effects on DM digestibility (P<0.05), VFA and propionate production (P<0.001). No plant decreased gas production, substrate digestibility, fermentation efficiency or pH (P>0.05).
4. Discussion The ‘Rumen-Up’ collection, mainly foliage, included a wide range of plant species although it was restricted to plants that grew, or could be grown, in one of the countries of the European Union. Samples were considered as possible candidates to manipulate ruminal fermentation based on traditional uses, known phytochemical composition, agronomic properties, or a combination of these factors. It is known that the activity of plant secondary compounds depends on their chemical nature and concentration. ‘Rumen-Up’ plant samples were collected at several geographical locations, under different weather conditions and at different times of the year. All these factors could have influenced the activity and concentration of their phytochemical compounds (Wink, 1999). Processing and storage of samples can also effect persistence of volatile or labile compounds, and the concentration and activity of secondary metabolites would different among samples, suggesting that a different level of addition for each plant tested would be required to reach an adequate dose of active compounds and to observe effects on ruminal fermentation. However in initial screening, all plants were examined at the same level of addition. The goal of screening assays is to examine as many samples as possible in as short period of time as possible. This condition restricts the number of samples of each plant species that can be examined thereby increasing the probability of overlooking positive samples. However, a compromise between the number of samples to be examined and the number of replicates for each sample is necessary. Limitations, such as small experimental replication, possible false negatives and positives, are inherent to screening assays. However once some activity was detected in the initial screening, assuming that for a sample to be considered a positive, the magnitude of the effect had to be at least 20% in methane production versus the control, a second portion of the same material used in the primary screening was used in the confirmation assay (Borris, 1996). The concentration of secondary compounds may have been low in the plant samples, and so a considerable amount of material was added (100 mg/g of total DM in the flask). Most of it was organic matter, which can be degraded to some extent. Hence, the amount of DM incubated was the same in all cases, as 550 mg of substrate were added in the control cultures. Therefore, since almost 900 mg/g of the substrate was identical in all bottles, we assumed that variation due to different degradabilities of the plant samples would not be important. In spite of the limitations of the screening assay, six plant species were identified as potential antimethanogenic agents, being: C. pycnocephalus, P. tremula, Prunus avium, Q. robur, R. nobile and S. caprea. These plants were examined in a confirmation assay, and all depressed methane production to some degree. Our objective was just to identify plant species (from a large collection) with antimethanogenic activity when used as feed additives to manipulate ruminal fermentation in vitro, and investigation of mode of action
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of these plants was not in the scope of the study. Information in the literature relative to antimethanogenic effects of some plant species and plant secondary metabolites, and on the secondary metabolites in these species, may be of interest for interpretation of results, and to address future research to identify chemical components responsible for antimethanogenic activity. Several secondary compounds from some medicinal plants have been reported to decrease methane production from ruminal fermentation, including garlic, clove, fennel (Patra et al., 2006b), Acacia angustissima, Sesbania sesban (Zeleke et al., 2006), Sapindus spp., Populus tremuloides, Syzygium zromaticum, Psidium guayaba, Terminalia chebula (Kamra et al., 2006), horsetail and sage (Broudiscou et al., 2000). Identification of the phytochemical fractions involved in reduction of methane production is difficult (Scehovic, 1999), as tannins (Hess et al., 2003), saponins (Lila et al., 2003), flavonoids (Broudiscou and Lassalas, 2000), thymol, carvacrol or eugenol (Chiquette and Benchaar, 2005) have all been suggested to be responsible. Tannins reduced rumen methanogenesis in sheep and cattle (Woodward et al., 2001; Waghorn et al., 2002) probably due to both direct effects on activity of methanogenic Archaea and indirect effects on fibre digestion. Saponins have also decreased methanogenesis in vitro (Lila et al., 2003), although their mode of action remains unclear. Newbold and Rode (2006) suggest that the effect may be related to anti-protozoal activity. Flavonoids are known to interact with ruminal microorganisms, either positively or negatively, and degradation products of flavonoids can also modify microbial metabolism in the rumen (Broudiscou and Lassalas, 2000). Reductions in methane production have been related to adverse effects on substrate degradation (e.g., Beauchemin and McGinn, 2006). However, some plant species decrease methane production and, at the same time, stimulate microbial metabolism (Broudiscou et al., 2000, 2002). Lack of effects on substrate degradation in response to some plant extracts ´ that have reduced methane production has also been reported (Sliwi´ nski et al., 2002). In our experiment, candidate plants did not cause substantial modifications in any fermentation parameter, apart from methane production, suggesting that these materials did not affect substrate degradation, and were not toxic to ruminal microbes, at the doses used. Results suggest potential to use them to enhance ruminal fermentation with these compounds. Several secondary metabolites have been isolated from Carduus spp., including essential oils such as hexadecanoic acid (Esmaeili et al., 2005). Bain and Desrochers (1988) suggested flavonoids as some of its principal active compounds, and a flavone glycoside from C. pycnocephalus has been reported to have antimicrobial activity (El Lakany et al., 1997). R. nobile accumulates a substantial quantity of flavonoids and anthranoid derivatives (Iwashina, 2003; Iwashina et al., 2004). Pharmacological actions, including anti-bacterial and anti-viral properties, of different bioactive compounds of Rheum species have been investigated (Cyong et al., 1987; Babu et al., 2003). Several flavonoids have been isolated from Prunus avium (Vinciguerra et al., 2003), as well as N-containing secondary compounds, such as non-protein amino acids or cyanogenic glycosides (Harborne, 1991). Aspens (Populus spp.) and willows (Salix spp.) appear to contain structurally related secondary phenolics and isoprenoids, which are generally accepted to be their main defence against herbivores (Palo, 1984; Ikonen et al., 2002). Lignans, another kind of phenolic
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compounds with a wide range of biological activities, including fungal growth inhibition, are present in Salix spp. and Quercus spp. (Gottlieb and Yoshida, 1989). Glycosides, such as salicin, occur in relatively large amounts in many Populus (Lee et al., 1993) and Salix species (Rowell-Rahier, 1984; Tahvanainen et al., 1985; Lee et al., 1993; Jassbi, 2003). Tannins are also secondary metabolites present in S. caprea (Juntheikki, 1996), while Q. robur has been reported to contain tannins, flavonoids such as quercitin, terpenoids (Harborne, 1991; Iwashina, 2003), and triterpene saponins (Arramon et al., 2002). A patent was filed (patent no. WO/2005/099729) for protection of intellectual property of information obtained on effects of these plants on ruminal methanogenesis.
5. Conclusion The screening of a collection of 450 plants for effects on ruminal methane production showed six species (i.e., C. pycnocephalus, P. tremula, Prunus avium, Q. robur, R. nobile and S. caprea) with potential and, within them, R. nobile, C. pycnocephalus and P. tremula were particularly promising. Plant material from these species had no inhibitory effects on total gas and volatile fatty acids production and on dry matter digestibility in vitro. It seems important to elucidate the underlying mechanism of inhibition, to assess persistence of their antimethanogenic effects, to characterize the chemical nature of the active compounds responsible for such effects and to validate their usefulness and applicability under practical conditions.
Acknowledgements The authors are grateful to Dr. A. Calleja and Dr. R. Garc´ıa for their advice on botanical identification of plants. This work was part of the project RUMEN UP supported by the EU commission (QLK5-CT-2001-00992). All partners of the RUMEN UP consortium (Rowett Research Institute, UK; University of Hohenheim, Germany; University of Le´on, Spain; University of Reading, UK; Alltech, Ireland; and Crina, Switzerland) are gratefully acknowledged for cooperation, plant supply and valuable discussion. The Rowett Research Institute is funded by the Scottish Executive Environment and Rural Affairs Department. References Arramon, G., Saucier, C., Colombani, D., Glories, Y., 2002. Identification of triterpene saponins in Quercus robur L. and Q. petraea Liebl heartwood by LC-ESI/MS and NMR. Phytochem. Anal. 13, 305–310. Babu, K.S., Srinivas, P.V., Praveen, B., Kishore, K.H., Murty, U.S., Rao, J.M., 2003. Antimicrobial constituents from the rhizomes of Rheum emodi. Phytochemistry 62, 203–207. Bain, J.F., Desrochers, A.M., 1988. Flavonoids of Carduus-Nutans and Carduus-Acanthoides. Biochem. Syst. Ecol. 16, 265–268. Beauchemin, K.A., McGinn, S.M., 2006. Effects of various feed additives on the methane emissions from beef cattle. Int. Congr. Ser. 1293, 152–155. Blummel, M., Makkar, H.P.S., Becker, K., 1997. In vitro gas production: a technique revisited. J. Anim. Physiol. Anim. Nutr. 77, 24–34.
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Wink, M., 1999. Introduction: biochemistry, role and biotechnology of secondary metabolites. In: Wink, M. (Ed.), Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology. Sheffield Academic Press, Sheffield, UK, pp. 1–16. Woodward, S.L., Waghorn, G.C., Ulyatt, M.J., Lassey, K.R., 2001. Early indication that feeding lotus will reduce methane emission from ruminants. Proc. N.Z. Soc. Anim. Prod. 61, 23–26. Zeleke, A.B., Clement, C., Hess, H.D., Kreuzer, M., Soliva, C.R., 2006. Effect of foliage from multi-purpose trees and a leguminous crop residue on in vitro methanogenesis and ruminal N use. Int. Congr. Ser. 1293, 168–171.
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In vitro screening of the potential of numerous plant species as antimethanogenic feed additives for ruminants夽 R. Bodas a , S. L´opez a,∗ , M. Fern´andez a , R. Garc´ıa-Gonz´alez a , A.B. Rodr´ıguez a , R.J. Wallace b , J.S. Gonz´alez a a
Departamento de Producci´on Animal, Universidad de Le´on, E-24071 Le´on, Spain b Rowett Research Institute, Greenburn Road, Aberdeen AB21 9SB, Scotland, UK Accepted 30 April 2007
Abstract A screening experiment was conducted to evaluate the potential of 450 plant species as antimethanogenic additives in ruminant feeds. Effects of addition of these plants, which were incorporated to the fermentation substrate as a dry powder, on ruminal fermentation, fibre digestion and methane production were studied in vitro in batch cultures of mixed rumen microorganisms. Serum bottles containing 500 mg of substrate (500 g alfalfa hay/kg, 400 g grass hay/kg and 100 g barley grain), 50 mg of the plant additive tested and 50 ml of buffered rumen fluid (10 ml sheep rumen fluid + 40 ml culture medium) were incubated at 39 ◦ C for 24 h. After incubation, gas and methane production, pH and volatile fatty acid (VFA) concentration in the incubation medium and dry matter and neutral detergent fibre disappearance were recorded. Of the 450 samples tested, 35 decreased methane production by more than 15% versus those with corresponding control cultures and, with 6 of these plant additives, the depression in methane production was more than 25%, with no adverse effects on digestibility, total gas and VFA production. With these six samples, incubations were repeated to confirm their effects on methane production in vitro. Some candidates, in particular Rheum nobile Abbreviations: DM, dry matter; NDF, neutral-detergent fibre; VFA, volatile fatty acids This paper is part of a special issue entitled “Enzymes, Direct Fed Microbials and Plant Extracts in Ruminant Nutrition” guest edited by R. J. Wallace, D. Colombatto and P. H. Robinson. ∗ Corresponding author. Tel.: +34 987 291 291; fax: +34 987 291 311. E-mail address: [email protected] (S. L´opez). 夽
0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.04.015
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and Carduus pycnocephalus, consistently decreased methane production without adversely affecting other parameters of the rumen fermentation. © 2007 Elsevier B.V. All rights reserved. Keywords: Methane; Plant additive; Botanicals; Plant secondary compound; Rumen fermentation
1. Introduction Many plants produce secondary metabolites, a group of chemicals that are not involved in the primary biochemical processes of plant growth and reproduction but are important to protect the plants from insect predation or grazing by herbivores. Several thousand plant secondary metabolites have been reported (Kamra et al., 2006). Some of them, for example phenolic compounds, essential oils and sarsaponins (Chesson et al., 1982; Wallace et al., 1994; Kamel, 2001) have antimicrobial activity (Cowan, 1999). Accordingly, it has been suggested that secondary metabolites could be used as alternatives to antibiotics in ruminant feeds (Greathead, 2003), as they may modify ruminal fermentation thereby enhancing the efficiency of utilization of feed energy while decreasing methane emissions (Garcia-Gonzalez et al., 2006). Manipulating the rumen microbial ecosystem to enhance digestibility of fibrous feeds, and reduce methane emission and N excretion by ruminants to improve their performance are some of the most important goals for animal nutritionists. Methane is a greenhouse gas many times more potent than CO2 . Its concentration in the atmosphere has doubled over the last century and continues to increase (Gibbs et al., 1989; Crutzen, 1995). Ruminants are major contributors to biogenic methane formation, and it has been estimated that preventing methane formation from domesticated ruminants could contribute to stabilising atmospheric methane concentrations (Gibbs et al., 1989; Crutzen, 1995; Johnson and Johnson, 1995). However, researchers in this area have failed to find a chemical inhibitor of ruminal methane formation whose effectiveness persists for more than several days (Clapperton, 1977; Van Nevel and Demeyer, 1996). The only effective method in common use is ionophores which inhibit H2 formation by species that provide H2 to the methanogens (Nagaraja et al., 1997), and decrease methane emissions by up to 25% (Van Nevel and Demeyer, 1996). There is a need for feed additives with the potential to reduce ruminal methanogenesis. Plant extracts with high concentrations of secondary compounds are potential candidates to achieve this objective (Teferedegne, 2000), as they are available in large number, and on an industrial scale, while considered as natural products that are generally authorised for human consumption by national regulatory agencies. Some plant extracts have been examined for their effects on ruminal microbial fermentation, including sarsaponins (Ryan et al., 1997), phenolic compounds (Evans and Martin, 2000) and essential oils (Cardozo et al., 2004; Busquet et al., 2005). Some plant extracts having high content of flavonoids decrease methane production and stimulate microbial metabolism leading to an increase in degradability of crude protein (CP) and neutral detergent fibre (NDF), and in the efficiency and yield of microbial biomass production (Broudiscou et al., 2000, 2002).
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Tannins have also been shown to reduce ruminal methane production (Woodward et al., 2001). However, the effectiveness of plants and plant extracts with high levels of saponins, flavonoids and tannins varies depending on their source, type and concentration (Patra et al., 2006a). Screening of natural products is an early step in discovery and development of new compounds and feed additives. Research to identify new compounds or novel uses for existing natural products is expensive, but is essential to discover new active compounds given the high level of molecular diversity in these products (Borris, 1996). The aim of this study was to identify plants that decrease ruminal methanogenesis, as one of the objectives of the EU project ‘Rumen-Up’ (QLK5-CT-2001-00992) which was commissioned to explore and identify plant-based alternatives to antibiotic growth promoters for ruminants.
2. Materials and methods 2.1. Plant material Plant samples were derived from the ‘Rumen-Up’ collection of 450 samples of plant parts (mainly foliage) from species listed at http://www.rowett.ac.uk/rumen up/. After collection, plants were freeze-dried, ground to pass through a 1 mm sieve, and stored in tightly closed glass jars in a dry, dark and cool place. 2.2. In vitro batch cultures 2.2.1. Screening assay Effects of plant samples were examined in vitro using cultures of mixed ruminal microorganisms. Substrate used for the batch cultures was a mixture of alfalfa hay (500 g/kg), grass hay (400 g/kg) and barley grain (100 g/kg) which was ground to pass a 1 mm sieve. Contents of organic matter, NDF and CP were 921, 450 and 133 g/kg dry matter (DM), respectively. Three adult rumen cannulated sheep were used as donors for rumen fluid. Ewes were fed alfalfa hay once a day, and rumen fluid was collected before morning feeding, strained through four layers of cheesecloth and kept under flushing CO2 . Incubations were completed in 120 ml serum bottles in which 500 mg DM of substrate and 50 mg DM of each plant were weighed, and 10 ml strained rumen fluid and 40 ml medium (according to Menke and Steingass, 1988) were dispensed anaerobically. Bottles were sealed and placed in an incubator at 39 ◦ C, with three replicates/plant incubated, and blanks (i.e., no substrate no plant) and controls (i.e., with either 500 or 550 mg DM of substrate and without any plant additive) were used. In order to examine all plant samples, 16 incubation batches were completed. After 24 h of incubation, accumulated head space pressures and gas volumes were measured using a pressure transducer (Bailey & Mackey Ltd., Birmingham, UK) and by collecting the gas in a calibrated syringe as described by Theodorou et al. (1994). A sample of the gas collected was transferred to a 10 ml vacuum tube (Venoject® , Terumo Europe N.V., Leuven, Belgium) for methane analysis. Bottles were swirled in ice to stop fermentation,
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and then opened to measure pH in the incubation medium. A sample of supernatant (0.8 ml) was added to 0.5 ml of deproteinising solution (20 g metaphosphoric and 4 g crotonic acids/l of 0.5N HCl) for volatile fatty acids (VFA) analysis. All contents remaining in the bottle were filtered through sintered Pyrex® crucibles (pore number 1), with the residue dried (100 ◦ C for 48 h) and weighed. A sample of the dry residue was weighed in F57 ANKOM filter bags to be extracted with ND (100 ◦ C for 1 h) in an ANKOM fibre analyzer (ANKOM Technology, Macedon, NY, USA). 2.2.2. Confirmation assay Based on the analysis of the frequency distribution, the criteria used for selection of candidates for anti-methane effects were a decrease in methane production by more than 20%, and no adverse effects on DM digestibility, total gas and VFA production and consistent changes in the propionate to acetate ratio. After assessment of results, further in vitro experiments were completed to investigate the consistency of the response in the screening experiment. In vitro incubations were completed using the same experimental procedure described above to obtain 16 observations per plant additive. Thus, four vials for each plant sample selected and six vials for the control were incubated in each of four incubations. 2.3. Chemical analysis The NDF (expressed inclusive of residual ash) was determined according to Van Soest et al. (1991), using sodium sulphite, but not ␣-amylase, in boiling ND solution. Methane concentration was determined by gas chromatography (GC) using a Shimadzu GC-14 B GC (Shimadzu, Tokyo, Japan) equipped with a CarboxenTM 1000, 45/60, 2 m × 1/8 in. column (Supelco, St. Louis, MO, USA) and flame ionization detector (FID). Temperatures were 170, 200 and 200 ◦ C in column, injector and detector, respectively. Carrying gas (He) flow was adjusted to 24 ml/min. A sample of 0.5 ml of gas was manually injected using Pressure-Lok® syringes A-2 series of 500 l (Supelco). Methane content was calculated by external calibration, using a certified gases mixture with (l−1 ): 100 ml CH4 , 250 ml N2 , 50 ml H2 and 600 ml CO2 (Carburos Met´alicos, Valladolid, Spain). The VFA were determined by GC using a Perkin Elmer Autosystem XL GD (Perkin Elmer Inc., Waltham, MA, USA), equipped with a semi-capillary, TR-FFAP, 30 m × 0.53 mm × 1 m column (Supelco), FID and auto-sampler. Temperatures were 140, 250 and 250 ◦ C in the column, the injector and the detector, respectively. Carrier gas (He) flow was adjusted to 13 ml/min. Each sample was injected automatically with a split ratio of 1/3. Chromatograms were integrated using Star Chromatography Workstation 6.2 software (Varian Inc., Palo Alto, CA, USA). 2.4. Calculations and statistical analysis Parameters studied were total gas, methane and VFA production after 24 h of incubation (expressed in mmol/g DM incubated or mmol/g DM digested), methane proportion in gas as mmol methane/mol gas, propionate to acetate ratio, DM disappearance after 24 h of in vitro incubation (i.e., DM digestibility), pH and fermentation efficiency (i.e., mg DM digested/ml gas produced).
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Volumes of gas and methane (ml) were converted to mmol assuming 1 mol is equivalent to 25.4 l of gas under the atmospheric pressure and temperature conditions of gas measurement in our laboratory. Fermentation efficiency was calculated as described by Blummel et al. (1997). Comparisons for each parameter in the screening assay were established using Student’s t-test (Steel and Torrie, 1980) between the average value of the three bottles containing any plant species and the average value of the control bottles incubated in the same batch. Effects of each plant additive on any fermentation parameter (i.e., difference from the control) was the relative increase (positive) or decrease (negative) in relation to the average value of the controls for that incubation batch. In the confirmation assay, results were analysed using a one-way analysis of variance with plant sample as the treatment factor and incubation run as a blocking factor. Multiple comparisons among mean values used Duncan’s test (Steel and Torrie, 1980).
3. Results 3.1. Screening assay Plant samples were derived from the ‘Rumen-Up’ collection of 450 samples of plant species that were screened for their effects on ruminal fermentation, in particular methane production. The relative effect of each plant sample on methane production (i.e., mmol/g DM incubated) after 24 h incubation is in Fig. 1. Most points are spread within the interval between −10% to +10% of change relative to control values. Few plants caused an increase greater than 20% and not many points can be seen below −20% effect, indicating that not many plants had a large effect on methane production.
Fig. 1. Effect as percent of increase (positive) or decrease (negative) of each plant on methane production (mmol/g DM incubated).
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Fig. 2. Histogram of grouped frequency (a) and grouped cumulative frequency (b) of effects (increase or decrease of control values) on methane production (mmol/g dry matter incubated) on the horizontal axis (values grouped into class intervals) and frequency (number of plants) on the vertical axis.
Fig. 2a is the histogram of grouped frequency of all plant additives such that each bar represents the number of plants for each class interval that caused a range of change in methane production (increase or decrease in relation to the corresponding control value). The frequency distribution plot is “bell-shaped”, showing that data approached normal distribution. A similar distribution occurred for total gas and VFA production, propionate to acetate ratio and DM digestibility. Effects on methane production, expressed either per mol of gas produced or per unit DM digested, did not differ. When a histogram of grouped cumulative frequency distribution was plotted (Fig. 2b), 35 plant samples (8% of the total examined) decreased methane production (mmol/g DM incubated) by more than 15%. Only a few samples (12, or 3% of the total) reduced methane production by more than 20% (values lower than −20%) and had no effect on VFA production (Fig. 3), VFA molar proportions or DM digestibility (data not shown). Effects on DM digestibility and VFA
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Fig. 3. Relative changes (increase or decrease of control values) in methane production (mmol/g dry matter incubated) and total volatile fatty acids production (VFA, mmol/g dry matter incubated) after 24 h incubation in response to the addition of plant samples.
production, of those samples that reduced methane production by more than 20%, are in Fig. 4. Based on this information, plant species represented by the points enclosed within a circle in Fig. 4 were selected as the most promising candidates and their effects were validated in subsequent confirmation assays. These plant species were Carduus pycnocephalus L., Populus tremula L., Prunus avium (L.) L., Quercus robur L., Rheum nobile Hook. f. & Thoms. and Salix caprea L. 3.2. Confirmation assays Effects of selected candidates on methane and gas production, DM digestibility, fermentation efficiency, VFA production, propionate proportion, propionate to acetate ratio and pH after 24 h of incubation are in Table 1. All plants depressed methane produc-
Fig. 4. Relative changes (increase or decrease of control values) in total volatile fatty acids production (VFA, mmol/g DM incubated) and dry matter (DM) digestibility in response to the addition of plant samples for those plants which showed a decrease in methane production lower than 20%.
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Methane (mmol/g DM incubated) Methane (mmol/g DM digested) Methane (mmol/mol gas) DM digestibility Gas (mmol/g DM incubated) Fermentation efficiency (mg DM digested/ml gas) Propionate to acetate ratio VFA (mmol/g DM incubated) Propionate (mmol/mol VFA) pH
Control
Carduus
Populus
Prunus
Quercus
Rheum
Salix
S.E.D.
P
1.094 a 1.664 a 174 a 0.662 b 6.31 ab 4.14
0.999 b 1.500 b 163 bc 0.666 b 6.14 b 4.29
1.001 b 1.495 b 160 c 0.671 ab 6.23 ab 4.28
1.043 b 1.544 b 163 bc 0.673 ab 6.40 a 4.15
0.991 b 1.502 b 161 bc 0.664 b 6.13 b 4.29
0.941 c 1.388 c 151 d 0.678 a 6.22 b 4.32
1.041 b 1.566 b 168 ab 0.668 ab 6.20 b 4.26
0.0247 0.0390 3.3 0.0054 0.084 0.071
<0.001 <0.001 <0.001 0.036 0.034 0.130
0.295 b 4.95 b 206 b 6.68
0.295 b 4.99 b 206 b 6.60
0.290 bc 4.90 b 204 bc 6.64
0.283 cd 5.01 b 201 cd 6.65
0.278 d 4.92 b 198 d 6.60
0.316 a 5.16 a 216 a 6.59
0.288 bc 4.90 b 202 c 6.65
0.036 0.069 1.7 0.020
<0.001 <0.001 <0.001 0.466
Means with different letters differ P<0.05.
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Table 1 Effects of Carduus pycnocephalus, Populus tremula, Prunus avium, Quercus robur, Rheum nobile and Salix caprea on rumen fermentation parameters in vitro after 24 h incubation
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tion (P<0.001). R. nobile caused the greatest, and most consistent, decrease in methane production (P<0.001), with positive effects on DM digestibility (P<0.05), VFA and propionate production (P<0.001). No plant decreased gas production, substrate digestibility, fermentation efficiency or pH (P>0.05).
4. Discussion The ‘Rumen-Up’ collection, mainly foliage, included a wide range of plant species although it was restricted to plants that grew, or could be grown, in one of the countries of the European Union. Samples were considered as possible candidates to manipulate ruminal fermentation based on traditional uses, known phytochemical composition, agronomic properties, or a combination of these factors. It is known that the activity of plant secondary compounds depends on their chemical nature and concentration. ‘Rumen-Up’ plant samples were collected at several geographical locations, under different weather conditions and at different times of the year. All these factors could have influenced the activity and concentration of their phytochemical compounds (Wink, 1999). Processing and storage of samples can also effect persistence of volatile or labile compounds, and the concentration and activity of secondary metabolites would different among samples, suggesting that a different level of addition for each plant tested would be required to reach an adequate dose of active compounds and to observe effects on ruminal fermentation. However in initial screening, all plants were examined at the same level of addition. The goal of screening assays is to examine as many samples as possible in as short period of time as possible. This condition restricts the number of samples of each plant species that can be examined thereby increasing the probability of overlooking positive samples. However, a compromise between the number of samples to be examined and the number of replicates for each sample is necessary. Limitations, such as small experimental replication, possible false negatives and positives, are inherent to screening assays. However once some activity was detected in the initial screening, assuming that for a sample to be considered a positive, the magnitude of the effect had to be at least 20% in methane production versus the control, a second portion of the same material used in the primary screening was used in the confirmation assay (Borris, 1996). The concentration of secondary compounds may have been low in the plant samples, and so a considerable amount of material was added (100 mg/g of total DM in the flask). Most of it was organic matter, which can be degraded to some extent. Hence, the amount of DM incubated was the same in all cases, as 550 mg of substrate were added in the control cultures. Therefore, since almost 900 mg/g of the substrate was identical in all bottles, we assumed that variation due to different degradabilities of the plant samples would not be important. In spite of the limitations of the screening assay, six plant species were identified as potential antimethanogenic agents, being: C. pycnocephalus, P. tremula, Prunus avium, Q. robur, R. nobile and S. caprea. These plants were examined in a confirmation assay, and all depressed methane production to some degree. Our objective was just to identify plant species (from a large collection) with antimethanogenic activity when used as feed additives to manipulate ruminal fermentation in vitro, and investigation of mode of action
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of these plants was not in the scope of the study. Information in the literature relative to antimethanogenic effects of some plant species and plant secondary metabolites, and on the secondary metabolites in these species, may be of interest for interpretation of results, and to address future research to identify chemical components responsible for antimethanogenic activity. Several secondary compounds from some medicinal plants have been reported to decrease methane production from ruminal fermentation, including garlic, clove, fennel (Patra et al., 2006b), Acacia angustissima, Sesbania sesban (Zeleke et al., 2006), Sapindus spp., Populus tremuloides, Syzygium zromaticum, Psidium guayaba, Terminalia chebula (Kamra et al., 2006), horsetail and sage (Broudiscou et al., 2000). Identification of the phytochemical fractions involved in reduction of methane production is difficult (Scehovic, 1999), as tannins (Hess et al., 2003), saponins (Lila et al., 2003), flavonoids (Broudiscou and Lassalas, 2000), thymol, carvacrol or eugenol (Chiquette and Benchaar, 2005) have all been suggested to be responsible. Tannins reduced rumen methanogenesis in sheep and cattle (Woodward et al., 2001; Waghorn et al., 2002) probably due to both direct effects on activity of methanogenic Archaea and indirect effects on fibre digestion. Saponins have also decreased methanogenesis in vitro (Lila et al., 2003), although their mode of action remains unclear. Newbold and Rode (2006) suggest that the effect may be related to anti-protozoal activity. Flavonoids are known to interact with ruminal microorganisms, either positively or negatively, and degradation products of flavonoids can also modify microbial metabolism in the rumen (Broudiscou and Lassalas, 2000). Reductions in methane production have been related to adverse effects on substrate degradation (e.g., Beauchemin and McGinn, 2006). However, some plant species decrease methane production and, at the same time, stimulate microbial metabolism (Broudiscou et al., 2000, 2002). Lack of effects on substrate degradation in response to some plant extracts ´ that have reduced methane production has also been reported (Sliwi´ nski et al., 2002). In our experiment, candidate plants did not cause substantial modifications in any fermentation parameter, apart from methane production, suggesting that these materials did not affect substrate degradation, and were not toxic to ruminal microbes, at the doses used. Results suggest potential to use them to enhance ruminal fermentation with these compounds. Several secondary metabolites have been isolated from Carduus spp., including essential oils such as hexadecanoic acid (Esmaeili et al., 2005). Bain and Desrochers (1988) suggested flavonoids as some of its principal active compounds, and a flavone glycoside from C. pycnocephalus has been reported to have antimicrobial activity (El Lakany et al., 1997). R. nobile accumulates a substantial quantity of flavonoids and anthranoid derivatives (Iwashina, 2003; Iwashina et al., 2004). Pharmacological actions, including anti-bacterial and anti-viral properties, of different bioactive compounds of Rheum species have been investigated (Cyong et al., 1987; Babu et al., 2003). Several flavonoids have been isolated from Prunus avium (Vinciguerra et al., 2003), as well as N-containing secondary compounds, such as non-protein amino acids or cyanogenic glycosides (Harborne, 1991). Aspens (Populus spp.) and willows (Salix spp.) appear to contain structurally related secondary phenolics and isoprenoids, which are generally accepted to be their main defence against herbivores (Palo, 1984; Ikonen et al., 2002). Lignans, another kind of phenolic
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compounds with a wide range of biological activities, including fungal growth inhibition, are present in Salix spp. and Quercus spp. (Gottlieb and Yoshida, 1989). Glycosides, such as salicin, occur in relatively large amounts in many Populus (Lee et al., 1993) and Salix species (Rowell-Rahier, 1984; Tahvanainen et al., 1985; Lee et al., 1993; Jassbi, 2003). Tannins are also secondary metabolites present in S. caprea (Juntheikki, 1996), while Q. robur has been reported to contain tannins, flavonoids such as quercitin, terpenoids (Harborne, 1991; Iwashina, 2003), and triterpene saponins (Arramon et al., 2002). A patent was filed (patent no. WO/2005/099729) for protection of intellectual property of information obtained on effects of these plants on ruminal methanogenesis.
5. Conclusion The screening of a collection of 450 plants for effects on ruminal methane production showed six species (i.e., C. pycnocephalus, P. tremula, Prunus avium, Q. robur, R. nobile and S. caprea) with potential and, within them, R. nobile, C. pycnocephalus and P. tremula were particularly promising. Plant material from these species had no inhibitory effects on total gas and volatile fatty acids production and on dry matter digestibility in vitro. It seems important to elucidate the underlying mechanism of inhibition, to assess persistence of their antimethanogenic effects, to characterize the chemical nature of the active compounds responsible for such effects and to validate their usefulness and applicability under practical conditions.
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