Effect of supplementation of herbal extracts on methanogenesis in ruminants

S J Bunglavan*, C Valli, M Ramachandran** and V Balakrishnan

Department of Animal Nutrition, Madras Veterinary College, TANUVAS, Chennai-7
valliviba@yahoo.co.in

Abstract

Methanogenesis from ruminants is one of the major cause of global warming and methanogenesis reduces the   efficiency of nutrient utilization hence manipulation of rumen microbial ecosystem for reducing methane emission is very vital. Hence a study was designed, to evaluate the anti methanogenic activity of herbs / herbal extracts in ruminants. The herbs tested were Acacia concinna pods, Emblica officinalis seeds, Allium sativum bulbs, Zingiber officinale rhizomes, Ferula assafoetida resin, Psidium guajava leaves, Terminalia chebula seeds and Azadirachta indica seed kernels. In vitro gas production studies were carried out using Hohenheim in vitro gas production test to rank the herbs / herbal extracts / residues according to their capacity in lowering the per cent methane production.

Acacia concinna pods methanol extract ranked one (13.3 %) followed by Acacia concinna pods methanol residue (13.34 %), Allium sativum bulbs water residue (15 %), Zingiber officinale rhizomes water residue (15.02 %) and Psidium guajava leaves methanol residue (15.1 %). In a second in vitro trial the first five ranked treatments were studied at graded inclusion levels of 30, 40, 50, 60 and 70 mg per 500 mg  substrate . Acacia concinna pods methanol extract, Acacia concinna pods methanol residue, Allium sativum bulbs water residue, Zingiber officinale rhizome water residue and Psidium guajava leaves methanol residue inclusion at 50, 30, 30, 50 and 50 mg respectively exhibited maximum inhibition of methanogenesis. These selected herbs in their selected levels were validated through an in vitro rumen fermentation experiment (RUSITEC) using complete feed having roughage concentrate ratio of 60: 40. All the selected herbal extracts / residues studied in this experiment significantly (P<0.05) lowered methane production. Maximum reduction was brought about by Acacia concinna pods methanol residue. The per cent in vitro dry matter degradability (IVDMD) remained unaltered.

This study therefore establishes the scope of use of plant extracts in ruminant rations to reduce methane production without adversely affecting IVDMD.

Keywords: Antimethanogenic activity, Hohenheim in vitro gas production test, IVDMD, plant extracts, rumen fermentation, RUSITEC

Introduction

Methane emission from ruminants reduces the efficiency of nutrient utilization.  Manipulation of rumen microbial ecosystem for reducing methane emission by ruminants to improve their performance is one of the most important goals for animal nutritionists. Reduction in methane emission from ruminants enhances the efficiency of nutrient utilization and augments productivity and also reduces methane impact on global warming. There are several methods to reduce methane emission from the rumen. These methods include processing of feeds, altering the type of ration, supplementation of unsaturated fatty acids (Johnson and Johnson  1995), defaunation (Van Nevel and Demeyer 1996), organic acids (Asanuma et al 1999), halogenated methane analogues (Haque 2001), ionophores (Kobayashi  et al 1992), microbial feed additives (Mutsvangwa et al 1992), non ionic surfactants (Lee and Ha  2003), sulphates (Kamra et al 2004) and herbal products (Patra et al 2006). Herbal preparations have been used for centuries for various purposes  because of their antimicrobial properties (Davidson and Naidu 2000) and because most of them are categorized under GRAS (Generally Recognized as Safe) for human consumption (FDA 2004).The use of herbal preparations appears as one of the most natural alternatives to the antibiotic use in animal nutrition. Plant secondary metabolites, have been shown to modulate ruminal fermentation to improve nutrient utilization in ruminants (Hristov et al 1999).These compounds  possess antimicrobial activity that is highly specific, which raises their possibility to target methanogens.

Keeping these in view a study was designed, to identify the potent methane reducing herbs or herbal extracts and determine their optimum level of inclusion in dairy cattle ration to reduce the methane emission from the in vitro rumen conditions..

Materials and methods 

This study was carried out using eight different plant materials hereafter to be referred to as herbs. The herbs collected for testing were Acacia concinna pods(shikakai), Emblica officinalis seeds(amla), Allium sativum bulbs(garlic), Zingiber officinale rhizomes(ginger), Ferula assafoetida resin(assafoetida), Psidium guajava leaves(guava), Terminalia chebula(kadukka) seeds and Azadirachta indica (neem) seed kernels. Three samples of each of the herbs were collected, dried in a hot air oven at a temperature of 55-60˚C to constant weight and ground to pass through 1mm sieve. Water soluble (water extract), water insoluble (water residue), methanol soluble (methanol extract) and methanol insoluble (methanol residue) fractions of these herbs were prepared as per the procedure adopted by Patra et al (2006) Twenty grams of each herb was mixed with 100 ml of methanol (98%) / 100 ml water in conical flasks and were stoppered and incubated at 39˚C on a rotary shaker for 24 hours and filtered through Whatman 1 filter paper.

Filtrates (methanol/water extracts) and residues (methanol/ water residues) were collected separately in pre-weighed glass crucibles and dried in hot air oven at a temperature of 55˚C to constant weight. The dried methanol/water  extracts and residues were weighed and stored in air tight containers for further use and tested for their potency to reduce methanogenesis. The quantity of extract / residue that was obtained was quantified and  was expressed in terms of per centage  yield of water/methanol extract for further calculation of the amount of water / methanol extract and water / methanol residue incubated in in vitro gas production studies.     .

In vitro gas production studies

The potency of herbs / herbal extracts to reduce methanogenesis was tested by using Hohenheim in vitro gas production studies as per the procedure of Menke and Steingass (1988). Two experiments were carried out in this direction. Experiment I was primarily a screening test that aimed to identify the herb / extracts / residues having greater ability to reduce methanogenesis and experiment II was carried out to identify the minimum inclusion level of the selected herbs / extracts / residues that significantly reduced methanogenesis. Both the experiments were carried out in triplicate with dried Hybrid Napier (Pennisetum typhoides x Pennisetum purpureum) CO3 variety grass used as substrate. The total quantity of substrate taken for in vitro gas production study was 500 mg as described by Blummel and Becker (1997).

The quantity of testing materials included in the screening test was 50mg of herb or weight of extract / residue of water / methanol that would have been generated from 50 mg of respective herb.

 The weight of water / methanol extract and residue was calculated as follows.

Weight of water/methanol extract incubated =

Weight  of water/methanol residue incubated = 50 mg - Weight  of water/methanol extract incubated

The trial included a blank, where only strained rumen liquor and media buffer solution (Menke and Steingass 1988) was taken and a control, where substrate, rumen liquor and media buffer solution was taken. All the 40 treatments viz. water / methanol extract, water / methanol residues of the herbs and herbs as such of all the herbs selected for the study with blank and control were subjected to gas production studies. Net gas volume was calculated by subtracting the recorded gas volume from blank as advocated by Menke and Steingass (1988). The total gas was partitioned as carbon dioxide and methane as per the procedure of Fievez et al (2005). Gas samples were drawn from the total gas produced. With the use of 10 M NaOH solution the methane and carbon dioxide from total gas was fractioned. Two ml of gas sample was drawn from the total gas produced  with the help of syringe and needle. It was then injected through the hub fixed to the nozzle of another syringe containing 2 ml of 10 M NaOH solution. This displacement of 10 M NaOH indicates the volume of methane, as carbon dioxide is soluble in the solution (Fievez et al 2005). From the proportion of methane to carbon dioxide in total gas, the percentage of methane in the total gas was calculated. The percentage of methane in the total gas was calculated from the proportion of methane to carbon dioxide in total gas.  In order to carry out a meaningful interpretation, ranking of the herbs / herbal extracts / residues was done based on their capacity in reducing per cent methane production from the total gas production.

Based on the results of experiment I that is on the  capacity of  reducing  percent methane production from the total gas producton experiment II was carried out. Since the level of herb and extract / residue included in screening test was 50 mg or its equivalent extract / residue, the promising herbs / extracts / residues were included at five levels viz. 30, 40, 50, 60 and 70 mg per 500 mg substrate  in experiment II and triplicate measurements were made. All the procedures followed were same as that described in experiment I.

Validation studies using RUSITEC

Based on the results of experiment II the identified levels of the selected five herbal extracts / residues were included in complete feed for medium yielding dairy cattle to study the rumen fermentation pattern using RUSITEC (Czerkawski and Breckenridge 1977). The complete feed had a crude protein of 14 per cent and TDN of 62 per cent.

The ingredient composition and nutritive value of the complete feed is given in Table 1.

Altogether there were five complete feeds supplemented with respective herbal extracts / residues and one complete feed without any herbal extracts / residues which acted as control. The parameters measured were total gas, methane and carbon dioxide production, pH and in vitro dry matter degradability. Two RUSITEC each equipped with eight fermenters with a volume of 1000 ml for each fermenter was used.  Two fermenters were allotted for each treatment and two were allotted for the control. Inoculum (rumen cud and liquor) was collected from three cows immediately on slaughter from the slaughter house. The pooled contents were transferred to a thermos cud transport container with flushing of carbon dioxide and brought to the laboratory.   

Ruminal fluid was filtered through 4 layers of muslin cloth and stored in pre‑warmed thermos container at 39^°C till its use. In vitro semi continuous culture in RUSITEC was initiated within half an hour from collection.  To begin the experiment the fermenters were filled with 500 ml of strained rumen liquor and 200 ml of artificial saliva (Czerkawski and Breckenridge 1977).  Eighty grams of solid digesta (solid inoculum) and 10 g of test feed (DM basis) were taken in nylon bags and placed in feed container. The pore size of nylon bag was 100 m as suggested by Carro et al (1995).  The solid inoculum bag was replaced by a feed bag with complete feed after 24 hours. During the change of bags the fermenters were flushed with CO₂ to maintain anaerobic condition. Artificial saliva was infused continuously into the fermenters at a flow rate of 500 ml/day.  Five days of adaptation was followed by collection period. The feed was incubated for period of 24 hours in the fermenters of RUSITEC.

At  the end of  incubation period, the total gas produced in each fermenter was collected in gas bags and quantified by displacement of water. Gas samples drawn from the total gas produced was fractioned for methane and carbon dioxide (Fievez et al 2005). Gas samples were drawn from the total gas produced. With the use of 10 M NaOH solution the methane and carbon dioxide from total gas was fractioned. Two ml of gas sample was taken from the gas bag with the help of syringe and needle. It was then injected through the hub fixed to the nozzle of another syringe containing 2 ml of 10 M NaOH solution. This displacement of 10 M NaOH indicates the volume of methane, as carbon dioxide is soluble in the solution (Fievez et al 2005). From the proportion of methane to carbon dioxide in total gas, the percentage of methane in the total gas was calculated. From the proportion of methane to carbon dioxide in total gas, the percentage of methane in the total gas was calculated. Rumen fluid samples were collected with the help of a syringe that was inserted through a three-way tap in the fermenter and its pH was recorded immediately. At the end of incubation period the bags were removed, washed and oven dried at 60^°C to constant weight to determine the in vitro dry matter degradability.

All the experiments adopted a completely randomized design (CRD).The percentage of in vitro   methane production,in vitro dry matter degradability and pH data were statistically  analysed using one way  analysis of variance (One Way - ANOVA)  to compare the means as per the procedure of statistical analysis system (SAS/ SPSS 1999 version 10.0 for windows). When significant difference (P<0.05) were detected the multiple range test was used to separate the mean value.

Results and discussion 

Among the 41 treatments including control, Acacia concinna pods methanol extract recorded the least per cent methane production (13.3 %) and ranked 1 followed by Acacia concinna pods methanol residue (13.34 %), Allium sativum bulbs water residue (15 %), ginger rhizomes water residue (15.0 %) and Psidium guajava leaves methanol residue (15.1 %) at the ranking of 2, 3, 4 and 5 respectively (Table 2).

Patra et al (2006) also had reported that methanol extract of seed pulp of Terminalia chebula and methanol, ethanol and water extracts of bulbs of Allium sativum reduced methane production significantly in rumen liquor of buffaloes

Total gas production under in vitro conditions on its own does not reflect the extent of efficient utilization of the substrate. High total gas production indicates that a majority of substrate has gone into gas production thereby reducing the production of VFA and other beneficial end products. Similarly low gas production can be due to inadequate fermentation of substrate or the fact that fermentation has taken place in the favour of VFA rather than gas (Czerkawski and Breckenridge 1977). Thus assessing the total gas production alone will not truly indicate the potency of the herb or its extract or its residue in bringing about a change in fermentation.

Though the carbon dioxide production was also measured, its data alone will not help us in elucidating a meaningful interpretation for ranking the herbs / extracts / residues for reduction in methane production.

While using reduction in methane production in terms of its volume for identifying the herb / extract / residue, the fact that a low total gas production will also reveal lower methane has to be considered. Therefore to arrive at a meaningful interpretation methane production was calculated as a percentage to select the herbs / herbal extracts / residues.

A significant finding in this study was that the active principles involved in reducing methane production were not water soluble as evident from the top five ranks held by methanol extract / methanol residue / water residue in their capacity in reducing per cent methane production.

Though Acacia concinna pods methanol extract and Acacia concinna pods methanol residue were ranked 1 and 2 respectively in their capacity in reducing methanogenesis, Acacia concinna herb as such ranked 10 in reducing methanogenesis indicating that the active principle responsible for reducing methanogenesis in Acacia concinna is a compound partially solubilised by methanol and / or activated in the presence of methanol (Table 3).

Pods of Acacia concinna have been found to contain triterpenoids, steroids, saponins, acacidol, acacic acid and sonumin (Chevallier 1996) and methane inhibiting effect of methanol extract / residue of Acacia concinna could be attributed to saponin. Saponins from different sources have been found to be toxic to protozoa and have been identified as possible defaunating agents (Newbold et al 1997). The antiprotozoal effects of saponins is due to their capacity to form irreversible complexes with the cholesterol in protozoal cell membrane causing break down in the membrane leading to cell lysis and death (Francis et al 2002).

Allium sativum bulbs water residue was capable of significantly reducing methane production and was ranked 3. Allicin is an active principle present in crushed and processed Allium sativum and preliminary studies using real time polymerase chain reaction (PCR) suggested that this allicin had a direct effect on reducing the number of methanogens with no effect on the total bacterial population in fermentor of RUSITEC (Hart et al 2006). The antimethanogenic activity of Allium sativum and its components was the result of direct inhibition of Archaea micro organisms in the rumen (Busquet et al 2005). The stability of cell membrane of Archaea micro organisms depends on glycerol linked long chain isoprenoid alcohols (De Rosa et al 1986) and the synthesis of these isoprenoid units in methanogenic Archaea is catalysed by HMG CoA (Hydroxy methyl glutaryl CoA) reductase. Organo sulphur compounds in Allium sativum are strong inhibitors of HMG CoA reductase and may be possibly inhibiting methanogenesis.

Ranked 4 in the ability to reduce methane production was Zingiber officinale rhizomes water residue. Zingiber officinale rhizomes are rich in camphene (14.1%), β-bisabolene (22.1%) and ar-curcumene (14.5%) (Chao et al 2000) that could have contributed to its methane reducing capacity.

Psidium guajava leaves methanol residue ranked 5 in inhibiting methane production. The inhibitory action could have occurred due to presence of phytochemical constituents viz. alkaloids, saponins, steroidal rings and deoxy sugars. Further Psidium guajava leaves extract have shown antimicrobial activities (Elekwa et al 2009).

The first five ranked treatments were selected for experiment II. The in vitro total gas (ml), carbon dioxide (ml) and methane production (ml and per cent) on incubation of these selected herbal extracts / residues is presented in Table 4.

Significantly (P<0.05) lowest total gas and carbon dioxide production for Acacia concinna pods methanol extract was observed at 70 mg inclusion level. Whereas, methane volumes and per cent methane production was significantly (P<0.05) lowest when it was included at 50 mg.

Acacia concinna pods methanol residue when included at 60 mg revealed significantly (P<0.05) lowest total gas, carbon dioxide, and methane and per cent methane production. However the reduction in methane percentage was comparable when included at 30 mg itself. Therefore the lower level itself was considered for further studies.

In the case of Allium sativum bulbs water residue 70 mg inclusion revealed significantly (P<0.05) lowest total gas, carbon dioxide and methane production. However the reduction in methane percentage was comparable when included at 30 mg itself. Therefore the lower level itself was considered for further studies.

Zingiber officinale rhizomes water residue when included at 60 mg revealed significantly (P<0.05) lowest total gas, carbon dioxide, and methane production. Whereas, per cent methane production was significantly (P<0.05) lowest when it was included at 50 mg.

Total gas production was significantly (P<0.05) lower at both 50 and 60 mg inclusions for Psidium guajava leaves methanol residue. All inclusions revealed no significant difference from control with respect to carbon dioxide production. However the reduction in methane percentage was comparable when included at 50, 60 and 70 mg. Therefore the lower level (50 mg) was considered for further studies.

Pen et al (2006) also had reported that the rate and extent of methane production were reduced (P<0.001) by Yucca schidigera extract addition in a dose dependent manner led to methane reduction upto 42 per cent. Garcia et al (2008) also had stated that Frangula alnus bark and Rheum officianale root resulted in dose dependent linear decrease in methane production.

Though substantial reports exist on the effect of plant based extracts on rumen fermentation only a few of them have reported dose response studies. Differences in the activity of the secondary compounds of the plants are not only due to their different chemical nature, but also to different factors that can influence the concentration and activity of secondary metabolites within a given plant species. Some of the influential factors are origin, botanical variety, conditions of cultivation and harvesting, climatic and atmospheric factors, topographic factors, phenological state of the plant, part of the plant used, processing and handling of the product (Wenk 2003). Thus, to reach an adequate dose of active compounds, a different level of addition for each plant additive tested might be required to observe an effect. High doses of plant extracts are not safe to be recommended in spite of their antimethanogenic activity because they may be detrimental in rumen microbial fermentation leading to reduced TVFA concentration. Ammonia nitrogen concentration could also decrease (Busquet et al 2006). Therefore identifying the minimum level of extract / residue becomes all the more vital.     

Thus from the study it was inferred that for Acacia concinna pods methanol extract inclusion at 50 mg, Acacia concinna pods methanol residue inclusion at 30 mg, Allium sativum bulbs water residue inclusion at 30 mg, Zingiber officinale rhizome water residue inclusion at 50 mg and for Psidium guajava leaves methanol residue inclusion at 50 mg exhibited maximum inhibition of methanogenesis. These treatments were further validated using RUSITEC.

Validation studies using RUSITEC

The results of the fermentation parameters studied in RUSITEC are presented in Table 5.

All the herbal extracts / residues significantly (P<0.05) lowered per cent methane production compared to that of control. Acacia concinna pods methanol residue was able to reduce methane production to a maximum (16.7 ± 1.05 %). Rumen pH remained unaltered compared to control in all the herbal extracts / residues except for ginger rhizomes water residues wherein the pH (6.72 ± 0.04) was significantly (P<0.05) lower than that of control. The per cent IVDMD for all the herbal extracts / residues did not reveal any significant change compared to control. Concurring with the findings of this study Wang et al (1998) observed that supplying Yucca schidigera extract (0-5 mg/ml) in buffer in RUSITEC did not affect IVDMD, gas production or total VFA concentration. Reductions in methane production have been mostly related to adverse effect on substrate degradation (Beauchemin and Mc Ginn 2006). However, Broudiscou et al (2002) reported that some plant species decrease methane production and at the same time stimulate micro organisms. Sliwinski et al (2002) also reported lack of effect on substrate degradation in response to plant extracts that reduced methane production. Bodas et al (2008) also reported that the plants did not cause any substantial modification in any fermentation parameter apart from methane production. Cardozo et al (2004) on evaluating Cinnamon cassia essential oil on microbial fermentation reported no effect on rumen fermentation pattern. Significantly reduced residual protozoal population but no effects on total bacterial numbers have been reported on feeding Yucca schidigera extract thus having antiprotozoal effect for ruminants (Pen et al 2007) but being less toxic to other ruminal microbial populations.

Conclusions 

• Active principles of herbs having capacity to reduce methane production differ in their property of solubility. The active principles involved in reducing methane production are not water soluble. Acacia concinna pods methanol extract ranked 1 Acacia concinna pods methanol residue ranked 2, Allium sativum bulbs water residue ranked 3, Zingiber officinale rhizomes water residue ranked 4 and Psidium guajava leaves methanol residue ranked 5 in their capacity to inhibit methanogenesis. Herbal extracts / residues at inclusion between 30 -50 mg reduced per cent methane production significantly (P<0.05) lower than control without adversely affecting the ruminal pH and in vitro dry matter degradability (IVDMD).

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Received 20 July 2010; Accepted 19 September 2010; Published 1 November 2010

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