Promising Plant Secondary Metabolites for Enteric ...

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Promising Plant Secondary Metabolites for Enteric Methane Mitigation and Rumen Modulation S.K. SIROHI, N. SINGH and A.K. PUNIYA

Methanogenesis occurring in the rumen is a significant cause of global warming. Methane generated during this process is an important greenhouse gas (GHG) which contributes 4-9% to the total greenhouse effect (Patra, 2012). Its concentrations have more than doubled over the last 150 yrs. Enteric fermentation accounts for 89.2% of the total GHG emissions, followed by manure-originated methane (9.49%; Patra, 2012a). Methane remains in the atmosphere for only 10 yrs, but trap 21 times more heat than CO2. Therefore, it is considered a highly potent GHG and requires immediate interventions to restrict its alarming emissions from livestock. Livestock populations in India witnessed increase from 292.8 million in 1951 to 529.7 million in 2007 (ICAR, 2012). GHG emissions from livestock production are likely to increase unless suitable abatement measures are implemented. Enteric methane emissions are estimated to be 10.65 million tonne (mt) from Indian livestock (Chhabra et al., 2009). The contributions of dairy cattle, non-dairy cattle, buffaloes, goats, sheep and other animals (Yak, Mithun, Donkeys, Horse, Pigs and Poultry) to total GHG were 30.52, 24.0, 37.7, 4.34, 2.09 and 3.52%, respectively in 2007 (Patra, 2012a). Although the total GHG emissions from livestock in India increased in year 2007 but there was a decreasing trend in GHG production per kg of milk production or animal products. This indicates the anthropogenic methane could be reduced through proper animal management. Enteric methane is the result of microbial fermentation of dietary carbohydrates in the rumen. Cellulolytic bacteria, anaerobic fungi and ciliated protozoa produced hydrogen during fibre digestion (Krause et al., 2003; Sirohi et al., 2012). Hydrogen does not accumulate in the rumen, because formation of methane by strict anaerobic archaea or

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methanogens keeps the partial pressure of hydrogen within physiological range. Hydrogen is also utilized for synthesis of volatile fatty acids and microbial biomass in the rumen. Methane is produced in the rumen by obligate anaerobic methanogens belonging to phylum Euryarchaeota, order Methanobacteriales usually predominate in the rumen (Hook et al., 2010; Sirohi et al., 2012). Only seven species of methanogens, i.e., Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanomicrobium mobile, Methanosarcina barkeri and Methanoculleus olentangii have been cultured from ruminant and non-ruminant herbivores (Janssen and Kirs, 2008; Hook et al., 2010; Sirohi et al., 2012). Their number ranges from 107 to 109 cells/ml in rumen content depending upon diet. Being extremely sensitive to oxygen, most of these survive and grow only under strict anaerobic environments on substrates like hydrogen, formate, methanol, methylamine, acetate, etc. (Sirohi et al., 2010). Methanogens convert CO2 to CH4 using electrons acquired from the oxidation of H2 or formate. Methanogenesis pathways involves seven coenzymes and eight enzymes (Ferry, 2002). In recent years, researchers have focussed on decreasing methane emissions by manipulating the rumen ecosystem which can simultaneously offer improvement in livestock performance. In this regard, plant secondary metabolites (PSM) or phytochemicals have been considered as a viable option by animal nutritionists worldwide. PSM are group of diverse molecules that are involved in the adaptation of plants to their environment but are not part of the primary biochemical pathways. PSM are represented by alkaloids, glycosides, steroids, triterpenoids, phenolics, phenolic glycosides, quinolizidines, tannins, saponins, lignins, polysaccharides and essential oils (Wallace, 2004; Hartmann, 2007). The types and content of these active principles differ among plants species. The synthesis of PSM often restricted to single organ such as roots, fruits or leaves. Thereafter they can be transported around the plant via the phloem or xylem or by symplastic or apoplastic transport and stored in number of different tissues. The site of storage often depends on the polarity of compounds, with hydrophilic compounds such as alkaloids, glucosinolates and tannin being stored in vacuoles or idioblasts, whilst lipolic compounds stored in resin ducts of thylakoid membranes or on the cuticle. Defense barriers such as alkaloids, flavonoids, glycosides, coumarins etc are stored in


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Promising Plant Secondary Metabolites for Enteric Methane Mitigation

the epidermis. Studies have established that PSM can modulate rumen fermentation favorably if used at lower concentration. Among the various PSM, essential oils, saponins and tannins received extensive attention due to their widespread occurrence in plant species.

Saponins The name ‘saponin’ is derived from the Latin word ‘sapo’ meaning soap as these usually produces foam in water. These are high molecular weight, amphiphilic and surface active compounds with detergent, wetting, emulsifying and foaming properties (Sarnthein-Graf and La Mesa, 2004). Saponins possses a fat-soluble nucleus, the aglycone (either triterpenoid, or neutral or alkaloid steroids) and sugar side chains called glycones linked through ether or ester linkages to the aglycone (Sparg et al., 2004). Triterpenoid saponins naturally occur as saponin or free aglycone forms, while steroid saponins exclusively occur in saponins form. The aglycone components are termed either genin or sapogenin. Saponins are widely distributed in the plant kingdom and occur in 754 species of central Asian plants (Gubanov et al., 1970). The soap bark tree (Quillaja saponaria), alfalfa (Medicago sativa), soapwort (Saponaria oficinalis), mojave yucca (Yucca schidigera), gypsophila (Gypsophila paniculata), and sarsaparilla (Smilax regelii) contains high contents of saponins (Sparg et al., 2004; Vincken et al., 2007; Hill and Connolly, 2012). In addition, siris, mahua, reetha and shikakai are also found to be rich source of saponins. Triterpenoidal saponins are predominately found in beans, peas, soybean, lucerne, tea, spinach, sugar beet, horse chestnut, and ginseng whereas, steroidal saponins are reported from yucca, oat, capsicum pepper, aubergine, tomato, alliums, asparagus, yam, fenugreek and ginseng (Sparg et al., 2004). Saponin content in different plants species varies widely and is influenced by age, physiology, environment as well as agronomic factors (Malik, 2007). The two major commercial sources of saponins are Yucca schidigera, which grows in the arid Mexican desert, and Quillaja saponaria, a tree that grows in arid areas of Chile. Apart from plants, marine invertebrates also synthesize triterpene glycosides. Takada et al. (2002) isolated a triterpenoidal saponin nobiloside from the marine sponge Erylus nobilis, whereas Van Dyck et al. (2010) isolated saponins from sea cucumber Holothuria atra, Holothuria leucospilota, Pearsonothuria graeffei and Actinopyga echinites.



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Impact of saponins In vitro studies: The in vitro studies revealed the effects of saponins or saponins-rich extracts on rumen microbes (Table 1). At lower doses, saponins are reported to improve the digestibility of feed by stimulating the growth of cellulolytic bacteria. However at high doses, saponins cause defaunation and significantly modulate rumen fermentation. Yucca schidigera extracts were found to inhibit the growth of Butyrivibrio fibrisolvens (Wallace et al., 1994), Ruminococcus spp. and Fibrobacter succinogens (Wang et al., 1998); stimulated the growth of Prevotella ruminicola. Similarly, Y. schidigera saponins inhibited Gram-positive amylolytic bacteria whereas Gram-negative species were either unaffected or stimulated (Wang et al., 2000). Goel et al. (2012) determined the anti-methanogenic potential of saponins isolated from Achyranthus aspara, Tribulus terrestris and Albizia lebbeck at 3-9% (DM basis) concentrations. Highest methane reduction (49.6% in term of mM/gDDM) was exhibited by A. aspara at 3 and 6% levels. A. lebbeck was effective in enhancing propionate production at 6% level. No significant variation was found in DM digestibility with three plants. Patra et al. (2012) found dose-dependent modulation of ruminal microbial communities by quillaja and yucca saponins at doses of 0-0.6 g/L. There was an increase in the archaeal abundance but total bacteria remained unaltered. Quillaja saponin decreased the abundance of Ruminococcus flavefaciens but not affected Fibrobacter succinogenes and Prevotella. In contrast, yucca saponin significantly increased the abundance of R. flavefaciens, Prevotella and F. succinogenes. However, both the saponins failed to reduce methane emissions. Saponins were found to be most effective against protozoa among different rumen microbial groups (Francis et al., 2002). These have been successfully utilized as defaunating agent (Newbold et al., 1997; Hristov et al., 1999). Rumen fungi were found to be very sensitive to saponins. Neocallimastix frontalis and Piromonas rhizinflata were found to be highly sensitive to yucca saponins even at a concentration as low as 2.25 mg/ml (Wang et al., 2000). Similarly, Muetzel et al. (2003) reported inhibitory effect of saponins on ruminal fungi with saponin-containing Sesbania leaves when included in the fermentation at >20% of the substrate. Ttriterpenoid saponin from Camellia sineis exhibited 79% reduction in relative abundance of anaerobic fungal populations (Guo et al., 2008). Goel et al. (2008a) observed 20-60% decrease in ruminal fungal population by saponin-rich fractions



Table 1. Effect of different saponins containing plant extracts on in vitro methanogenesis and rumen fermentation parameters

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TVFA: Total volatile fatty acids; A/P: Acetate: propionate ratio; IVDMD: in vitro DM digestibility.



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of leaves of Carduus, Sesbania, Knautia and fenugreek seeds. Fenugreek was the most active against fungi. Saponins have shown anti-methanogenic activity by reducing the numbers of protozoa harbouring methanogens as reported by Sharp et al. (1998). In another study, 78, 22 and 21% reduction in methanogen’s population, respectively by Sesbania sesban, fenugreek and Knautia saponins was reported (Goel et al., 2008b). Wina et al. (2005a) observed anti-methanogenic effect of saponins from methanolic extract of Sapindus rarak at a dose range of 0.25- 4.0 mg/ml. Whereas protozoa are completely inhibited at 1 mg/ml, methanogens were inhibited only at a dose >2 mg/ml. Kamra et al. (2008) found saponins containing extracts of Sapindus mukorossi and Yucca schidigera inhibited in vitro methanogenesis by more than 25% accompanied by a sharp decline in methanogen numbers and ciliate protozoa. Reduction of nearly 50% in CH4 production with addition of saponins rich plant extracts has been reported (Jouany and Morgavi, 2007; SzumacherStrabel and Cieslak, 2010). In vivo studies: The ruminal protozoal numbers were depressed on feeding saponins to the ruminants [(Wallace et al., 1994; Klita et al., 1996), (Table 2)]. However, a significant increase in cellulolytic and total bacteria in the rumen of sheep was observed when fed with S. saponaria fruit (Diaz et al., 1993) or on feeding methanol extract of S. rarak (Thalib et al., 1996). Yucca extract failed to show any change in apparent ruminal digestibility of OM or ADF due to intra ruminal administration of yucca extract at lower level (9g/d) in dairy cows (Wu et al., 1994). Antiprotozoal activity of saponins depends upon its source, dose, and nature of the basal diet. Intra ruminal administration of yucca extract (20g/d) in heifers fed barley grain based diet decreased protozoal numbers by 42% while a higher dose of 60g/d did not further depress protozoal population (Hristov et al., 1999). Kamra et al. (2000) observed about 70% reduction in total protozoal population when soapnut (80g/d) was fed to buffaloes. In another study, quillaja extracts fed to cattle @ 60g/d reduced protozoal count by 61% (Baah et al., 2002). Ivan et al. (2004) revealed that the dietary supplementation of E. cyclocarpum foliage decreased protozoal numbers by 49 to 75% during 4 days, but their level gradually increased to the level of control group by day 20. Sarsaponins from Y. schidigera and triterpenoidal saponins from Q. saponaria have been most extensively studied for their potential to reduce or inhibit CH4 production in vivo (Wang et al., 2009; Holtshausen et al., 2009). Administration of 5 g/kg of S.



A/P: acetate to propionate ratio; ND: not determined

Table 2. Effects of saponins on rumen microbial population, methanogenesis and rumen fermentation parameters in different ruminants

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saponaria fruits to sheep for 21 days reduced CH4 release by 6.5% (Hess et al., 2004) while the supplementation of Y. schidigera plant (6% saponins) for 28 days to dairy cows did not influence CH4 production significantly (Holtshausen et al., 2009). It has been shown that saponins decreased the expression of genes in methanogens which are involved in CH4 synthesis (Hess et al., 2003a; Guo et al., 2008). The antimethanogenic activities of saponins are found to be dependent on type of solvents used for extraction of saponins and composition of diets. Ethanol, water and methanol extracts of S. murkossi showed the 96, 39.4 and 20% CH4 depression, respectively in comparison with controls (Agarwal et al., 2006). Saponins of S. sesban and fenugreek were more effective in animals fed concentrate based diets compared to those fed roughage-based diets (Goel et al., 2008a). Inclusion of saponins in ruminant diets showed no adverse effects on feed intake (Hess et al., 2004; Mao et al., 2010). Interestingly, increase in feed intake was observed in dairy cows (Holtshausen et al., 2009) and sheep (Abreu et al., 2004). However, few studies reported decrease in digestibility of nutrients following feeding of saponins (Klita et al., 1996; Hess et al., 2004; Holtshausen et al., 2009). Santoso et al. (2004) and Wang et al. (2009) found that saponin extracts or saponincontaining plants did not alter digestibility, but decreased CH 4 production. At low dose, saponins exhibit anti-methanogenic effects without affecting digestibility, while at higher doses, decrease in both digestibility and methanogenesis was observed (Hu et al., 2005; Holtshausen et al., 2009d). Saponins have shown variable effects on VFA production, but most studies indicated an increase in the proportion of propionate and a reduction in acetate, butyrate and branched chain VFA (Patra and Saxena, 2009; Castro-Montoya et al., 2011). Further, the effects of saponins are more pronounced at low pH (Hristov et al., 1999; Lila et al., 2003; Hess et al., 2003a). The effect of saponins on rumen VFA levels was shown to be influenced by diet (Hess et al., 2003a; Lila et al., 2003). Saponins derived from Quillaja saponaria reduced VFA levels whereas sarasaponin enhanced VFA levels. However, A. concinna, Enterolobium cyclocarpum and tea saponins did not showed any appreciable effect on VFA concentrations (Patra et al., 2006; Pen et al., 2007; Guo et al., 2008). Studies also revealed that the dietary incorporation of saponin containing extracts enhances propionate production in the rumen (Hristov et al., 1999; Lila et al., 2003). Effect of yucca saponin (De-Odorase) was investigated on TVFA in sheep by Ryan et al. (2003). At 500 mg/day dose level, it



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showed increase in ruminal TVFA levels by 9.4% in hay fed animals whereas in straw fed sheep, it decreased TVFA by 8.7%. Wang et al. (2000) observed an increase in microbial protein synthesis when yucca steroidal saponins was added at 15 µg/ml dose to mixed ruminal microbes in the presence of pound barley grain as substrate. However, higher concentration of yucca extract (225 µg/ ml) reduced microbial protein synthesis. Microbial efficiency (g of microbial nitrogen/kg of OM apparently fermented in the rumen) significantly increased due to supplementation of S. saponaria fruit in sheep fed grass hay as sole diet (Abreu et al., 2004).

Tannins Tannins are high molecular weight phenolic PSM which have been used by human being since pre-historic times. Their molecular weight ranges from 500 kDa to >20000 kDa. These form reversible and irreversible complexes with proteins, nucleic acids, cellulose, hemicellulose and alkaloids (McMahon et al., 2000; Schofield et al., 2001; Frutos et al., 2004; Hassanpour et al., 2011; Goel and Makkar, 2012). Tannins have traditionally been divided into two groups: the condensed and the hydrolysable tannins. Hydrolysable tannins (HT) are made up of a carbohydrate core (MW 500-3000 Da) whose hydroxyl groups are esterified with phenolic acids (mainly gallic and hexahydroxydiphenic acid). The condensed tannins (CT) also termed as proanthocyanidins, are non-branched polymers made of flavonoids units (flavan-3-ol, flavan-3,4-diol), and usually have MW 1000-20000 Da (Mueller-Harvey, 1999; Frutos et al., 2004). Tannins are widely distributed throughout pteridophytes, gymnosperms and angiosperms. Plants which contains high content of tannins are betula, ceratonia, cistus, juniperus, castanea, quercus, cytisus, medicago, onobrychis, trifolium, genista, lathyrus, lotus, sorghum, triticum, salix etc (Frutos et al., 2004). Tannins have astrigent taste and often used as skin hides. Commercially, tannin has been produced and extracted from bark of Acacia mearsnii or Acacia mimosa. As the case with saponins, the concentration of tannins in plant species is influenced by environmental stress, rainfall, light, temperature, soil fertility and infection (Espirito-Santo et al., 1999).

Impact of tannins In vitro studies: Tannins have been extensively evaluated in vitro for their effects on fermentation parameters, digestibility and methane


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reduction (Table 3). Sliwinskii et al. (2002a) found no effect on methane production with chestnut extract in vitro. Makkar et al. (1995) reported decrease in protozoal population in the presence of quebracho tannins in vitro. However, McSweeny et al. (2001) reported no change in protozoa population when calliandra tannins were tested. Methane production was inhibited by 90% by methanol extract of Terminalia chebula (Patra et al., 2010). Min et al. (2005) found that quebracho tannin (75% CT) included at concentrations of 1-2 g in fermentation medium decreased methane production by 12.3-32.6%. Similarly, feeding of quebracho tannins at 10-20 g/kg DM intake to cattle grazing wheat grass in reproductive stage with rumen liquor collected from them for testing methane production in vitro caused a decrease in methane by 25-51% (Min et al., 2006). Bhatta et al. (2009) found linear inhibition of methane production (13-45%) with increasing doses of quebracho tannins (5-25% of substrates). It has been suggested that the action of CT on methanogenesis may be attributed to the direct inhibitory effects on methanogens depending upon the chemical structure of CT and also indirectly by decreasing fiber degradation (Patra and Saxena, 2010; Patra, 2012). Jones et al. (1994) found that sainfoin CT had profound effects on 5 strains of proteolytic rumen bacteria. Molan et al. (2001) studied the effect of CT derived from L. corniculatus and L. pedunculatus on the growth of four strains of proteolytic rumen bacteria. Both the sources of CT inhibited the growth of all the four strains of bacteria. However, growth of Eubacterium sp., Prevotella bryantii and Butyrivibrio fibrisolvens was inhibited more by CT of L. pedunculatus than by L. corniculatus. McSweeny et al. (2001) observed that total number of cellulolytic bacteria including Fibrobacter succinogens and Ruminococcus flavifaciens was lower in sheep supplemented with calliandra forage rich in CT while fungal population was less affected. In vivo studies: Acacia mearnsii tannin extracts suppressed methane production in sheep and cattle by 10% (Carulla et al., 2005) and 30% (Grainger et al., 2009), respectively (Table 4). Hess et al. (2006) reported that supplementation with a tannin-rich legume (25 g/kg DM) decreased methane emission by 13%. Quebracho tannin extract failed to reduce methane emission in beef cattle when fed at 10-20 g /kgDM intake (Beauchemin et al., 2007). Tannins from Sericea lespedeza, Calliandra calothyrsus and Flemingia macrophylla have also reduced methane emission in goat (Animut et al., 2008a,b). Methane production was inhibited when Terminalia chebula was fed to sheep at a dose of


Table 3. Effect of tannins containing plant extracts on in vitro rumen fermentation parameters and methane production


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TVFA: Total volatile fatty acids; A/P: Acetate: propionate ratio; IVDMD: in vitro DM digestibility; HGT: Hohenheim gas test system; CT: Condensed tannins; APES: Automated pressure evaluation system; SCFA: Short chain fatty acids

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Table 4. Effects of tannins containing plant extracts on rumen fermentation parameters, nutrient digestibility, microbial population and methane production in different ruminant species


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OM: Organic matter; A/P: Acetate to propionate ratio; SCFA: Short chain fatty acids; TVFA: Total volatile fatty acids; CT: Condensed tannins;@: Inhibition of methane production compared with control relative to DM or OM digested

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10 g/kg of DM intake (Patra et al., 2010). Similarly, Chanthakhoun et al. (2011) found that Phaseolus calcaratus condensed tannins (2.8%) reduced methane gas production in swamp buffalo. An increase in protozoal numbers was observed by Terill et al. (1992) in sheep grazing sulla which contain CT. However, quebracho powder fed to cattle at 0.6% of dietary DM reduced protozoal numbers (Baah et al., 2002). In another study, Ruiz et al. (2004) observed low numbers of endodiniomorph protozoa and absence of holotrichs in the rumen of goats fed olive leaves alone. However, when animals received a diet composed of olive leaves, barley and faba beans, holotrichs appeared and endodiniomorph populations increased. Hydrolysable tannins from Castanea saliva wood extract did not influence protozoal count both in vitro (Sliwinski et al., 2002a) and in vivo (Sliwinski et al., 2002b). Tannins exhibit inhibitory effects on carbohydrate and nitrogen metabolisms in the rumen. Makkar et al. (1995) reported decrease in TVFA and acetate but increase in propionate in the presence of quebracho tannins as compared to tannic acid. Reduction in NH3-N concentrations was also observed by Singh et al. (2001) using tannic acid as well as by Sliwinski et al. (2002a) with HT of Castanea sativa wood extract (2.5 g/kg DM) using RUSITEC.

Essential oils The essential oils (EOs) are complex mixtures of volatile and aromatic lipophilic secondary metabolites (Turek and Stintzing, 2013). These have molecular weight