sem final (4)

Slide 2:

The estimated CH4 emission from enteric fermentation is 17–30% of global production. According to Johnson and Johnson (1995), cattle can produce 250–500 l of methane per day per animal. Cattle typically lose 2–15% of their ingested energy as eructated methane (Giger-Reverdin and Sauvant 2000). Thus, mitigatin methane losses from cattle has two important benefits. Firstly, less methane means a lower concentration of greenhouse gases (GHGs) in the atmosphere. Secondly-less methane means increased efficiency of livestock production and increased income for farmers. The ability of methane to retain heat (global warming potential) is 21 times more than carbon dioxide (EPA 2008).

Mechanisms of methanogenesis in the rumen:

Mechanisms of methanogenesis in the rumen In the anaerobic conditions prevailing in the rumen, the oxidation reactions required to obtain energy in the form of ATP release hydrogen. The amount of hydrogen produced is highly dependant on the diet and type of rumen microbes as the microbial fermentation of feeds produces different end products that are not equivalent in term of hydrogen output. For instance, the formation of propionic acid consumes hydrogen whereas the formation of acetic and butyric acids releases hydrogen . Methanogenesis is the mechanism favoured by the rumen to avoid hydrogen accumulation. Free hydrogen inhibits dehydrogenases and affects the fermentation process.

Slide 4:

The utilisation of hydrogen and CO2 to produce CH4 is a specificity of methanogenic archaea. The methanogens interact with other rumen microorganisms enhancing the energy efficiency and extent of feed digestion. Positive interactions have been described for cellulolytic (Ruminococcus albus and R. flavefaciens) and noncellulolytic bacteria (Selenomonas ruminantium), protozoa, and fungi (reviewed by McAllister et al., 1996).

Mitigation :

Mitigation Through biotechnologies :- A vaccine against three selected methanogens decreased methane production by nearly 8% in Australian sheep (Wright et al., 2004). Additive:- A bacteriocin obtained from a rumen bacterium, bovicin HC5, decreased methane production in vitro up to 50% without inducing methanogens’ adaptation (Lee et al., 2002). Probiotics:- The use of probiotics or the stimulation of rumen microbial populations capable to decrease methane emissions.

Defaunation :

Defaunation Elimination of protozoa Hydrogen is one of the major end products of the rumen protozoa metabolism. Methanogens associated extra- and intra-cellularly to ciliate protozoa have been estimated to contribute between 9 to 37% of the rumen methanogenesis (Finlay et al., 1994, Newbold et al., 1995). The removal of protozoa from the rumen (defaunation) has been shown to reduce CH4 production by up to 50% depending on the diet (reviewed by Hegarty, 1999).

Mitigation through additives:

Mitigation through additives Ionophores and organic acids Plant extracts Mitigation through feeding -Increased proportion of concentrates in the diet -Adding lipids in the diet

Plant - metabolite :

Plant - metabolite Primary Metabolites (PMs) The universal compounds found in all plants: the known sugars, protein amino acids, purines, purimidiness of nucleic acids, chlorophylls, etc. Secondary Metabolites (SMs) All other plant chemicals that vary in plant species and also do not appear to have an essential role in metabolism: Alkaloids, terpenoids, phenolics,

Plant secondary metabolite:

Plant secondary metabolite The term plant secondary metabolite is used to describe a group of chemicals present in plants that are not involved in the primary biochemical processes of plant growth and reproduction. Provide protection against predators, pathogens and invaders because of their anti-microbial activity. The majority of these compounds fall into the category of lignins, tannins, saponins, volatile essential oils, alkaloids, etc. ( D.N. Kamra,Neeta Agarwala and L.C. Chaudhary)

Slide 16:

Three different groups of plant materials, rich in saponins, tannins and essential oils, respectively, have been evaluated for their anti-methanogenic and anti-protozoal activit. The anti-microbial activity of these compounds is highly specific and therefore may be used for the manipulation of rumen fermentation by selective inhibition of a microbial group of the ecosystem. Different parts of plants like seed pulp, leaves and spices extracted in water, methanol and ethanol have been evaluated in in vitro gas production test for their anti-methanogenic and anti-protozoal activities. The ethanol extract of soapnut (Sapindus mukorossi) seed pulp completely inhibited in vitro methane production along with a significant reduction in protozoa count and acetate/propionate ratio.

Slide 17:

The methanol extract of seed pulp of harad (Terminalia chebula), leaves of poplar (Populus tremuloides), flower buds of cloves (Syzygium aromaticum), ethanol extract of guava (Psidium guayaba) leaves and both the ethanol and methanol extracts of garlic (Allium sativum) strongly inhibited in vitro methanogenesis. The effect on ciliate protozoa was variable with these plant extracts and there was no correlation between methane and protozoa inhibition. The presented in vitro results indicate that plant secondary compounds seem to have a potential to be used as feed additives for rumen manipulation to reduce methane emission

Tannins:

Tannins Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins and various other organic compounds including amino acids and alkaloids. They are water soluble and their multiple phenolic hydroxyl groups lead to the formation of complexes primarily with proteins and to a lesser extent with metal ions, amino acids and polysaccharides.

Effects of tannins on rumen fermentation:

Effects of tannins on rumen fermentation Dietary tannins can adversely affect fermentation by bacteriostatic and bactericidal activities and by inactivating ruminal enzymes (Faixova and Faix, 2005). Methanol extract of harad (T. chebula) decreaseed methane production in vitro by 95% when given at the level of 0.25ml/30ml incubation medium and the complete inhibition was observed at double this level (Patra et al., 2006a). In another study with Harad (30mg) and Poplar (10mg) replaced in the feed supplementation in vitro, Patra et al. (2006b) observed reduced methane production by 32% and 20%, respectively Tannins can influence the protein digestion in the rumen or the protein availability in the intestine. The high level of tannin resulted in poor nitrogen turn over. Dey et al. (2006 )

Slide 20:

The CT content in diet influences cellulose degradation in the rumen. Tannins could reduce fibre digestion by complexing with lignocellulose and preventing microbial digestion or by directly inhibiting celluloytic microorganism or both. The extent of fibre digestion in the rumen was reduced in animals fed Lotus pedunculatus (9.5% condensed tannin) or the tropical legume C. alothyrsus (6% free condensed tannins) (Barry et al., 1986; Waghorn et al., 1987; Palmer and McSweeney, 2000).

Tannins and rumen micro-organism:

Tannins and rumen micro-organism In addition to the effect on the other nutritional contents of the feed in rumen polyphenolics are reactive with the cell wall of bacteria and the extracellular enzymes secreted. Some of the bacteria showing sensitivity while some of them are resistant to the tannin addition upto certain level (Odenyo and Osuji, 1998). But they are unable to degrade the condensed tannins (Makkar et al. , 1995). Condensed tannins from the pasture legume Lespedeza cuneata have been demonstrated to be inhibitory to pectinase and cellulase in rumen fluid (Smart et al. , 1961 and Bell et al ., 1965).

Slide 22:

The effects of tannins on rumen protozoa are variable. Patra et al . (2006a) observed reduced protozoa count in vitro with the addition of tannin rich Harad at the level of 0.5ml/30ml incubation medium b ut not at 0.25ml level. In contrary to this, Patra et al . (2006b) did not find any effect on the protozoa count when supplemented with Harad and Poplar or their mixture along with Garlic. Newbold et al. (1997) investigated that tannins were not responsible for the antiprotozoal activity of Sesbania sesban.

Slide 23:

Fibre degrading ability of rumen fungi may be less sensitive to the inhibitory effects of condensed tannins compared with cellulolytic bacteria (McSweeney et al., 1998). The ability of the cellulolytic ruminal fungus Neocallimastix patriciarum to degrade cellulose was not affected by exposure to 100 µg/ml condensed tannins from L. corniculatus (McAllister et al., 1994). Muhammad et al. (1995) tested the effects of tannic acid, ellagic acid, gallic acid and catechin on rumen fungus Neocallimastix frontalis strain RE1. All these compounds inhibited the cellulolysis and zoospore attachment to cellulose by this fungus. Gallic acid, ellagic acid and catechin were more inhibitory to cellulolysis than tannic acid.

Saponins :

Saponins Chemically, saponins are high molecular weight glycosides in which sugars are linked to a triterpene or steroidal aglycone moiety. A large number of saponins could be possible depending upon the modifications of ring structure of aglycone moieties and number of sugars added to it. It covers a wide variety of chemical compounds and mankind has used it as soap substitutes for many years.

Effect of saponins on rumen fermentation:

Effect of saponins on rumen fermentation There are inconsistent results on the effect of saponins on methane production. Agarwal et al. (2006a) observed reduced methane production in vitro with water, ethanol and methanol extracts of Soapnut (Sapindus mukorossi) with highest that of ethanol extract (96%). They observed lower gas production with water extract (22.68% inhibition) and ethanol extract (11.48%), whereas methanol extract did not affect it. Addition of Tea Saponin at 2, 4, 6 and 8 mg against 200 mg mixture in vitro decreased methane concentration by 13, 22, 25 and 26%, respectively (WeiLian et al 2005).

Slide 26:

Hess et al. (2001) using high saponin doses of S. saponaria, noted a decline of in vitro methane release by about 20%. While Sliwinski et al. (2002) applied a lower dose of saponins and found no affect on methanogenesis. Addition of 8% fruit, 5% pericarp or 1.2% semipurified saponins extract of Sapindus sapinaria had no effect on methanogenesis in RUSITEC (Abreu et al., 2003). Lila et al. (2003) studied effect of different concentrations of sarsaponins on different substrates. As the concentration of sarsaponin increased from 1.2 to 3.2 g/L, fermentation of soluble potato starch, corn starch and hay plus concentrate decreased methane production from 20 to 60% (6h) and 17 to 50% (24 h), 21 to 58% (6 h) and 18 to 52% (24 h) and 23 to 53% (6 h) and 15 to 44% (24 h), respectively.

Saponins and ruminal micro-organisms:

Saponins and ruminal micro-organisms Microbial activity may be affected by the use of saponins. The rumen microbial population increased by low saponins supply (Sen et al., 1998), but decreased when doses became excessive (Wallace et al., 1994; Cheeke, 1996 and Sen et al., 1998). Saponin containing plants appear to be useful as a means of suppressing the bacteriolytic activity of rumen ciliate protozoa and thereby enhancing the total microbial protein flow from the rumen. In particular, protozoal counts in rumen fluid decreased with higher saponin doses, with tea saponin (WeiLian et al 2005), sarsaponin from Y. schidigera (Valdez et al., 1986; Wang et al., 1997; Hristov et al., 1999), quillaja saponin (Makkar and Becker, 1996) and with saponin rich plants or fruits (Navas-camacho et al., 1994; Klita et al., 1996; Teferedegne et al., 1999; Hess et al., 2001; Abreu et al, 2003). Agarwal et al. (2006a) found detrimental effects of methanol extract of Sapindus mukorossi.

Slide 28:

In the same line, Patra et al. (2006a) observed suppressed protozoa count with the inclusion of A. concinna (Shikakai) and A. indica (Neem seed) extracts at 0.25 and 0.50ml/30ml incubation medium in vitro. The effect of these plant extracts could be due to the presence of saponin. However, Sliwinski et al. (2002) did not find any effect on protozoal numbers by saponin-rich products containing sarsaponin. In long term trial with the sheep at the level of 0.24, 0.48, 0.72g Sapindus rarak saponins /kg body weight, protozoa numbers were decreased but not in short term trial of 7 days.

Slide 29:

Saponins possibly bind with sterol of cell memberanes of protozoa and change the permeability of cell membrane (Patra et al ., 2006a). Saponin are also known to influence both rumen bacterial species composition and number through specific inhibition, or a selective enhancement of growth of individual species (Sen et al ., 1986).

Slide 30:

saponins affect bacteria with gram positive ultrastructure more than gram-negative organisms. Wang et al. (2000) reported that steroidal saponins reduced the growth of S. bovis, P. bryanti and Ruminobacter amylophilus but the growth of S. ruminantium was enhanced. They also reported that steroidal saponins inhibited the ruminal cellulolytic bacteria ( Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus ) and fungi ( Neocallimastix frontalis and Piromyces rhizinflata ). They attributed methane suppressing effect of sarsaponin to the inhibition of these cellulolytic bacteria.

Essential oils:

Essential oils Essential oils (EO) are natural products that give plants and spices their characteristic odor and color. These are mixtures of may lipophilic acids which are volatile and often associated with terpenoid compounds present in higher plants (Kohlert et al., 2000) and are obtained by steam and or water distillation (Losa, 2001). The antimicrobial activity of spices is considered to be due to the presence of essential oils which are also responsible for their characteristic aroma.

Mode of action:

Mode of action Acamovic and Brooker (2005) suggested that EO, interact with a wide variety of cellular components and can modulate a response at their targets, these compounds have the ability to modulate a large number of cellular targets. It is believed that most EO exert their antimicrobial activities by interacting with processes associated with the bacterial cell membrane, including electron transport, ion gradients, protein translocation, phosphorylation, and other enzyme-dependent reactions ([Ultee et al., 1999] and [Dorman and Deans, 2000]). Helander et al. (1998) showed that thymol from thyme oil (Thymus vulgaris) and carvacrol from oregano oil (Origanum vulgaris) both disrupt the cell membrane thereby decreasing the intracellular ATP pool and increasing the extracellular ATP pool in E. coli.

Slide 33:

Essential oils have a high affinity for lipids of bacterial cell membranes due to their hydrophobic nature, and their antibacterial properties are evidently associated with their lipophilic character. Burt (2004) suggested that Gram-positive bacteria appear to be more susceptible to the antibacterial properties of plant EO compounds than Gram-negative bacteria. This may be expected as Gram-negative bacteria have an outer layer surrounding their cell wall that acts as a permeability barrier, limiting the access of hydrophobic compounds. However, Helander et al. (1998) reported that the phenolics thymol and caravacrol also inhibited growth of Gram-negative bacteria by disrupting the outer cell membrane. It appears that the small molecular weight of EO allows them to penetrate the inner membrane of Gram-negative bacteria ([Nikaido, 1994] and [Dorman and Deans, 2000]). Trombetta et al. (2005) reported that the monoterpenes linalyl acetate, menthol, and thymol were active against Gram-positive Staphlococcus auras and Gram-negative E. coli., and suggested that antimicrobial effect of these monoterpenes is due to the disruption of the plasma membrane of bacteria, thereby interfering with membrane permeability causing intracellular leakage.

. Effects on rumen microbial fermentation:

. Effects on rumen microbial fermentation Nagy and Tengerdy (1968) observed that EO extracted from Sagebrush (Artemesia tridentata) markedly inhibited activity of ruminal bacteria in vitro. Oh et al. (1967) showed that EO extracted from Douglas fir needles (Pseudotsuga menziesii) exerted a general inhibitory effect on ruminal bacteria activity in vitro. The degree of inhibition depended, however, on the chemical structure of the EO compound added. Of the compounds evaluated, oxygenated monoterpenes, particularly monoterpene alcohols and aldehydes, strongly inhibited growth and metabolism of rumen microbes, whereas monoterpene hydrocarbons slightly inhibited and, sometimes, stimulated activity of rumen microbes . These findings were some of the first to demonstrate that the chemical composition of EO greatly influences their effects on activity of ruminal microorganisms .

Slide 35:

In a study with Garlic replaced in the feed supplementation (30mg in 200mg) in vitro, Patra et al. (2006) observed reduced methane production by 14%. The strong smelling joice of Garlic contains a mixture of aliphatic mono and polysulphides and its chief constituents are allicin, diallyl aisulphide oxide and volatile oils. Patra et al. (2006c) in an in vitro experiment with spices showed significant reduction in methane emission by adding ethanol and methanol extracts of fennel, clove and garlic in the incubation medium. Maximum inhibition (83%) was observed with methanol extract of clove. This finding is supported with the changes in the TVFA production. Higher gas production was associated with the increased TVFA with addition of extracts of garlic and onion. Decrease in acetate level along with increased propionate resulted in decreased acetate to propionate ratio with garlic extract addition. Whereas, clove addition produced no change in acetate but decreased propionate, resulting in increased acetate to propionate ratio. With clove extract, there was significant reduction in protozoa count but garlic increased it. As mentioned previously, the effect of Poplar on rumen fermentation is inhibitory on methanogenesis (Patra et al., 2006b). The poplar also contains essential oils along with the phenolic and flavonoid compounds, which might be the contributing factors in the modulating effects.

Slide 36:

Agarwal et al. (2006b) assessed peppermint oil on fermentation of feed and methanogenesis in in vitro gas production test and observed reduced methane production at the level of 30 and 60 µl/30 ml reaction mixture but the level of total volatile fatty acids was reduced with the fall in population density of total bacteria, fungi, protozoa and methanogens. Growth of the methanogen M. smithii was unaffected by EO concentrations of upto 160 ppm although inhibition occurred at 1,000 ppm (McIntosh et al., 2003). However, Beauchemin and McGinn (2006) did not observe any effect of essential oil on methanogenesis in growing beef cattle fed a diet supplemented with essential oil and spice extract (1 g/d).

Other plant secondary metabolites:

Other plant secondary metabolites Flavanoids which consist of flavones, flavonols, flavonones and isoflavones have been suspected to have anti-nutritional characteristics for ruminants (Van Soest, 1994) and also antibacterial effects, which were found to be more effective against gram-positive bacterial strains than gram-negative ones (Mirzoeva et al., 1997). Broudiscou et al. (2000) studied the effect of thirteen dry plant extracts, selected for their high flavonoid contents on rumen fermentation and methanogenesis in continuous cultures of rumen microbes. Lavandula officinalis and Solidago virga-aurea stimulated fermentation, whereas methanogenesis was decreased with Equisetum arvense and Salvia officinalis

Slide 38:

In another experiment Broudiscou and Lassalas (2000) studied the effects of Lavandula officinalis and Equisetum arvense dry extracts, and of isoqercitrin, a flavonoid present in Equisetum arvense on fermentation of diets varying in forage contents by rumen microbes in batch culture. The amounts of acetate and propionate produced from 100% hay diet were increased by the plant extracts, strongly by L. officinalis (60% and 37%, respectively) and E. arvense (59% and 40%, respectively), to a lesser degree by isoquercitrin (29% and 15%, respectively). In dual outflow fermenters receiving a 50:50 hay-barley diet for seven days, the addition of L. officinalis appeared to increase the amount of fermented organic matter, while E. arvense tended to inhibit methanogenesis (Broudiscou and Lassalas, 2000). Achillea millefolium appeared to induce extensive stimulation of microbial metabolism as it increased both degradability of CP and cell wall constituents and the efficiency and yield of biomass production (Broudiscou et al., 2002).

9,10-anthraquinone:

9,10-anthraquinone In other studies with continuous and batch cultures, increasing levels of 9,10-anthraquinone (0.5, 1.0, and 5.0 ppm) reduced total VFA concentration and the molar proportion of acetate, and increased propionate, butyrate and valerate. Increasing levels of 9,10-anthraquinone caused linear and quadratic decreases (P < .05) in methane production, and increases (P < .05) in hydrogen (Garcia-Lopez et al., 1996).

Sinigrin:

Sinigrin Sinigrin (allyl glucosinolate) is present in many plants of Brassica family. It is converted to allyl isothiocyanate by enzyme myrosinases (Simon et al., 1984) in mustard seeds and horseradish root. Lila et al. (2003) reported that synthetic allyl isothiocyanate inhibited in vitro methane production and ruminal methanogenic bacteria. Tyagi and Singhal (1998) reported that mustard oil and glucosinolate decreased ruminal methane production in in vitro. They speculate that biohydrogenation of mustard oil diverted hydrogen from methane generation. Mohammed et al. (2004) studied the effect of horseradish oil coated with cyclodextrin on methane production. They found decreased methane production on horseradish oil suplementation at 0.17, 0.85 and 1.7 g/L of buffered medium by 19, 41 and 90%, respectively ,

Slide 46:

Globally, ruminants produce 80 MMT of methane annually. (NRC, 2002) India has largest livestock population in the world & emit about 10.8 MMT of CH 4 annually or 405.75 x 10 8 Kcal / day from enteric fermentation. (Singh and Sikka, 2007) Dairy animals are most popular livestock enterprises in the country and account for nearly 60% of these enteric emissions. (Singhal et al., 2005) From agricultural sector, ruminants contribute major 49 % CH 4 in India. (NATCOM, 2004) In ruminants, 87% CH4 is produced in rumen & remaining 13% from hindgut fermentation. ( Moss et al ., 2000) Methane Production

:

Method Extent of Reduction (%) Increase in Concentrate mixture 20– 32 Ionophore supplementation i. Maintenance diet 14-23 ii. Medium production diet 23-32 iii. High producing diet 14-25 Supplementation of UMB 10-11 Supplementation of green fodder 11-27 Processing feeds i. Grinding and pelleting 20 ii. Steam-flaking 40 (Singh and Sikka, 2007) Results of Dietary manipulation

Methane Abatement Options in Ruminant:

Methane Abatement Options in Ruminant Dietary Manipulation Concentrate proportion Grazing manegment Additives Molasses / UMNB Fats,Oil Direct Inhibitors: bromochloromethane amichloral, chloroform,chloral hydrate etc Tannins, saponins Manegement Rumen manipulation Animal numbers Forage quality Alternative livestock system Efficiency/ Less RFI Genetic engineering Defaunation Antibiotics Bacteriocin Vaccines Acetogenes Archaeal Viruses Probiotics Longevity of dairy animals Ionophores: monensin , lasalocid, salinomycin Propionate enhancers / Orga nic acids: fumarate, malate Essential oils digestibility of forage Methane oxidizers Conc. type Animal Productivity Leguminous fodder