Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

ISSN: 2319-7706 Volume 3 Number 1 (2014) pp. 684-706 http://www.ijcmas.com

Review Article Sheep and goat production: basic differences, impact on climate and molecular tools for rumen microbiome study

A.R.Agrawal1, S. A. Karim2, Rajiv Kumar2, A.Sahoo2 and P. J. John1

1Department of Zoology, University of Rajasthan, Jaipur, Rajasthan, India 2Central Sheep and Wool Research Institute, Avikanagar, Tonk, Rajasthan, India *Corresponding author e-mail:

A B S T R A C T

Small are able to utilize the lingo-cellulosic materials and convert them to animal products of high nutritional value viz. meat, milk, wool/fur, hide and manure. However ruminants are also contributing towards green house gas emission. Like large ruminants, these fore gut fermenters also harbor a dense and diverse microbial population belonging to different group of flora and fauna. The mammalian system is K e y w o r d s devoid of enzymes cellulase to degrade structural carbohydrate while ruminants are able to degrade them by the enzymes elaborated by symbiotic microbes inhabiting in Sheep; the rumen. Importance of small ruminants in Indian economy lies in their smaller size, Goats; Rumen which is easy to graze and manage. Sheep and goat production is an integral microbiome; component of rural economy of India and serves as major source of economic Green House sustenance for weaker segments of the society in the hot semiarid and arid region. gas; Sheep and goats are closely related since both belong to subfamily Caprinae whereas Differences they are separate species. However, minor differences also exist in their anatomy, in sheep and physiology, behavior, etc. Sheep as individuals and breeds are more sensitive Goatd; to environmental changes than other domestic animals but as a species they thrive Modern everywhere. The inherent characteristics of goats such as resistance to dehydration, Biotechnology wider choice of vegetation, and wide-ranging feeding habits with preference for browse tools. species, enable them to perform better than sheep in regions of scanty rainfall. Furthermore, goats appear to digest fiber more efficiently than sheep. Studies are required to delineate differences in microbial population between sheep and goat which might be helpful in enhancing productivity of the species reared under changing climatic conditions. Modern biotechnology based tools are widely applied to unravel complexities of microbial communities harboring rumen and their functions and interaction among different microbes.

Introduction

Herbivores, particularly ruminants, animals involved in production of food for constitute large share of the domestic human consumption. They are able to

684 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 utilize the lingo-cellulosic materials and employment to small holder farmers and convert them to animal products of high other weaker sections of society including nutritional value viz. meat, milk, wool/fur, women and landless laborers. Livestock is hide and manure. Moreover ruminants do the major source of cash income for not compete with human and subsistence of the livestock owners in livestock species for feed resources. They critical zones of the country. In today s are fore gut fermenters harboring a dense competitive economy driven environment, and diverse microbial population which the survival of small scale sustainable break down the lingo-cellulosic fibrous animal production has some inherent feeds. The mammalian system is devoid of constrains. Moreover with economic enzymes cellulase to degrade structural empowerment simultaneous greater carbohydrate while ruminants are able to demand is being placed on limited and degrade them by the enzymes elaborated finite natural resources resulting in chaotic by symbiotic microbes inhabiting in the situation. It is now essential to ensure that rumen medium. However rumen, also the farmers not only produce for their own called false stomach/fore-stomach, itself is needs but also to meet the expanding devoid of glandular tissues and as such do demand of the market. This is possible not secret any enzyme for digestion of only by application of appropriate ingested food. In rumen fermentation technological interventions besides hydrogen and CO2 are contributed by ensuring better access to credit and ciliate protozoa, bacteria and fungi while market. Unfortunately, there exist a wide methanogenic bacteria utilize the gap in demand and supply of nutrients for hydrogen to reduce CO2 to produce the Indian livestock which is the major methane. cause of concern in realizing optimal and sustained production. Therefore, in the Among the herbivores, the ruminants, present scenario, judicious utilization of particularly sheep, goat, and feed resources from agriculture, Common buffaloes are dominant livestock species Property Resources (CPR) and forest for human consumption. Animal resource is of vital concern to meet the husbandry has made sizeable contribution ever-increasing demand of animal derived to human being in the past century. protein. Animal products provide one-sixth of human food energy and more than one- Importance of small ruminants in Indian third of the protein on global basis economy lies in their smaller size, which (Bradford, 1999). Livestock production in is easy to graze and manage. Sheep and India is an integral part of traditional goat production is an integral component mixed farming (crop-livestock) and play of rural economy of India and serves as important role in the agrarian economy. major source of economic sustenance for The importance of livestock increases with weaker segments of the society in the hot aridity of the environment since traditional semiarid and arid region. The species is crop production is a gamble in the region traditionally reared by small and marginal due to unfavorable agro-climatic farmers and land less laborers under condition. The livestock contribute milk, extensive range management on CPR with meat, fiber, manure, fuel, farm power and top feed supplementation during lean rural transport and thus have major role in season. As per last Census (2007), India rural economy by providing income and was host to 71.5 and 140.5 million (m)

685 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 sheep and goats which increased to 74.0 has revealed that sheep are most important and 154.0 m , respectively by 2010 (FAO, domestic animals, harboring huge genetic 2010). As per literature (Karim, 2008; diversity (40 descript breeds in India) and Karim and Sankhyan, 2009) the slaughter substantial prospects for continued rate of sheep and goats is 32% and 36 % breeding (Acharya, 1982) to further boost hence a total of 23.68 and 55.44 m heads meat and wool production for rising are slaughtered annually. Considering population. Human have molded sheep to average carcass weight of 10 kg the total suit diverse environments and to enhance meat production by sheep and goats comes the specialized production of meat, wool to 237 and 554 m kg respectively and milk. Earlier studies have identified (Gadekar et al, 2011). particular regions of the sheep genome that appear to have changed rapidly in Hoofed mammals like sheep and goats response to selection for genes controlling belong to the highly successful order traits such as coat color, coat cover, body Artiodactyls, family Bovidae with wide size, reproduction and especially, the lack geographical distribution. The main of horns: one of the earliest goals of difference between ruminants and selective breeding (Kijas et al, 2012). monogastric animal is that have Sheep (Ovis aries) are range fed ruminant four parts in stomach i.e. rumen, and like all ruminants are even-toed reticulum, abomasum and omassum. Food ungulate. Domestic sheep are relatively mixed with saliva in rumen and reticulum smaller sized ruminants and depending on and separated in two layers solid and breed, sheep exhibit a wide range of liquid. Then solid part of food regurgitated heights and weights usually with a and mixes with saliva to break down in crimped hair called wool or hairy. Their small particles. Fiber particles of food rate of growth, mature weight and break in hexose monomers and further fecundity are heritable traits that are often degraded by rumen microbes into volatile used in selection for breeding. Ewes fatty acids like acetic acid, propionic acid, worldwide weigh between 45 and 100 kg butyric acid etc. Polymers of amino acids and rams between 45 and 160 kg whereas and non structural carbohydrates are in India the average weight of sheep is fermented to propionates. Then digested around 30-35 kg with coat cover ranging part of food moves towards stomach and from hairy to wooly with fiber diameter of from stomach it moves towards small 20- 55 µ (Melinda et al, 2004). intestine where the digestion is effected by the enzymes elaborate by the system Sheep and goats are closely related since followed by absorption of nutrients. both belong to subfamily Caprinae whereas they are separate species. Visual Sheep rearing is one of the oldest differences between sheep and goats professions closely associated with human include the beard and divided upper lip of civilization wherein the domestication of goats. Sheep tails also hang down, even animals was carried out during neolithic when short or docked, while the short tails times along with the cultivation of cereals. of goats are held upwards. Sheep breeds First sheep and goat, followed by cattle are also often naturally polled (either in and pig, and finally draft animals such as both sexes or just in the female), while horse and asses were domesticated. Sheep naturally polled goats are rare (though ancestry status over the past 11,000 years many are polled artificially). Sheep are

686 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 primarily herbivorous mammals: most There is not dominant role of lignin, lipid, breeds are close grazers on surface vitamins and minerals to produce energy. vegetation and short height herbage, Rumen microbial ecosystem is a complex avoiding the taller woody parts of plant medium of different microbial groups that goats readily consume. Sheep are living in a symbiotic relationship with the predominantly grazers while the goats are host and capable of degrading structural browsers and in the process of carbohydrates (Wolin and Miller, 1988). grazing/browsing both the species resort to Rumen microbial ecosystem is specialized intensive selection on plant parts rich in and buffered in a narrow range of pH thus nutrients and pick up a wide range of stabilizing the system (Kamra, 2005). vegetation from the range land. Both lambs and kids are mono-gastric animals Rumen protozoa play important role in at birth while coming in contact with feed fermentation particularly in surrounding environment microbes inhabit . The rumen protozoa are their rumino-reticulum within two months categorized into two broad heads viz. transformation, lambs and kids became holotrichs and entodiniomorphhids and small ruminants. they can also be classified as soluble sugar/starch degrader and ligno-cellulose The first chamber in alimentary canal is hydrolyser. The ability of sheep and goats rumino-reticulum of small ruminants and to utilize wide range of conventional feed, it is also biggest part of rumen. Microbial top feed and otherwise toxic herbage is fermentation of food occurs in it. possible because of some microbial Reticulum is the smaller part and adaptation in rumino-reticulum. In process continuous with rumen. In rumino- of rumen microbial degradation, 12-15 % reticulum the digestion is a very complex digestible energy is wasted as rumen gases process and occurs through fermentation particularly CH4. In rumen fermentation by microbes and digestion of cellulose and hydrogen and CO2 are contributed by other carbohydrates proceeds here. ciliate protozoa, bacteria and fungi while methanogenic bacteria utilize the There are different prokaryotes hydrogen to reduce CO2 to produce (Eubacteria, archea), eukaryotes (Protozoa methane. Hence elimination of organism and fungi) and viruses harbors the rumen. contributing to synthesis of hydrogen in Protozoa engulf other microbes through the rumen or methane synthesizing phagocytosis for their nutrient microbes will save digestible energy loss requirement. Hydrolization of structural in waste full fermentation and increase carbohydrate and non carbohydrates digestible energy availability to the host occurs by microbial enzymes and these are animals for better production. However fermented to volatile fatty acids and rumen bacteria cannot be effectively polymer of amino acids breaks into eliminated by chemical treatments because peptides and amino acids through of associated adverse effects to the host microbial enzyme and stores as cell animals hence alternatively the elimination biomass. Nitrogen sources present in feed of protozoa and fungi by suitable used directly by microbes with in small treatments without deleterious effects is quantity. If nitrogen is required in excess the method of choice. amount, then proteins are also fermented to produce energy and yield ammonia.

687 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

Climate change is no longer a distant characteristics of goats such as resistance threat as inter-governmental panel on to dehydration, wider choice of vegetation, climate change (IPCC) has amply and wide-ranging feeding habits with demonstrated that we are already preference for browse species, enable experiencing climate change induced by them to perform better than sheep in human activities. Industrial revolution regions that receive less than 750 mm of based on combustion of hydrocarbons rainfall (Khan et al, 2003). However (initially coal followed by petroleum and proliferation of sheep has provided breeds natural gases) has increased emission of or types adapted to almost every climate, Green House Gases (GHG) leading to from snow-covered hills to hot accumulation of carbon dioxide in the semiarid/arid environment, but sheep are atmosphere precipitating global warming essentially grazers and prefer to graze on and climate change. Methane is a green short plants/ground coverage as a result house gas whose atmospheric they thrive best on rangelands with a low- concentration has increased dramatically growing plant population that usually over the last century. CH4 is emitted from occurs in the drier areas. variety of both human related (anthropogenic) activities and natural Various features of sheep and goats sources. Human related activities include fossil fuel production, animal husbandry Sheep may be distinguished from goats by (enteric fermentation from livestock and the presence of a beard, strongly manure management), rice cultivation, odoriferous tail-glands of the male, the biomass burning and atmosphere. It is absence of facial glands and lachrymal pits estimated that 60 % global CH4 emission in the skull, the absence of foot glands in is related to human activities. Natural the hind feet. Domestic goats generally sources of CH4 include wasteland, gas carry their tails up, while these are hydrates, permafrost, termites, ocean, hanging in sheep. The body cover differs fresh water bodies, wasteland, soils and widely between the two species, hair in sources such as wild fires. The domestic case of goats and hair/hairy wool or wool animal population has increased by 0.5- in sheep. Horn direction and spirals also 2.0 % annually during last century and one exhibit variation in sheep and goat and of the results of this population increase is skeletal differences also exist (Khan et al, that emission from livestock has become a 2003). significant source of . In fact, domestic animals currently Sheep as individuals and breeds are more account for about 15 % of annual sensitive to environmental changes than anthropogenic methane emission. other domestic animals but as a species they thrive everywhere. Worldwide Worldwide distribution of sheep indicates distribution of sheep indicates their ability their ability to adapt to a variety of to adapt to a variety of environments. environments. However, the preferred However, the preferred environment is environment is on the lighter sandy soils in lighter sandy soils in hot semiarid and hot semiarid and arid, drier tropics, rather arid, drier tropics, rather than in the wet than in the wet humid tropics: in India humid tropics: in India they perform best they perform best and thrive well in large and thrive well in large numbers in the dry numbers in the dry tropics. The inherent tropics. The inherent characteristics of

688 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 sheep/goats such as resistance to Goatcher and Church, 1970). Sheep and dehydration, wider choice of vegetation, goats behave differently on rangeland: and wide-ranging feeding habits with goats are naturally curious and preference for browse species, enable independent, while sheep tend to be more them to thrive in regions that receive less distant and aloof. Sheep have a stronger than 750 mm of rainfall. Proliferation of flocking instinct and become very agitated sheep has provided breeds or types if they are separated from the rest of the adapted to almost every climate, from flock. It is easier to keep sheep inside a snow-covered hills to hot semiarid/arid fence than goats. Goats will seek shelter environment, but sheep are essentially more readily than sheep. grazers and prefer to graze on short plants/ground coverage as a result they Differences in digestion between grazers thrive best on rangeland with a low- and browsers growing plant population that usually occurs in the drier, but not the driest areas. First, differences between grazers and As is the case with goats, sheep adapted to browsers exist in the structure of molars, the humid environment appear to be which would be expected to influence smaller in size than those adapted to the chewing rates and longevity of teeth. drier climatic regions. The most telling Grazers like sheep tend to have wide difference, though not visible, is that sheep muzzles, with lower incisors of similar have 54 chromosomes and goats have 60. size that project forward in a spatulate Male goats have a characteristic smell, fashion (Janis and Ehrhardt, 1988). The which is quite different from the smell of a greater incisor width of grazers should ram. The rams have a secretary gland on serve to maximize bite size (and thus the hind feet which goats do not possess. harvest rate) when feeding on a continuous Among the similarities the important distribution of grasses (Illius and Gordon, features are: both are ruminants, ungulates, 1987; Janis and Eharhardt, 1988). cloven-hooved, similar dentition, both However, wider muzzles reduce the have horned and hornless breeds and both grazer s ability to select the smaller, more species have some dairy breeds. In nutritious portion of grasses (Janis and addition, both sheep and goats have been Eharhardt, 1988). Australian workers domesticated for thousands of years. (Wilson et al, 1975) used esophageal Sheep are grazing animals while goats are fistula to study the food preferences of essentially browsing animals. Goats have a captive feral goats compared with sheep at competitive advantage over sheep in three grazing pressures (0.5, 0.25 and 0.17 woodland and shrub land, are generally animals per hectare). At low stocking more active, selective, walk longer rates, sheep ate 80 % herbs and 20 % distances in search of feed and relish browse, while goats preferred the reverse. variety of feeds (Devendra, 1990). Sheep At medium and high stocking rates, are less selective and utilize pasture more availability of herbs governed intake. effectively. Another feature of the feeding Goats tended to select diets with behavior of goats is their discerning ability appreciably higher nitrogen content than to taste. Goats can distinguish between sheep, but in vitro digestibility of the bitter, sweet, salty and sour tastes, and nitrogen was not always as high. The show a higher tolerance for bitter taste significantly higher rumen volume in goats than do sheep and cattle (Bell, 1959; confirms a similar report from Australia

689 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

(Watson and Norton, 1982), which was As regards rumen contents, sheep are accompanied by a longer mean retention almost exclusively grazers. In goats, time. With high quality forages, it was Thomson's gazelle and Impala, grass concluded that there may be little accounted for about 70 % of all plant parts differences between-species in partitioning identified. In Grant's gazelle, browse of nutrients, digestion of dry matter, including Acacia seed which constituted neutral detergent fiber and non-ammonia 68 % of rumen ingesta. The preferred food nitrogen (Alam et al, 1985). In the same selected from range land would in turn study it was also reported that there were decide the types of microbial dominance no major differences between species in in reticulo-rumen. Sheep band together protein digestion. In the study comparing and stay together when grazing for sheep and goats on anatomical and protection. This instinct is stronger in fine physiological differences (Singh et al, wooled sheep such as the Rambouillet and 1980) reported that the gut length of sheep decreased in black faced sheep like the was more than goats while total retention Suffolk, but it is there to some degree in time of digesta in the GI tract was more in all sheep (Cobb, 1999). Grazers tend to goats than sheep. have larger, more muscular, subdivided rumen/reticulum, and a smaller opening Dry matter intake (DMI) and total water between the reticulum and omasum than intake is generally higher in sheep than do browsers (Shipley, 1999). This goats (Quick and Dehority, 1986). adaptation may serve to retard the However in another study, goats shown fractional passage rate of digesta to lower greater digestible organic matter intake tract, giving more time for fermentation of (DOMI) that sheep (Alam et al, 1985). plant fiber. Because a greater proportion of Differences between browsers and grazers grass cell is cellulose hence this adaptation extend beyond diet selection: they include would presumably allow grazers to digest specialization within the digestive tract the cell wall more thoroughly and obtain that may allow grazing and browsing more energy per unit of food. Moreover herbivores to better extract nutrients from during higher water intake in terms of their preferred forage class. Grazers and DMI result in faster rumino-reticulum browsers have measurable differences in wash out which is reflected in lower fiber the morphology of the foregut (rumen, digestibility (Odashima et al, 1991). reticulum, abomasums and omasum), the Although the total length of GI tract is hindgut, salivary glands, mouth, teeth, higher in sheep than goats, still greater liver, and body mass that may influence fractional retention time beyond the their ability to consume grasses and pyloric end till ceacum in goats leads to browses and its digestion. All fore gut higher total retention time in GI tract of fermenters have a pouch goats than sheep. In contrast, grazers have (rumen/reticulum) that lies before the true fewer, uneven papillae that limit the (acid-pepsin) stomach (abomasum) in absorptive capacity of the rumen. which the bulk of fermentation occurs. Browsers have a proportionately large The longer plant fiber is retained in the abomasum, or true stomach, a larger rumen, the more complete the digestion of hindgut (ceacum and colon), and the cellulose and other structural ventricular groove in the rumen/reticulum carbohydrates (Demment and Soest, which allow some cell contents to escape 1985). inefficient rumen fermentation in favor of

690 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 direct digestion in the abomasum and Physiological differences: pH, water lower digestive tract. requirement and saliva secretion

In contrast, most browses contain less cell An animal s anatomy and physiology wall and fiber within their cell wall are clearly affect its food choices. more lignified and indigestible, so the Characteristics of food, in turn, are one of smaller rumen of browsing animals should the primary forces that shape animal allow indigestible food particles to flow behavior, physiology and anatomy (Alam more rapidly through the tract (Shipley, et al, 1964). Present evidence suggests that 1999). This rapid flow should promote a sheep are more selective, have a higher higher food intake. Browsers (goats) tend intake, rumen volume/gut fill, salivary to have extensive dense papillae in all secretion and rumen ammonia/urea parts of the rumen, enlarging the surface recycling but lower water intake and area by 22 times, which may allow turnover rate compared to goats. Increased efficient absorption of VFA s from the salivary function and urea recycling may rapidly-fermenting cell contents of the be associated with their ability to have a browse plants. Anatomical differences in higher tolerance for tannins. On the other sheep and goat digestive tract, digesta hand goats appear to digest fiber more retention time and gut contractibility efficiently than sheep (Gihad, 1976). indicate that goats have greater ability to Precise reasons for these and a much better adapt to a wide range of conditions understanding of the real differences merit (Coblentz, 1977; Soest, 1982) but the more thorough investigations on particle causes of that ability are not clear. A size, salivary secretion and microbial higher digestive capacity has been found activity. Rumen pH was not significantly in goats in comparison with sheep when different between species, with mean consuming roughages with low nitrogen values ranging between 6·0 and 6·3. and high lignin contents (Gihad et al, Besides differences in the structure of the 1980; Doyle et al, 1984; Howe and Barry, gastrointestinal tract, grazers and browsers 1988). These differences have been also differ in the relative size of the ascribed to special ability of goats for parotid salivary glands and composition of selecting the morphological parts of the saliva. Parotid salivary gland weight plants with the highest nutritive value increases linearly with body mass in both (Morand-Fehr et al, 1991), which become grazers and browsers, but averages 4 times pronounced when food availability is larger in browsers than in grazers scarce (Soest, 1982; Pfister and Malechek, (Robbins et al, 1995). Although 1986; Bato and Sevilla, 1988), greater (Hofmann, 1989) suggested that larger retention time of the digesta in the rumen parotid salivary glands yield greater flow of goats (Watson and Norton, 1982; of liquids to the digestive tract and buffer Domingue et al, 1991) and to interspecies fermentation. Robbins et al. (1995) did not differences in the rumen environment, find differences in rate of saliva such as a higher production of microbial production between grazers and browsers. protein in goats (Hadjipanayiotou and Cattle and sheep saliva is thin and watery Antoniou, 1983) or a higher number of compared to mule/ saliva which is cellulolytic bacteria in goats than in sheep viscous and gelatinous. These observations (Gihad et al, 1980). suggest that the larger parotid salivary glands of browsers produce tannin binding

691 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 salivary proteins that may prevent tannins saccharides. Then these monosaccharides in browses from greatly reducing protein and di-saccharides are transported to digestibility (Austin et al, 1989; Robbins microbes or fermented to VFAs like acetic et al, 1995). Hofmann (1989) also noticed acid, propionic acid, butyric acid, lactic that browsers have up to 100 % more liver acid and valeric acid to yield energy for tissue for their body size than grazers the microbial cell. Rumino-reticulum wall because plant secondary metabolites and absorbs most VFAs and transferred them toxic chemicals present in browses may be to blood stream where these are used as detoxified in the liver (Foley et al, 1995), substrate for energy production and a large liver might be an additional biosynthesis and hydrolyzed product of adaptation to the chemicals in browse protein are transferred across the microbial species which do not commonly occur in cell wall for assimilation into cell biomass. grasses. A little amount of nitrogen containing source like peptide, amino acid and Microbial population in ruminants ammonia are used directly by microorganism without hydrolyzing. If the The rumen is composed of several nitrogen for microbial growth is in excess muscular sacs, the cranial sac, ventral sac, amount then protein and its derivatives can ventral blind sac and reticulum. The lining also be fermented to produce energy and of the rumen wall is covered with small yield ammonia. fingerlike projections called papillae. The reticulum (derived from the Latin for net) If glycerol is present in lipids, than it is is lined with ridges that form a hexagonal fermented otherwise it is partly hydrolyzed honeycomb pattern. The hexagons in the and hydrogenated. Proteins and some reticulum increase the surface area of the carbohydrates may be used for de novo rumino-reticulum wall, facilitating the synthesis of microbial lipid. Unsaturated absorption of VFAs. Despite the lipids are poisonous for microbes present differences in the texture of the lining of in the rumen and these are also the two parts of the reticulo-rumen, it suppressing fermentation activity. Fungi represents one functional space. In play an important role to solublise rumino-reticulum digestion of feed is very phenolic compounds like lignin. Minerals complex process and it occurs through which are necessary for growth are also microbes by fermentation rather than absorbed by microbes. Microbes also enzymes which are secreted by animal synthesize many vitamins like system. Digestion of cellulose and other cyanocobalamine (Vit. B12) in large structural and non structural carbohydrates quantity which is often enough to sustain occurs in rumino-reticulum. Not only the ruminant even when vitamins are starch, sugar and pectin are the non highly deficient in the diet. structural carbohydrates which are digested here but also structural Ruminants, the fore gut fermenters, have carbohydrate like cellulose, hemi-cellulose developed a microbial symbiosis to digest as well as nitrogen containing compounds fiber in ingested feeds (Dehority, 1997). like amino acids and polymer of amino Rumen flora and fauna belonging to acids (protein) are also well digested in it. diverse families from bacteria, protozoa, Here microbial enzymes are used to fungi and phages (Kamra, 2005). Among hydrolyse monosaccharides and di- the ruminal micro biota, members of the

692 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 domain Archea, which occupy < 4 % of al, 2012). These microbes play significant the entire microbial population, play a role in the biological breakdown of vital role in microbial fermentation (Lin et organic matter in the anaerobic al, 1997; Sharp et al, 1998; Ziemer et al, environments to methane which is 2000). The majority of the Archaea in the produced by domesticated ruminants. rumen are methanogens which utilize H2 However, methane is a very potent as the energy source to reduce CO2 to CH4 greenhouse gas that is estimated to be 23 and provide oxidized reducing factors times more potent than carbon dioxide. (e.g., NAD+) to other microbial metabolic Hence, the growing interest in isolating pathways (Hungate et al, 1970; Wolin, and identifying methanogens from various 1979). Methanogenesis provides environments, especially those from thermodynamically favorable conditions ruminants would be helpful in developing for ruminal microbial fiber degradation strategy to reduce GHG emission. Studies (Zinder, 1993). However, the released CH4 are required to delineate differences in results in loss of dietary energy (Johnson microbial population between sheep and and Johnson, 1995) and contributes to goat which might be helpful in enhancing agricultural greenhouse gas emissions productivity of sheep and goats reared (Enviornment Canada, 2002; International under changing climatic conditions. Panel on Climate Change, 2001). Consequently, a better understanding of Green House Gases (GHG) and methanogens may facilitate mitigating ruminants production of enteric CH4: a significant contributor to greenhouse gases. The As per 1994 estimates of GHG, all methanogenic archaea are a together 1228 m MT CO2 equivalent (CH4 morphologically diverse group of strict 21 and N2O 310 times) emission took anaerobes that can be extremely place from all anthropogenic sources in thermophilic, moderately thermophilic, or India which amounted to 3 % of global mesophilic. Although they resemble emission. Out of this about 794 m MT i.e. bacteria, existing as cocci, spirillum, and 63 % of CO2 equivalent was emitted as rods, methanogenic archaea are CO2 while 33 % of emission (18 m MT) phylogenetically and physiologically was in form of CH4 and the rest 4 % (178 distinct from bacteria. Methanogens use m MT) was N2O. The higher contribution hydrogen to reduce carbon dioxide to of CO2 was due to transformation methane gas, hence their common name activities, fuel combustion in transport, methanogens . However, some cement and steel production. Likewise the methanogens use methyl compounds or enhanced CH4 production was due to acetic acid instead as alternatives to emission from enteric fermentation from hydrogen and carbon dioxide for livestock and rice cultivation. The major methanogenesis. Several species of contribution of N2O came from agriculture methanogens have been isolated from the due to fertilizer application. In summary gastrointestinal tract of ruminants and CO2 contributed 61 %, agriculture 28 % other vertebrates, as well as from and 21 % was contributed by other sources invertebrates, marine and bog sediments, viz. industrial processes, waste generation, lakes rich in decaying vegetation, sewage land use system, forestry etc. Although the sludge and hydrothermal vents (Garcia, compound annual growth rate of CO2 1990; Wright and Pimm, 2003; Eecke et equivalent emission is relatively higher in

693 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

India still it is 1/6 of USA. Moreover while in ruminants the fore gut digestion is during the year 2000 per capita CO2 effected by symbiotic microbes inhabiting equivalent emission was 15.3 % higher in in rumen. In order to facilitate microbial USA than India. Agriculture contributes degradation of feed consumed, the digesta about 21- 25, 60 and 65- 80 % of total is retained in foregut for longer period. In anthropogenic emission of CO2, CH4 and mono gastric digestion the end product is a N2O, respectively. Agriculture is thought monomer which is absorbed and to contribute 95 % NH3, 50 % of CO and assimilated in the body while in ruminants 35 % N2O released in to atmosphere as a the monomers are further degraded to result of human activity. An emission of VFAs and fermentation gases. The VFA total 205-245 m MT CH4 from agricultural diffuse in to the system and the sector is contributed from enteric fermentation gases primarily CH4 and CO2 fermentation (80), paddy rice production are eructed in to environment. It is (60-100), biomass burning (40) and animal established that under Indian conditions waste (25). this loss will amount of 8-28 g CH4/kg dry matter intake depending on species, Methane emission by livestock species production level, physiological state and type of feed consumed by the animals. Global warming and climate change is Hind gut fermentation is another source of unequivocal as evident from increase in CH4 accounting for 10-30 % which is average global and ocean temperature, mainly absorbed in to the system excreted melting of polar ice cap and rise in sea through lungs and a fraction of it is passed level(IPCC, 2007; ). Such climate change off per rectum as flatus. Depending on the has lead to droughts/excessive rain in level of feeding, composition of the diet certain pockets damaging crop cycles and and digestibility of nutrients it is estimated livestock production. Man made global that 2-15 % of gross energy of diet is lost Green House Gases (GHG) emission has as CH4. increased since pre industrial times with an increase of 70 % between 1970 and Rumen methanogenic bacteria utilize H, 2004. The global concentrations of CO2, CO2 or formate, acetate, methylamine and CH4 and N2O have increased markedly as methanol for production of methane a result of human activity since 1970 and (Zeikus, 1977). However the major now far exceed the pre industrial values. substrate for formation of methane in Global increase of CO2 is primarily due to rumen is H and CO2 while minor substrate extensive use of fossil fuel while land use is formate and acetate. The major factor changes also contribute marginally to the affecting rumen fermentation of CH4 are phenomenon. rumen pH and turnover rate of digesta while both in turn are affected by diet and, Methane emission is characteristic of fore level of feed intake, feeding strategy, R: C gut fermenters. In adult ruminants, the ratio and quality. Cattle and buffaloes are expanded fore gut (reticulo-rumen the most important source of CH4 from generally termed as rumen) represents 85 enteric fermentation in India because of % of total GI tract volume amounting to the large population, large body size, 10-20 % of animal weight. In mono gastric predominantly roughage bases feeding animals, the fore gut digestion is effected system and ruminant digestion. Methane is by enzymes elaborated by the system also produced from decomposition of

694 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 animal excreta under aerobic conditions Rumen methane production particularly where the animals reared under intensive system and under A wide variety of methanogenic Archea confinement. Methane emission from inhabit in rumen medium (Kim, 2012) out manure management is relatively smaller of which only 10 % have been in quantity than enteric emission and of characterized employing gene sequencing. significance in intensive production Until recently it was thought that only system. The principal factors affecting three genera of methanogens are present in CH4 from animal manure are amount of the rumen (Methanobravibacter, manure produced and proportion of Methanomicrobium and Methanosercina) manure allowed to decompose an while of late, the total known genera of aerobically and climate of the location. methanogenic Archea has increased to 22 One of the mitigating factors in manure (Leahy et al, 2010) hence it is likely that management under Indian condition is the more genera especially more sub species in vogue production system. Majority of of rumen methanogens will be identified. livestock are reared under extensive Modulation of rumen microbes by production system where the feces voided development of vaccines may be effective are dispersed in grazing area which is on some methanogens but not others rapidly desiccation under hot sun without (Wilams et al, 2009). Moreover further decomposition. development of vaccines has inherent limitation that it cannot be effective without knowing the exact identity of During the last century global domestic target organism. Lack of information on animal population have increased by 0.5 to predominant species of rumen 2.0 % and one of the consequences of methanogens in Indian ruminants is a population growth is the increase in serious limitation for developing emission of CH4. Cattle and buffalo on an biological approach to emission control. average consume 4.0 kg DM/day while it The biochemical pathways and the is 0.8 kg/day in sheep and goats emitting avenues for interventions in rumen an average of 78.4 and 19.6 g CH4/day, methanogenesis are well defined and the respectively. Accordingly among the pathways and enzyme system which livestock species cattle, buffalo, goat and enables methanogens to generate ATP sheep contribute 54.72, 30.37, 9.70 and from CO2 and H have been intensively 5.38 % of total 9.31 Tg amounting to 12.4 studied. The biochemical pathways for % of total world emission of 75 Tg methanogenesis are not the limitation in contributed by animals. It is generally development of protocol for emission agreed that domestic animals currently reduction but understanding the chemistry account for 15 % of annual anthropogenic of methanogens particularly glycoprotein CH4 emission (Crutzen et al, 1986). S-layers and their intensive study will However, CH4 emission levels and sources favor development of methanogen specific widely vary among the countries antibiotics (Deppenmeier, 2002). The depending on climatic conditions, methanogenesis serve as process of industrial and agricultural production, exhaust system in rumen fermentation to energy types and its usage and waste remove hydrogen gas which would management practices. otherwise inhibit the activity of bulk of other organism those do not contribute to

695 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 methane production. Hydrogen is preferences. The basis for this conclusion considered as currency of rumen is due to relatively large number of cases fermentation as transfer of hydrogen in which goats raised in harsh between organisms and its eventual environments were found to be superior to disposal is fundamental to freeing the other ruminants. Whenever goats were primary fermenting organism to ferment found superior to other ruminants, the fresh feed. The principal source of digestive physiology was under reference hydrogen is the bacteria and protozoa viz. goats had an extended retention time which produce acetic acid hence the of digesta in the gut (Devendra, 1990), and ruminants maintained on high fibrous diet higher cellulolytic activity in the rumen usually have higher protozoa population which could be partially related to a more and generally have higher methane efficient recycling of urea from blood to emission (Prins et al, 1977). Hence the rumen (Devendra, 1990; Tisserand et provision of alternate hydrogen sink in the al, 1991). The greater secretion of saliva in rumen or reduction of hydrogen goats in comparison to sheep (Dominigue production by manipulation of other et al, 1991; Seth et al, 1976), and the hydrogen contributing microbes or larger surface area for absorption from the defaunation will increase digestible energy rumen due to broad leaf like papillae availability, reduce emission of methane compared to narrow tongue like papillae in and improve product (Santra et al, 2003). sheep (Bhattacharya, 1980) is a general characteristic of intermediate feeders like Normally about 12-14 % digestible energy goats, in comparison to grass eaters, like is wasted in rumen fermentation as sheep. These characteristics may explain methane wherein the rumen protozoa more efficient urea recycling to the rumen contribute H+ which is utilized by because urea recycling is mediated via methanogens to convert it to methane. saliva secretion and via diffusion through About 37 % methanogenesis is due to the rumen walls. The process prevent fall rumen methanogens or rumen protozoa in rumen pH even at peak fermentation (Newbold et al, 1995). Therefore due to buffering by higher salivary flow elimination of rumen protozoa by which is the major contribution to rumen defaunating agents will cut off the source buffer capacity and relatively efficient of substrate for methanogenesis thereby absorption of VFAs through the rumen improving utilization of digestible energy wall also enhance the buffer capacity of to the tune of 12-14 % with proportionate the rumen (Silanikove et al, 1993). A improvement in livestock production capacity to maintain a spacious rumen (Santra et al, 2003). Hence defaunation helped the Bedouin goats to quench not only reduce emission in ruminant against reduction in the quality of the diet digestion but also improve energy (Silanikove et al, 1993) and to maintain availability for productive purpose. sufficient food intake under infrequent watering regimen (Silanikove, 1992; Based on the fore going observations it Silanikoe, 1994). The Mediterranean could be inferred that there are large spring green vegetation has high protein differences in the pattern of rumen content (>14%) and high in vitro and in fermentation between wild and domestic vivo (Cattle, sheep) digestibility (70/80 mixed-feeding ruminants which has %). Unlike sheep and cattle with abundant evolved due to their respective food grazing on leafy material during spring,

696

Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 browse constitutes at least 50 % of the This maintains their specific advantage in forage selected by goats (Kakabya, 1994; digesting the food that is available to them Kakabya et al, 1998; Lu, 1988; Mill, in large amounts all the year around. On 1990). the other hand, the ability of goats to survive prolong periods of water Rumen microflora of sheep and goat deprivation allows them to graze far from containing a large no. of bacteria from the watering site and to exploit desert which three strains of tannin tolrent pasture evenly and efficiently. As a result, bacteria have been isolated in medium cattle are much more susceptible to containing high concentration of crude changes than sheep and goats to tannin extract (Odenyo and Osuji, 1988). malnutrition, disease and death during Selenomonas ruminantium is capable of severe droughts. Goats in the tropics thrive growing on tannic acid as a sole energy well on diets composed of tree-leaves and source and has been isolated from feral shrubs (browse), which ensure a reliable goats browsing on high tannin containing and steady supply of food all year around, acacia species (Skene and Brooker, 1995). albeit, a low to medium quality food. This Transferring these micro-organisms from grazing strategy in combination with the feral goats to domestic goats and sheep fed anatomical and physiological adaptations tannin-rich foliage (Acacia aneura) renders the goat most efficient desert- increased feed intake and nitrogen dwelling species among domestic retention in inoculated animals compared ruminants. The most remarkable feature of with uninoculated ones. Inoculation also the intermediate selector ruminant is improved N digestibility and improved characterized by short-term or seasonal rate of live weight gain in sheep and anatomical acclimatization to changes in domestic goats (Miller et al, 1995). It is forage quality (Hofmann, 1989). The shown that acclimatization of the corresponding morpho-physiological microbial system in the rumen of goats adaptations are larger salivary glands, adapted to the Mediterranean scrubland higher surface area of absorptive mucosa forms a very important element in the than in grass and roughage eaters and capacity of these goats to utilize efficiently capacity to increase substantially the high-tannin tree leaves (Gilboa, 1996). volume of the foregut when fed high- Spring in the Mediterranean is very short, fibrous food. The results discussed suggest and after three months, the nutritional that the general characteristics of quality of the grass diminishes at an intermediate feeders are probably accelerated rate. Thus, much of the short- important for the development of superior term advantage from switching the grazing digestion and nitrogen conservation habits can be lost during the time capacities and for the efficient use of water necessary to regain the capacity required in goats. for digesting high-tannin browse sources. It seems that although goats take Biotechnology tools for assessment of advantage from the abundance of highly rumen microbial diversity digestible grass (increasing its proportion from approximately 10 % in winter to 40- Delineating the diversity of rumen 50 % in spring), they maintain the intake microbes are of prime importance to of browse sufficiently high to preserve intervene and manipulate rumen their acclimatization to tannin-rich food. fermentation with ultimate goal to enhance

697 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 productivity by improving utilization of study. Kang et al (2009) described the easily available low grade roughage diets. RNA extraction method for estimating gut However, it would be difficult to study microbial diversity. Popova et al. (2010) diverse population of rumen flora and were the first to report a protocol for high- fauna primarily due to requirement of quality DNA/RNA co-extraction from stringent conditions for in vitro culturing rumen ingesta, thus improving nucleotide and isolation in pure culture. The recent extraction efficiency. The success of advancement in molecular biology tools applying molecular approaches strongly have brought boom in this field of study relies on the DNA/RNA quality, and and made this task possible. proper nucleic acid isolation methods must Biotechnology based tools helps in studies be applied when studying different types of complexities of microbial communities of samples. Previously, DNA/RNA function and interaction among these extractions were mostly conducted microbes. Furthermore, molecular tools separately for rumen samples, because of proved effective in culture independent the varied requirements of these two types analysis of the rumen ecosystem of molecules (Guan et al, 2008). (Ghazanfar and Azim, 2009). Molecular ecological studies proved better in For molecular identification, PCR determining the metabolic role of amplification is the first essential step to uncultivated species of rumen (Edwards et enrich the DNA of microbial cells that are al, 2004; Wright et al, 2004, 2006 and present in low numbers. PCR-cloning- 2007). Advances in DNA extraction from sequencing methods are best suited for rumen fluid and low cost sequencing direct examination of the ruminal diversity technology has rendered analysis of of the microbiomes. microbial diversity within the reach of the small set up biotechnology laboratories PCR detection and enumeration of rumen worldwide including India. The microbes has been successfully applied to conventional culture based techniques for study the molecular diversity of rumen estimating rumen microbial diversity is microbiota (Koike and Kobayashi, 2001; being rapidly replaced by modern Koike et al, 2003). Quantitative PCR molecular techniques. High quality DNA chemistries have also been used with extraction from rumen liquor/contents, success to monitor and enumerate the PCR amplification, cloning and microbial populations in the rumen advancement in sequencing protocols (Tajima et al, 2001a; Sylvester et al, 2004; made this task possible. Sharma et al. Skillman et al, 2006b; Koike et al, 2007). (2003) described the DNA extraction Real time PCR is important tool in protocols from rumen contents and several quantitative analysis of different ruminal other workers have also mentioned the flora and fauna however sequencing of improved protocols for the same. Total conserved genes like ribosomal RNA or DNA representing the complete diversity other conserved gene like mcrA in of rumen microbial communities is methanogenes (Luton et al, 2002; necessary to determine the composition of Tatsuoka et al, 2004) will give better the microbial community and monitoring opportunity to identify species specificity. changes in population size. Advances in Pillard et al. (2007) has used molecular DNA extraction methods from rumen beacon chemistries for quantification of contents are instrumental in this field of ruminal microbial population and

698 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 monitored diet associated changes in the 1998; Kocherginskaya et al, 2001; Krause population. Stivension et al (2011) has et al, 1999; Neufield et al, 2004; used this technique to quantify the Regensbogenova et al, 2004a & 2004b). uncultured bacterial population from sheep These techniques have been used to rumen fed on different diets. Abundance monitor succession of microbes in rumen and population dynamics of uncultured with age and diet and also impact of a bacteria was quantified in the study. Real- particular feed/feed additive on microbial time PCR results complement the results diversity. DGGE results had shown greater of rRNA clone libraries. In another study, microbial diversity as compared to 16S Zhou et al (2012) has used real time PCR clone library (Rattray and Craig, 2007). to enumerate rumen methanogenes, Several other studies have shown the bacteria, protozoa and fungi. In this study effectiveness of these techniques in study small subunit ribosomal DNA (SSU) i.e. and identification of culturable and 16S rDNA gene for methanogenes and nonculturable microbes using ruminal bacteria and 18S rDNA gene for protozoa fluid DNA/RNA (Ivey et al, 2009). and fungi were used to quantify the Molecular diversity of rumen has been microbes. In contrast, RT-PCR is an investigated by 16S rRNA gene library approach developed to provide (Tajima et al, 1999; Tajima et al, 2001b; quantitative measurement of a target from Wright and Pimm, 2003; Wright et al, the early phase of PCR amplification. The 2004; Shin et al, 2004). Sequencing study detection threshold of RT-PCR is termed provide better ecological information the threshold cycle (Ct) which is the point about rumen microbes and functional where the amplification curve surpasses information on uncultured group of rumen the threshold line and enters an microorganism could be obtained by exponential phase. As a result, RT-PCR providing required stringent conditions for can measure the relative density of target culturing to characterize its enzyme molecules by comparing the Ct value with secreting ability for feed digestion. 16S a reference, or measure the absolute rRNA gene markers were used to quantify quantity of the targeted fragments by changes in the microbial population in the reference to an external standard. This rumen (Skilman et al, 2006a; method has been utilized to quantify the Perumbakkam et al, 2011). Since abundance of the archaeal community methanogens possess unique 16S rRNA (Ohene-Adjei et al, 2008; Hook et al, gene sequences, and produce CH4, 2009; Zhou et al, 2009 and 2010) and to both16S rRNA genes and genes coding compare specific members among cattle enzymes that are unique to methanogens fed different diets, and with different feed have been utilized to distinguish them efficiencies (Zhou et al, 2009). from other microorganisms. Early experiments to obtain PCR amplicons of Downstream to PCR like PCR-restriction methanogen specific genes from different fragment length polymorphism (RFLP), environments included amplification of PCR- denaturing gradient gel the methyl-coenzyme M reductase (mcrI) electrophoresis (DGGE) and PCR- gene of the family Methano sarcinaceae sequencing techniques have also been used (Springer et al, 1995) and the 16S rRNA successfully to characterize rumen gene from methanogens isolated from microbe diversity (Hiraishi et al, 1995; blanket bog peat samples (Hales et al., Withford et al, 1997 & 1998; Wood et al, 1996). Presently, the 16S rRNA gene and

699 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706 methyl-coenzyme M reductase A-subunit rumen fluids. Microarray technology has (mcrA) gene are the principal targets that been recognized as potentially valuable are amplified and used to characterize tool for high throughput, quantitative and methanogens from environmental samples. systematic studies of microbial An alternative to the 16S rRNA gene for communities (Palmer et al, 2006). Flow phylogenic analysis is the mcrA gene. cytometry (FCM) based analysis has also Methanogens have few morphological been successfully used in determining the traits thus making them difficult to phylogenetic position of uncultured rumen identify. They also have limited microbes (Yanagita et al, 2003). physiological diversity and some are either difficult to grow, or grow very slowly. Advent in sequencing technologies made With the advent of molecular technology, possible the preparation of rumen the methanogens were one of the first microbiome profile domesticated groups to have their taxonomy based upon ruminants. De novo assemblies of 16SrRNA gene comparisons (Balch et al, microbial genomes have been 1979). Thus, many species can be easily accomplished with new generation identified by their 16S gene sequence. sequencing (NGS) technologies. Consequently, a number of methanogen- Currently, 6 genome sequences belonging specific fingerprinting assays have been to 4 species are available for the family developed with the aim of being simpler Methanobacteriaceae. M. ruminantium is and rapid than conventional phenotypic the first methanogen from the rumen to characterizations. have a completely assembled genome sequence. Several other molecular methods like hybridization- and array-based techniques Acknowledgement have been successfully applied to study the microbial diversity in different Authors wish to thank Director, CSWRI ruminants (Dore et al, 1993; Lin et al, for allowing compilation of this work. 1997; Stahl et al, 1988; Dennis et al, 2000; Manefield et al, 2002; Ziemer et al, 2003; References Koike et al, 2010). 16S rRNA-targeting probes have been used to compare the Acharya, R. M. 1982. Sheep and goat breeds of rumen microbial ecosystem (Doreay et al, India. FAO Animal production and Health 2002). Fluorescent in situ hybridization Paper. 30, FAO of United Nations, Rome, (FISH) probes targeting 16S rRNA genes Italy. Alam, M. B., Poppi, D. P., and Sykes, A. R. 1985. are used to study the rumen microbial Comparative intake of digestible organic diversity of both cultured and uncultured matter and water by sheep and goats. P. populations. New Zealand Soc. Anim. Prod. 45, 107-l 11. DNA microarray platform offers the Alam, M. R., Borens, F., Poppi, D. P., and Sykes, A. R. 1964. Comparative digestion in sheep possibility to analyze biodiversity of and goats in: Baker, S.K. (Ed.), Ruminant microbial communities without Physiology-concepts and consequences. Univ. cultivation. DNA microarray technology W. Aust., pp 164. has ability to detect and measure Austin, P. J., Suhar, L. A., Robbins, C.T., and thousands of distinct DNA sequences Hagerman, A. E. 1989. Tannin binding present in DNA samples extracted from proteins in saliva in deer and their absence

700 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

in saliva of sheep and cattle. J. Chem. Ecol. Dore, J., Brownlee, A. G., Millet, L., Virlogeux, I., 15, 1335- 1347. Saigne, M., Fonty, G., and Gouet, P. 1993. Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. Ribosomal DNA-targeted hybridisation probes R., and Wolfe, R.S. 1979. Methanogens: for the detection, identification and reevaluation of a unique biological group. quantitation of anaerobic rumen fungi. Proc. Microbiol. Rev. 43, 260-296. Nutr. Soc. 52, 176A. Bato, R.V., and Sevilla, C. C. 1988. Plant Doreay, M. B., Fernandez, I., and Fonty, G. 2002. preference and diet selectivity of goats in three A comparision of enzymatic and molecular diets. Philippine Agriculturist. 71, approaches to characterise cellulytic microbial 229-239. ecosystems of the rumen and cecum. J. Anim. Battacharya, A. N. 1980. Research on goat Sci. 80, 790-796. nutrition and management in Mediterranean Doyle, P. T., Egan, J. K., and Jhalen, A. J., 1984. Middle East and adjacent area countries. J. Intake, digestion and nitrogen and sulphur Dairy Sci. 63, 1681-1700. retention in Angora goats and Merino Bell, F. R. 1959. Preference thresholds for taste sheep fed herbage diets. Austral. J. Exptl. determination in goats. J. Agric. Sci. 52, 1258. Agric. and Anim. Husb. 24, 165-169. Bradford, G. E. 1999. Contributions of animal Edwards, J. E., Mcewan, N. R., Travis, A. J., and agriculture to meeting global human food Wallase, R. J. 2004. 16S rDNA library-based demand. Livest. Prod. Sci. 59 (2) : 95-112. analysis of ruminal bacterial diversity. Anton. Cobb, R. 1999. An introduction to sheep behaviour Van Leeuw. 86, 263-281. Illini sheep net meat goat net. Eecke, H. C. V., Butterfield, D. A., Huber, J. A., www.livestocktrial.illinois.edu (access Lilley, M. D., Olsen, E. J., Roe, K. K., Evans, dated 28/10/2012). L. J., Merkel, A. Y., Cantin, H. V., and Coblentz, B. E. 1977. Some range relationships of Holden, J. F. 2012. Hydrogen limited growth feral goats on Santa Catalina Island, of hyper thermophilic methanogens at deep sea California. J. Range Manage. 30, 415- hydrothermal Vents. P. Natl. Acad. Sci. 109 419. (34) : 13674-13679. Crutzen, P. J., Aselmann, I., and Seiler, W. 1986. Environment Canada, 2002. Canada s greenhouse Methane production by domestic animals, wild gas inventory. 1990 2001. ruminants other herbivorous fauna and FAO. 2010. State of the world's animal genetic humans. Tellus.38B, 271-284. resources for food and agriculture. pp. 512. Dehority, B. A. 1997. Foregut fermentation, in: Foley, W. J., McLean, S., and Cork, S. J. 1995. Mackie, R. I., White, B. A. (Eds.), Consequences of biotransformation of plant Gastrointestinal Microbiology, Vol I, secondary metabolites on acid- Chapman & Hall, New York, USA, pp. 39-83. base metabolism in mammals: A final Demment, M. W., and Soest, P. J. V. 1985. A common pathway. J. Chem. Ecol. 21, 721- nutritional explanation for body size patterns 743. of ruminant and non-ruminant herbivores. Gadekar, Y. P.., Shinde, A. K., Bhatt, R. S., and Am. Naturalist. 125, 640-671. Karim, S. A. 2011. Restructuring of carcasses Dennis, P., Edwards, E. A., Liss, S. N., and of cull ewe by dietary incorporation of Fulthorpe, R. 2003. Monitoring gene rumen protected fat during pre slaughter expression in mixed microbial communities by fattening. Int. J. Meat Sci. 1, 117-123. using DNA microarrays. Appl. Environ. Garcia, J. L. 1990. Taxonomy and ecology of Microbiol. 69, 769- 778. methanogens. FEMS Microbiol. Rev. 87, Deppenmeier, U. 2002. The unique biochemistry of 297-308. methanogenesis. Prog. Nucleic Acid Res. Mol. Ghazanfar, S., and Azim, A. 2009. Metagenomics Biol. 71, 223-283. and its application in rumen ecosystem: Devendra, C. 1990. Comparative aspects of potential biotechnological prospects. Pak. J. digestive physiology and nutrition in goats and Nutr. 8(8), 1309-1315. sheep. in: Devendra, C., Imazumi, E. Gihad, E. A. 1976. Intake, digestibility and (Ed.), Ruminant Nutrition and Physiology in nitrogen utilization of tropical natural gram Asia, pp. 45-60. hay by goat and sheep. J. Anim. Sci. 43, Domingue, B. M. F., Dellow, D. W., and Barry, T. 879-883. N. 1991. Voluntary intake of rumen digestion Gihad, E. A., E1-Bedawy, T. M., and Mehrez, A. of low quality roughage by goats and sheep. Z. 1980. Fibre digestibility by goats and sheep. J. agric. Sci Camb. 117, 111-120. J. Dairy Sci. 63, 1701-1706.

701

Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

Gilboa, N. 1996. The negative effects of tannins Climate Change 2001: a Scientific Basis. and its neutralization in livestock, Ph.D. Cambridge University Press, Cambridge, UK. Thesis, The Hebrew University of Ivey, L. L. S., Silva, J. B., and Horvath, M. B. Jerusalem. 2009. Technical note: Biodiversity and Goatcher, W. D., and Church, D. C. 1970. Taste fermentation end products in the rumen fluid response in ruminants IV. Reaction of pygmy sample collected via oral lavage or rumen goats, normal goats, sheep and cattle to acetic cannula. J. Anim. Sci. 87, 2333-2337. acid and quinine hydrochloride. J. Anim. Sci. Janis, C. M., and Ehrhardt, D. 1988. Correlation of 31, 373-382. relative muzzle width and relative incisor Guan, L. L., Nkrumah, J. D., Basarab, J. A., and width with dietary preference in Moore, S. S. 2008. Linkage of microbial ungulates. Zool. J. Linn. Soc. 92, 267-284. ecology to phenotype: correlation of rumen Johnson, K. A., and Johnson, D. E. 1995. Methane microbial ecology to cattle s feed efficiency. emissions from cattle. J. Anim. Sci. 73, 2483- FEMS Microbiol. Lett. 288, 85-91. 2492. Hadjipanayiotou, M., and Antoniou, T. 1983. A Kababya, D., Perevololotsky, A., Bruckental, I., comparision of rumen fermentation patterns in and Landau, S. 1998. Selection of diets by sheep and goats given a variety of diets. J. Sci. dual-purpose Mamber goats in Mediterranean Food Agric. 34 (2), 1319-3122. woodland. J. Agric. Sci. Camb. 131, 221-228. Hales, B. A., Edwards, C., Ritchie, D. A., Hall, G., Kakabya, D. 1994. Grazing behaviour and nutrition Pickup, R. W., and Saunders, J. R. 1996. of goats in Mediterranean woodland. Master Isolation and identification of methanogen- Thesis, The Hebrew University of Jerusalem. specific DNA from blanket bog peat by PCR Kamra, D. N. 2005. Rumen microbial ecosystem. amplification and sequence analysis. Appl. Curr. Sci. 89 (1), 124-135. Environ. Microbiol. 62, 668 675. Kang, S., Denman, S. E., Morrison, M., Yu, Z., and Hiraishi, A., Kamagata, Y., and Nakamura, K. McSweeney, C. S. 2009. An efficient RNA 1995. Polymerase chain reaction amplification extraction method for estimating gut microbial and restriction fragment length polymorphism diversity by polymerase chain reaction. Curr. analysis of 16S rRNA genes from Microbiol. 58, 464 471. methanogens. J. Ferment. Bioeng. 79, 523- Karim, S. A. and Sankhyan, S. K. 2009. Present 529. status and future prospective of small ruminant Hofmann, R. R., 1989. Evolutionary steps of nutrition. Lead Papers in 13th Biennial ecophysiological adaptation and Conference on Diversification of animal diversification of ruminants: a nutrition research in the changing scenario, comparative view of their digestive system. NIANP, Bangaluru, 17-19 December, pp. 7- Oecologia. 78, 443-457. 21. Hook, S. E., Northwood, K. S., Wright, A. D., and Karim, S. A. 2008. Meat animal production in McBride, B. W. 2009. Long-term monensin India: an over view. Key Note Address in 3rd supplementation does not significantly affect Convention of Indian Meat Science the quantity or diversity of methanogens in the Association, National Symposium on Safe rumen of the lactating dairy cow. Appl. meat for good health and environment , July Environ. Microbiol. 75, 374 380. 4- 5, Veterinary College, Bangalore, pp. 2-8. Howe, J. C., and Barry, T. N. 1988. Voluntary Khan, B. B., Iqbal, A., and Mustafa, M. I. 2003. intake and digestion of gorse (I&.X Sheep and Goat production. Department of europaeous) by goats and sheep. J. Livestock Managment, University of Agric. Sci. 111, 107-114. Agriculture, Faisalabad, Pakistan. Hungate, R. E., Smith, W., Bauchop, T., Yu, I., and Kijas, J.W., Lenstra, J. A., Hayes, B., Boitard, S., Rabinowitz, J. C. 1970. Formate as an Neto, L. R., San Cristobal,. M., Servin, B., intermediate in the bovine rumen fermentation. McCulloch, R., Whan, V., Gietzen, K., J. Bacteriol. 102, 389 397. Paiva, S., Barendse, W., Ciani, E., Raadsma, Illius, A. W., and Gordon, I. J., 1987. The H., McEwan, J., and Dalrymple, B. 2012. allometry of food intake in grazing ruminants. Genome-wide analysis of the world s sheep J. Anim. Ecol. 56, 989-999. breeds reveals high levels of historic Intergovernmental Panel on Climate Change 2007: mixture and strong recent selection. PLoS Biol. Synthesis Report. Accessed from 10 (2), e1001258. http://www.ipcc.ch dated 11-11-201s2. Kim, C. C. 2012. Identification of rumen Intergovernmental Panel on Climate Change 2001. methanogens, characterization of substrate

702 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

requirements and measurement of hydrogen selection of goats. Small Rumin. Res. 1, 205- threshold. A master s thesis submitted to 216. Massey university. Luton, P. E., Wayne, J. M., Sharp, R. J., and Riley, Kocherginskaya, S. A., Aminov, R. I., and White, P. W. 2002. The mcrA gene as an alternative to B. A. 2001. Analysis of the rumen bacterial 16S rRNA in the phylogenetic analysis of diversity under two different diet conditions methanogen populations in landfill. Microbiol. using denaturing gradient gel electrophoresis, 148, 3521-3530. random sequencing, and statistical ecology Manefield, M., Whiteley, A. S., Ostle, N., Ineson, approaches. Anaerobe. 7, 119-134. P., and Bailey, M. J. 2002. Technical Koike, S., Handa, Y., Goto, H., Sakai, K., considerations for RNA based stable isotope Miyagawa E., Matsui, H., lto, S., and probing: an approach to associating microbial Kobayashi, Y. 2010. Molecular monitoring diversity with microbial community function. and isolation of previously uncultured Rapid Commun. Mass Spectrom. 16, 2179- bacterial strain from the sheep rumen. Appl. 2183. Environ. Microbiol. 76(6) : 1887-1894. Melinda J., 2004. Burrill Ph. D. Professor Koike, S., and Kobayashi, Y. 2001. Development Coordinator of Graduate Studies, Department and use of competitive PCR assays for the of Animal and Veterinary Sciences, California rumen cellulolytic bacteria: Fibrobacter State Polytechnic University. "Sheep". World succinogenes, Ruminococcus albus and Book. Mackiev. Ruminococcus flavefaciens. FEMS Microbiol. Morand-Fehr, P. 1981. Behavioural and digestive Lett. 204, 361-366. characteristics of goats. in: Nutrition and Koike, S., Yabuki, H. and Kobayashi,Y. 2007. Systems of Goat Feeding (Ed. Morand- Validation and application of real-time Fehr, P., Bourbouze, A., Simiane, and M. D.), polymerase chain reaction assays for ITOVIC-INRA, Tours, France, pp. 21-45. representative rumen bacteria. Anim. Sci. J. 78, Neufeld, J. D., Yu, Z., Lam, W., and Mohn, W. W. 135-141. 2004. Serial analysis of ribosomal sequence Koike, S., Yoshitanib, S., Kobayashi, Y., and tags (SARST): a high throughput method for Tanaka, K. 2003. Phylogenetic analysis of profiling complex microbial communities. fiber-associated rumen bacterial community Environ. Microbiol. 6, 131-144. and PCR detection of uncultured bacteria. Newbold, C. J., Lassalas, B., and Jouany, J. P. FEMS Microbiol. Lett. 229, 23-30. 1995. The importance of methanogens Krause, D. O., Smith, W. J. M., Ryan, F. M. E., associated with ciliate protozoa in ruminal Mackie, R. I., and McSweeney, C. S. 1999. methane production in vitro . Lett. Appl. Use of 16S-rRNA based techniques to Microbiol. 21 (4), 230 234. investigate the ecological succession of Odashima, M., Nam, K. T., Lee, S. L., Chiga, H., microbial populations in the immature lamb Kato, K., Otha, M., and Sasaki, T. 1991. rumen: Tracking of a specific strain of Seasonal changes in particulate panage inoculated Ruminococcus and interactions rate in gastro-intestinal tract and digestibility with other microbial populations in vivo. of Sika deer and sheep under restricted Microb. Ecol. 38, 365-376. feeding conditions. Anim. Sci. Technol. 62, Leahy, S. C., Kelly, W. J., Altermann, E., 308-313. Ronimus, R. S., Yeoman, C. J., Pacheco, D. Odenyo, A. A., and Osuji, P. O. 1988. Tannin- M., Li, D., Kong, Z., McTavish, S., Sang, C., tolerant ruminal bacteria from East African Lambie, S. C., Janssen, P. H., Dey, D., and ruminants. Canad. J. Microbiol. 44, 905- Attwood, G. T., 2010. The genome sequence 909. of the rumen methanogen Methanobrevibacter Ohene-Adjei, S., Chaves, A. V., McAllister, T. A., ruminantium reveals new possibilities for Benchaar, C., Teather, R. M., and Forster, R. J. controlling ruminant methane emissions. PLoS 2008. Evidence of increased diversity of One 5, e8926. methanogenic archaea with plant extract Lin, C., Raskin, L., and Stahl, D. A. 1997. supplementation. Microb. Ecol. 56, 234-242. Microbial community structure in Palmor, C., Bik. E. M., Eisen, M. B., Eckburg, P. gastrointestinal tracts of domestic animals: B., Sana, T. R., Wolber, P. K., Relman, D. A., comparative analysis using rRNA-targeted and Brown, P. O. 2006. Rapid quantitative oligonucleotide probes. FEMS Microbiol. profiling of complex microbial populations. Ecol. 22, 281-294. Nucleic Acids Res. 34(1), e5. Lu, C. D. 1988. Grazing behaviour and diet Perumbakkam, S., Mitchell, E. A., and Craig, A.

703 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

M. 2011. Changes to the rumen bacterial and chemical composition of parotid saliva in population of sheep with the addition of 2, 4, sheep and goats. Indian J. Anim. Sci. 46, 6-trinitrotolune to their diet. Antonie van 660-663. Leeuw. 99, 231-240. Sharma, R., John, S. J. Damgaard, D. M., and Pfister, J. A., and Malechek, J. C. 1986. The McAllister, T. A. 2003. Extraction of PCR- voluntary forage intake and nutrition of goats quality plant and microbial DNA from total and sheep in the semi-arid tropics of rumen contents. Biotechniqe. 34, 92-97. northeastern Brazil. J. Anim. Sci. 63, 1078- Sharp, R., Ziemer, C. J., Stern, M. D., and Stahl, D. 1086. A. 1998. Taxon-specific association between Pillard, D., Mckain, N., Rinon, M. T., Shingfield, protozoal and methanogen populations in the K. J., Givens, D. I., and Wallace, R. J. 2007. rumen and a model rumen system. FEMS Quantification of ruminal Clostridium Microbiol. Ecol. 26, 71-78. proteoclasticum by real time PCR using a Shin, E. C., Choi, B. R., Lim, W. J., Hong, S. Y., molecular beacon approache. J. Appl. An, C. L., Cho, K. M., Kim, Y. K., An, J. M., Microbiol. 103, 1251-1261. Kang, J. M., Lee, S. S., Kim, H., and Yun, H. Popova, M., Martin, C., and Morgavi, D. P. 2010. D. 2004. Phylogenetic analysis of archaea in Improved protocol for high-quality co- three fractions of cow rumen based on the 16S extraction of DNA and RNA from rumen rDNA sequence. Anaerobe. 10, 313 319. digesta. Folia Microbiol. (Praha) 55, 368 Shipley, L. A. 1999. Grasers abd browsers: how 372. digestive morphology affect diet selection. Prins, R. A., and Van Hoven, W. 1977. Presented in Grazing behavior of Carbohydrate fermentation by the rumen cilia livestock and wildlife accessed from Isotricha prostoma. Protistologica. 13, www.cnr.uidaho.edu. 549- 556. Silanikove, N. 1992. Effect of water scarcity and Qiuck, T. C., and Dehority, B. A. 1986. A hot environment on appetite and digestion in comparative study of feeding behavior and ruminants: a review. Livest. Prod. Sci. 30, digestive function in dairy goats, wool 175-194. sheep and hair sheep. J. Anim. Sci. 63, 1516- Silanikove, N. 1994. The struggle to maintain 1526. hydration and osmoregulation in animals Rattray, R. M., and Craig, A. M. 2007. Molecular experiencing severe dehydration and rapid characterisation of sheep ruminal enrichments rehydration: the story of ruminants. Exper. that detoxify pyrrolizidine alkaloids by Physiol. 79, 281-300. denaturing gradient gel electrophoresis. Silanikove, N., Nitsan, Z., and Perevolotsky, A. Microbial Ecol. 54, 264-275. 1994. Effect of daily supplementation of Regensbogenova, M., McEwan, N. R., Javorsky, polyethylene glycol on intake and P., Kisidayova, S., Michalowski, T., Newbold, digestion of tannin-containing leaves C. J., Hackstein, J. H., and Pristas, P. 2004a. A (Ceratonia siliqua) by sheep. J. Agric. Food re-appraisal of the diversity of the Chem. 42, 2844-2847. methanogens associated with the rumen Silanikove, N., Tagari, H., and Shkolnik, A. 1993. ciliates. FEMS Microbiol. Lett. 238, 307 313. Comparison of rate passage fermentation rate Regensbogenova, M., Pristas, P., Javorsky, P., and efficiency of digestion of high fiber diet in Moon-van der Staay, S. Y., Moon-van der desert black Bedouin goats as compared to Staay, G. W., Hackstein, J. H., McEwan, N. Swiss Saanen goats. Small Rumin. Res. R., and Newbold, C. J. 2004b. Assessment of 12, 45-60. ciliates in the sheep rumen by DGGE. Lett. Singh, M., More, T., and Rai, A. K. 1980. "Heat Appl. Microbiol. 39, 144-147 tolerance of different genetic groups of sheep Robbins, C. H., Spalinger, D. E., and Van Hoven, exposed to temperature conditions." J. W. 1995. Adaptation of ruminants to browse Agric. Sci. Camb. 94, 63-81. and grass diet: are anatomical-based browser- Skene, I. K., and Brooker, J. D. 1995. grazer interpretations valid? Oecologia. 103, Characterization of tannin acyl hydrolase 208-213. activity in the ruminal bacterium Selenomonas Santra, A., and Karim, S. A. 2003. Rumen ruminantium. Anaerobe. 1, 321-327. Manipulation to improve animal productivity. Skillman, L. C., Evans, P. N., Strompl, C., and Asian-Aust. J. Anim. Sci. 16 (5): 748-763. Joblin, K. N. 2006a. 16S rDNA directed PCR Seth, O. N., Rai, G. S., Yadav, P. C., and Pandey, primers and detection of methanogens in the M. D. 1976. A note on the rate of secretion bovine rumen. Lett. Appl. Microbiol. 42, 222-

704 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

228. Morand-Fehr, P., (Ed.), Goat Nutrition, Skillman, L. C., Toovey, A. F., Williams, A. J., and Pudoc Wageningen. Wright, A. D. G. 2006b. Development and Watson, C., and Norton, S. W. 1982. The validation of a real-time PCR method to utilisation of pang & grass by sheep and quantify rumen protozoa and examination of Angora goats. P. Australian Soc. Anim. variability between protozoal populations in Prod. 74, 467-470. sheep fed hay based diet. Appl. Environ. Whitford, M. F., Forster, R. J., Beard, C. E., Microbiol. 72, 200-206. Gong, J., and Teather, R. M. 1997. Soest, P. J. V. 1982. Nutritional Ecology of Phylogenetic analysis of rumen bacteria by Ruminant. O. and B. Books, Corvallis. comparative sequence analysis of cloned 16S Springer, E., Sachs, M. S., Woese, C. R., and rRNA genes. Anaerobe. 4, 153-163. Boone, D. R. 1995. Partial gene sequences for Whitford, M. F., Forster, R. J., Beard, C. E., Gong, the A subunit of methyl-coenzyme M J., and Teather., R. M. 1998. Phylogenetic reductase (mcrI) as a phylogenetic tool for the analysis of rumen bacteria by comparative family Methanosarcinaceae. Int. J. Syst. sequence analysis of cloned 16S rRNA genes. Bacteriol. 45, 554 559. Anaerobe. 4, 153-163. Stahl, D. A., Flesher, B., Mansfield, H. R., and Williams, Y. J., Popovski, S., Rea, S. M., Skillman, Montgomery, L. 1988. Use of phylogenetically L. C., Toovey, A. F., Northwood, K. S., and based hybridization probes for studies of Wright, A. D. 2009. A vaccine against rumen ruminal microbial ecology. Appl. Environ. methanogens can alter the composition of Microbiol. 54, 1079-1084. archaeal populations. Appl. Environ. Stiversion, J., Morrision, M., and Yu, Z. 2011. Microbiol. 75, 1860-1866. Population of select cultured and uncultured Wolin, M. J., and Miller, T. L. 1988. Microbe bacteria in the rumen of sheep and the effect of interactions in the rumen microbial ecosystem. diets and ruminal fractions. Int. J. Microbiol. in: Hobson, P. N. (Ed.), The Rumen Article ID 750613, 8 pages. Ecosystem. Elsevier Applied Science, New Sylvester, J. T., Karnati, S. K. R., Yu, Z., York. Morrison, M. and Firkins, J. L. 2004. Wolin, W. J. 1979. The rumen fermentation: a Development of an assay to quantify rumen model for microbial interactions in anaerobic ciliate protozoal biomass in cows using real- ecosystems. Adv. Microb. Ecol. 3, 49-77. time PCR. J. Nutri. 134, 3378-3384. Wood, J., Scott, K. P., Avgustin, G., Newbold, C. Tajima, K., Aminov, R. I., Nagamine, T. H., J., and Flint, H. J. 1998. Estimation of the Matsui, M., and Nakamura Benno, Y. 2001a. relative abundance of different Bacteroides Diet-dependent shifts in the bacterial and Prevotella ribotypes in gut samples by population of the rumen revealed with real- restriction enzyme profiling PCR-amplified time PCR. Appl. Environ. Microbiol. 67, 2766- 16S rRNA gene sequences. Appl. Environ. 2774. Microbiol. 64, 3683-3689. Tajima, K., Aminov, R. I., Nagamine, T., Ogata, Wright, A. D., Auckland, C. H., and Lynn, D. H. K., Nakamura, M. Matsui, H., and Benno, Y. 2007. Molecular diversity of methanogens in 1999. Rumen bacterial diversity as determined feedlot cattle from Ontario and Prince Edward by sequence analysis of 16S rDNA libraries. Island, Canada. Appl. Environ. Microbiol. 73, FEMS Microbiol. Ecol. 29, 159-169. 4206-4210. Tajima, K., Nagamine, T., Matsui, H., Nakamura, Wright, A. D., Toovey, A. F., and Pimm, C. L. M., and Aminov, R. I. 2001b. Phylogenetic 2006. Molecular identification of analysis of archaeal 16S rRNA libraries from methanogenic archaea from sheep in the rumen suggests the existence of a novel Queensland, Australia reveals more uncultured group of archaea not associated with known novel archaea. Anaerobe. 12, 134-139. methanogens. FEMS Microbiol. Lett. 200, 67- Wright, A. D., Williams, A. J., Winder, B., 72. Christophersen, C. J., Rodgors, S. L., and Tatsuoka, N., Mohammed, N., Mitsumori, M., Smith, K. D. 2004. Molecular diversity of Hara, K., Kurihara, M., and Itabashi, H. 2004. rumen methanogens from sheep in Western Phylogenetic analysis of methyl coenzyme-M Australia. Appl. Environ. Microbiol. 70(3), reductase detected from the bovine rumen. 1263-1270. Lett. Appl. Microbiol. 39, 257-260. Wright, A. G., and Pimm, C. 2003. Improved Tisserand, J. L., Hadjipanayiotou, M., and Gihad, strategy for presumptive identification of E. A. 1991. Digestion in goats, chapter 5, in: methanogens using 16S riboprinting.

705 Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 684-706

J. Microbiol. Meth. 55 (2), 337-349. Yanagita, K., Manome, A., Meng, X. Y., Handa, S., Kanagawa, T., Tsuchida, T., Mackie, R. J., and Kamagata, Y. 2003. Flow cytrometric sorting, phylogenetic analysis and in situ detection of Oscilliospira guillermondii a large morphologically conspicuous but uncultured ruminal bacterium. Int. J. Syst. Evol. Microbiol. 53, 1609-1614. Zeikus, J. G. 1977. The biology of methanogenic bacteria. Bacteriol. Rev. 41(2), 514-541. Zhou, M., Hernandez-Sanabria, E., and Guan, L. L. 2009. Assessment of the microbial ecology of ruminal methanogens in cattle with different feed efficiencies. Appl. Environ. Microbiol. 75, 6524 6533. Zhou, M., Hernandez-Sanabria, E., and Guan, L. L. 2010. Characterization of variation in rumen methanogenic communities under different dietary and host feed efficiency conditions, as determined by PCR-denaturing gradient gel electrophoresis analysis. Appl. Environ. Microbiol. 76, 3776-3786. Zhou, Y. W., Mcsweeney, C. S., Wang, J. K. and Liu, J. X. 2012. Effects of disodium fumarate on ruminal fermentation and microbial communities in sheep fed on high forage diets. Animal. 6(5), 815-823. Ziemer, C. J., Sharp, R., Stern, M. D., Cotta, M. A., Whitehead, T. R., and Stahl, D. A. 2000. Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA- targeted probes. Environ. Microbiol. 2, 632- 643. Zinder, S. H. 1993. Physiological ecology of methanogens. In: Ferry, J.G. (Ed.), Methanogenesis: Ecology, Physiology, Biochemistry and Genetics. Chapman and Hall, New York, pp.128 206

706