1. The paper describes the development and construction of an apparatus for maintaining a normal microbial population of the rumen under strictly controlled conditions over long periods of time, 2. The apparatus is simple to construct and operate. It is possible to do four replicate experiments at the same time.3. The results of three experiments are given. The experiments showed that when the steady-state was reached it could be maintained indefinitely, with the type and quantities of products of fermentation very similar to those in the rumen of donor animals, including the maintenance of normal protozoal populations for up to 49 d.4. It was found that within wide ranges, the digestibility of rations and the output of products were independent of dilution rate.5. Except for the lowest 'level of feeding', the digestibility was independent of the level of feeding. The output of products was proportional to the amount of food digested and was the same as would be expected in sheep on similar rations.6. An experiment in which a ration of hay was changed to a mainly concentrate ration showed that the fermentation characteristics were determined mainly by the food given.Many types of artificial rumen apparatus have been described (see review Czerkawski, 1976~). The apparatus constructed in this laboratory (Czerkawski & Breckenridge, 1969) was designed specifically for short-term work involving 6-8 h incubations and the possibility of measuring the gaseous exchanges was one of the main requirements. This apparatus and a small-scale version of it (Czerkawski & Breckenridge, 1970) have been used extensively in the studies of the mechanism of methane production and its inhibition. It was possible to 'trace out' several biochemical pathways and to develop new inhibitors of methane production.Several experiments in vivo (Czerkawski, Christie, Breckenridge & Hunter, 1975; Czerkawski, 19763) showed that the inhibition of methane production gives rise to gross changes in rumen metabolism. Some of these changes come about gradually over a period of weeks rather than days or hours and it is difficult to exert sufficient control or to interpret the results of experiments with animals. Therefore there was great need for a suitable longterm artificial rumen technique. A simple continuous culture procedure was discounted because it does not simulate the conditions in the rumen sufficiently (e.g., see Isaacson, Hinds, Bryant & Owens, 1975). Usually the protozoal concentrations decrease and often protozoa disappear altogether. Sometimes the bacterial metabolism bears little relation to that of the rumen of the donor animal and it is often necessary to stimulate the fermentation by infusing clarified rumen fluid. Another drawback in using a commercial fermentor is the provision of one or at most two reaction vessels; this limits severely the type of comparative work that can be undertaken.In designing the apparatus described here the following requirements had to be met: (a) the reaction vessels should contain solid phase (partly ...

Microbial fractions, comprising protozoa, large and small bacteria and whole particulate matter, have been isolated from rumen contents of sheep given a mainly concentrate diet, a mixture of hay and concentrate, and hay only. Samples of rumen contents were taken before and 2 h after feeding. The main components determined were: protein, lipid, nucleic acids, carbohydrate and ash. The amount of cell wall was estimated in terms of known cell wall constituents (diaminopimelic acid (DAP) and glucosamine). The concentration of some of the constituents varied with diet and with respect to the time of feeding. Many of the differences disappeared when the results were expressed on a polysaccharide-free basis. The amino acid composition of large and small bacteria was virtually the same. The amino acid composition of protozoa was similar except for the proportions of glutamic acid and lysine which were greater in protozoa, and alanine, glycine and DAP, the proportions of which were greater in bacteria. There were higher proportions of protein in large bacteria and protozoa than in small bacteria. Small bacteria contained more lipid, ash and DNA, and less RNA than the other two fractions. The polysaccharide content of protozoa and large bacteria increased from about 8 % before feeding to about 30 % after feeding, while the polysaccharide content of small bacteria increased only slightly after feeding.

I .Nine experiments, each with one of six sheep with cannulated rumens given a constant diet of dried grass, were made in which oleic, linoleic or linolenic acid was infused into the rumen and energy and lipid metabolism were measured. One experiment was made in which palmitic acid was given. 2. Judged by changes in the composition of isolated fatty acids, the unsaturated fatty acids were hydrogenated in the rumen. An increase in the excretion of lipid in the faeces occurred when the unsaturated acids were given. The heat of combustion of the faeces increased by 12.6 3.0 kcal/Ioo kcal fatty acid, of which 94 yo was accounted for by the additional lipid. 3. Methane production fell when the unsaturated fatty acids were infused, the decreases being 13.8 k 1.6 kcal CH,/IOO kcal oleic acid, 142 f 1-5 kcal CH,/IOO kcal linoIeic acid and 16.4+ 1.3 kcal CH,/IOO kcal Iinolenic acid. The introduction of a double bond into an n-alkyl acid was calculated to reduce methane production by 0.24 f 0.09 moles/ mole double bond. 4. Because the depression of methane production on infusing the fatty acids exceeded the increase in the heat of combustion of the faeces, the metabolizable energy of the fatty acids was 104.1 k5.3 % of their heat of combustion. 5. The efficiencies with which the fatty acids were used to promote energy retention were 74.6 5 7 % for oleic acid, 79.2 f 2.0 % for linoleic acid and 82.5 f 3.0 yo for linolenic acid. These efficiencies agreed with those noted in experiments by others with rats, horses and pigs given glycerides, but were higher than those noted by others when glycerides were added to the diets of ruminants.A part of the methane produced by micro-organisms in the digestive tract of ruminants arises from the reduction of carbon dioxide. This reduction accompanies the oxidation of formic acid in the rumen, indeed formic acid when added to rumen contents in vitro, or given to sheep leads to the production of methane, I mole formic acid giving rise to 0.25 moles CH, (Carroll & Hungate, 1955 ; Vercoe & Blaxter, 1965). Since the CO, reduced is identical with the CO, pool of the rumen (Williams, Hoernicke, Waldo, Flatt & Allison, 1963) it is possible that hydrogen acceptors other than CO, added to the rumen might reduce methane production. Accordingly, linolenic acid was given to a sheep by intraruminal infusion and it was found that the methane production of the sheep fell markedly. The fall in methane production, however, was considerably greater than that expected even assuming that all three double bonds of the linolenic acid had been hydrogenated. This paper deals with the primary observations made with linolenic acid and with similar experiments in which oleic and linoleic acid were given to sheep. E X P E R I M E N T A LAnimals. Six castrated male sheep each with a permanent cannula inserted in the rumen were used as experimental animals.Food and fatty acids. Artificially dried grass was given as the only solid food. The amounts given were either 900 or 1000 g daily in two meals and in any one exp...

No abstract

The normal process of methane production in ruminants is described and it is pointed out that about 8 % of the energy of the diet is inevitably lost to the animal as methane. Experiments are described which show that fatty acids and other alkyl compounds added to the diet or infused into the rumen reduce methane production by, in some instances, as much as two-thirds. Although methanogenesis is markedly depressed, with some compounds there is little concomitant depression of the digestion of cellulose or indeed of the non-lipid organic constituents of the diet. The implications of these findings are discussed.The ability of herbivorous mammals to obtain energy from the cellulose and hemicelluloses of plants is due to the activity of the microbial populations of their digestive tracts. Micro-organisms in the gut degrade cellulosic materials with the formation of characteristic end products, notably steamvolatile fatty acids with 1-5 carbon atoms, some dicarboxylic and tricarboxylic acids, carbon dioxide, methane and on occasions hydrogen. The microbial populations increase at the expense of the free energy of these degradations, synthesising large amounts of nucleic acids and proteins. Many of the organisms are swept on from the sites where the fermentation takes place to be digested by enzymes secreted by the host or to be expelled in the faeces. In addition, heat is generated.Generally speaking, the biologically useful energy which a herbivore obtains from its normal diet is a much smaller proportion of its total energy than is the proportion obtained by omnivores subsisting on diets rich in oligosaccharides which can be hydrolysed without microbial assistance. The reasons for this difference are, firstly, microbial degradation of cellulose is a slow process and rarely goes to completion: much of the cellulose is excreted in the faeces unchanged, particularly if the cellulose is lignified. Secondly, the end products of microbial digestion which are absorbed are relatively small molecules, and the energy required for their activation before they can enter the cycles of intermediary metabolism are a high proportion of the free energy they eventually provide.1 To activate one mole of acetic acid by forming acetyl-coenzyme A, involves the expenditure of 2 moles of high-energy bonds of adenosine triphosphate: to activate one mole of glucose involves but one. Thirdly, a proportion of the energy derived from the microbial fermentation is diverted to synthesis of compounds by the microorganisms which are subsequently lost to the animal. Thus purines synthesised by bacteria are converted after absorption to allantoin and excreted in the urine. Lastly, a considerable part of the energy of the cellulose which is degraded by microorganisms is lost to the animal as heat and as methane.This paper is concerned entirely with the extent of the loss of energy by herbivores as methane, and gives an account of investigations we have made with a view to its control in ruminant animals (Czerkawski et ~1 .~) ) . Methane production of ...