No abstract

Twenty male crossbred Texel lambs were used in a 2 × 2 factorial design experiment to assess the effect of dietary addition of nitrate (2.6% of dry matter) and sulfate (2.6% of dry matter) on enteric methane emissions, rumen volatile fatty acid concentrations, rumen microbial composition, and the occurrence of methemoglobinemia. Lambs were gradually introduced to nitrate and sulfate in a corn silage-based diet over a period of 4 wk, and methane production was subsequently determined in respiration chambers. Diets were given at 95% of the lowest ad libitum intake observed within one block in the week before methane yield was measured to ensure equal feed intake of animals between treatments. All diets were formulated to be isonitrogenous. Methane production decreased with both supplements (nitrate: -32%, sulfate: -16%, and nitrate+sulfate: -47% relative to control). The decrease in methane production due to nitrate feeding was most pronounced in the period immediately after feeding, whereas the decrease in methane yield due to sulfate feeding was observed during the entire day. Methane-suppressing effects of nitrate and sulfate were independent and additive. The highest methemoglobin value observed in the blood of the nitrate-fed animals was 7% of hemoglobin. When nitrate was fed in combination with sulfate, methemoglobin remained below the detection limit of 2% of hemoglobin. Dietary nitrate decreased heat production (-7%), whereas supplementation with sulfate increased heat production (+3%). Feeding nitrate or sulfate had no effects on volatile fatty acid concentrations in rumen fluid samples taken 24h after feeding, except for the molar proportion of branched-chain volatile fatty acids, which was higher when sulfate was fed and lower when nitrate was fed, but not different when both products were included in the diet. The total number of rumen bacteria increased as a result of sulfate inclusion in the diet. The number of methanogens was reduced when nitrate was fed. Enhanced levels of sulfate in the diet increased the number of sulfate-reducing bacteria. The number of protozoa was not affected by nitrate or sulfate addition. Supplementation of a diet with nitrate and sulfate is an effective means for mitigating enteric methane emissions from sheep.

1. An isotope tracer method for estimating methane production in sheep is described.2. The technique was used to estimate methane produced in both the upper and lower digestive tract and to determine the routes by which it was excreted.3. Four Merino ewes given lucerne chaff (33 g every hour) were used.4. Total methane production rate was 21±1.1 (se) ml/min; production in the rumen accounted for 87±1.2% of the total production; 95±1.4% of the methane produced in the rumen was excreted by eructation.5. Of the methane produced in the lower digestive tract, 89±2.3% was excreted through the lungs and 11% through the anus.

I. To obtain a quantitative model for nitrogen pathways in sheep, a study of ammonia and urea metabolism was made by using isotope dilution techniques with ['SN]ammonium sulphate and [Wlurea and [14C]urea.2. Single injection and continuous infusion techniques of isotope dilution were used for measuring ammonia and urea entry rates.3. Sheep were given 33 g of chaffed lucerne hay every hour; the mean dietary N intake was 23.4 gjd.4. It was estimated that 59 yo of the dietary N was digested in the reticulo-rumen; 29 yo of the digested N was utilized as amino acids by the micro-organisms, and 71 % was degraded to ammonia.5. Of the 14'2 g N/d entering the ruminal ammonia pool, 9.9 g N/d left and did not return to the pool, the difference of 4'3 g N/d represented recycling, largely within the rumen itself (through the pathways : ruminal ammonia + microbial protein + amino acids --z ammonia).6. Urea was synthesized in the body at a rate of 18.4 g N/d from 2.0 g N/d of ammonia absorbed through the rumen wall and 16.4 g N/d apparently arising from deaniination of amino acids and ammonia absorbed from the lower digestive tract. 7.In the 24 h after intraruminal injection of [15N]ammonium salt, 40-50 % of the N entering the plasma urea pool arose from ruminal ammonia; 26% of the lSN injected was excreted in urinary N.8. Although 5-1 g N/d as urea was degraded apparently in the digestive tract, only 1.2 g N/d appeared in ruminal ammonia; it is suggested that the remainder may have been degraded in the lower digestive tract. 9. A large proportion of the urea N entering the digestive tract is apparently degraded and absorbed and the ammonia incorporated in the pools of nitrogenous compounds that turn over only slowly. This may be a mechanism for the continuous supply to the liver of ammonia for these syntheses.10. There was incorporation of 16N into bacterial fractions isolated from rumen contents after intraruminal and intravenous administration of [15N]ammonium salts and [lSN]urea respectively. 11. A model for N pathways in sheep is proposed and, for this diet, many of the pool sizes and turn-over rates have been either deduced or estimated directly.

Nitrogen metabolism is reviewed with emphasis on methods for quantitating various nitrogen-transactions in the rumen of animals on a variety of diets. Ammonia kinetics, microbial cell synthesis, the inputs of endogenous nitrogen, degradation of dietary protein, and availability to the animal of dietary bypass protein are discussed. The efficiency of microbial protein from the rumen is discussed in relation to the ratio of protein to energy in the nutrients available to meet the requirements of the animal. The ratio is determined largely by the maintenance requirements of microbes and the breakdown of microbial materials, which result in the recycling of microbial nitrogen in the rumen. Emphasis is placed on the role of rumen protozoa in decreasing the ratio of protein to energy in absorbed nutrients in ruminants on diets that are marginally deficient in protein. Recent studies of the dynamics of protozoa in the rumen and their contribution to microbial protein outflow are summarized.

Abstract. Minimising enteric CH 4 emissions from ruminants is a current research priority because CH 4 contributes to global warming. The most effective mitigation strategy is to adjust the animal's diet to complement locally available feed resources so that optimal production is gained from a minimum of animals. This essay concentrates on a second strategy -the use of feed additives that are toxic to methanogens or that redirect H 2 (and electrons) to inhibit enteric CH 4 emissions from individual animals. Much of the published research in this area is contradictory and may be explained when the microbial ecology of the rumen is considered.Rumen microbes mostly exist in organised consortia within biofilms composed of self-secreted extracellular polymeric substances attached to or within feed particles. In these biofilms, individual colonies are positioned to optimise their use of preferred intermediates from an overall process of organic matter fermentation that generates end-products the animal can utilise. Synthesis of CH 4 within biofilms prevents a rise in the partial pressure of H 2 (pH 2 ) to levels that inhibit bacterial dehydrogenases, and so reduce fermentation rate, feed intake and digestibility. In this context, hypotheses are advanced to explain changes in hydrogen disposal from the biofilms in the rumen resulting from use of anti-methanogenic feed additives as follows.Nitrate acts as an alternative electron sink when it is reduced via NO 2 -to NH 3 and CH 4 synthesis is reduced. However, efficiency of CH 4 mitigation is always lower than that predicted and decreases as NO 3 -ingestion increases. Suggested reasons include (1) variable levels of absorption of NO 3 -or NO 2 -from the rumen and (2) increases in H 2 production. One suggestion is that NO 3 -reduction may lower pH 2 at the surface of biofilms, thereby creating an ecological niche for growth of syntrophic bacteria that oxidise propionate and/or butyrate to acetate with release of H 2 .Chlorinated hydrocarbons also inhibit CH 4 synthesis and increase H 2 and formate production by some rumen methanogens. Formate diffuses from the biofilm and is converted to HCO 3 -and H 2 in rumen fluid and is then excreted via the breath. Short-chain nitro-compounds inhibit both CH 4 and formate synthesis when added to ruminal fluid but have little or no effect in redirecting H 2 to other sinks, so the pH 2 within biofilms may increase to levels that support reductive acetogenesis. Biochar or activated charcoal may also alter biofilm activity and reduce net CH 4 synthesis; direct electron transfer between microbes within biofilms may also be involved. A final suggestion is that, during their sessile life stage, protozoa interact with biofilm communities and help maintain pH 2 in the biofilm, supporting methanogenesis.

I.A study of ammonia and urea metabolism in sheep was made using isotope dilution techniques with (lSNH4),S04, [lSN]urea and [Wlurea in order to determine quantitatively the movements of urea-N and NH,-N throughout the body of normal, feeding sheep.2. Single injections of 15N-labelled compounds were made into the rumen fluid NH,, caecal fluid NH, and the blood urea pools, in order to estimate the rates of flux through, and the transfer of N between, these and other nitrogenous pools in the body. fWr EDTA was injected into the rumen and caecum with (16NH&S0, to allow estimation of fluid volumes and to provide an indication of mixing, and of times of transit of isotopes between different sampling sites in the digestive tract.3. The sheep ate approximately 22 g lucerne chaffjh and the mean dietary N intake was 16.3 g/d.4. The rate of flux of NH, through the rumen NH, pool was 15.0 g/d (i.e. 90y0 of the dietary N ingested; however, this amount also included N from plasma urea (1.1 g/d) and other endogenous sources including NH, derived from caecal NH, (0.4 g/d).5. Only 40% of the N in isolated rumen bacteria was derived from NH,, indicating that a considerable proportion of their N requirements were obtained from compounds other than NH, (e.g. peptides and amino acids).6. There was evidence of recycling of N between nitrogenous pools in the rumen, probably through rumen NH, -+ microbial N --+ NH,. 7.It was estimated that 5-3 g blood urea-N/d entered the digestive tract: 20% of this urea was degraded in the rumen, 25 yo in the caecum and the remainder was apparently degraded elsewhere; there was evidence of urea degradation in the large intestine posterior to the caecum and it is suggested that urea degradation and absorption of the resultant NH, may occur in the ileum. 9.A large proportion of the NH, entering the caecal NH, pool (70% or 3.2 g N/d) was apparently derived from degradation of nitrogenous products, other than urea, including rumen microbial N (1.0 g N/d) passing undigested from the small intestine. 10. Less than half the NH,-N of caecal origin entering the rumen passed through the blood urea pool; the remainder was apparently transported as other nitrogenous compounds in the blood or body fluids.11. The results of the three experiments were combined in a general three-pool, opencompartment model which formally recognizes an unlimited number of other unspecified, interconnected pools together comprising the whole-animal system. Rates of flux through, and transfer of N between these and other nitrogenous pools in the body were calculated by solving this model and the information derived has been applied to whole-animal models with a view to subsequently using these models in computer simulation studies.

I . The entry rates of urea into the urea pool of the body fluids have been measured in sheep 2 . Results obtained with a single injection and with continuous infusions of [l'C]urea were 3. The difference between the entry rate and the rate of excretion of urea in the urine was 4. Plasma concentrations and urea entry rates were significantlj and linearly related. 5. The relationship between excretion rate and plasma urea concentration was best described by a cubic equation.6. Degradation of urea in sheep was found to be extensive in all the animals studied ; as the protein intake increased, the quantity of urea degraded also increased but the percentage of ureaentering the body pool thatwas degraded was decreased. Animals given a ration containing 3.5 yo crude protein degraded 76-92 yo of the urea entering the body pool. 7. A rectilinear relationship was found between pool size and plasma urea concentration. The urea space in animals given low-protein rations was significantly less than in animals on highprotein rations.8. The effects of starvation for 2, 4 and 6 days on urea metabolism in sheep were investigated. In a11 the sheep starved for 2 days there was a significant increase in urea pool size, but the entry rate was markedly depressed indicating a retention of urea in the body pool on starvation.9. A significant amount of nitrogen was found to g o through the system: rumen ammonia-+ portal blood ammonia-tblood urea-wumen ammonia.10. Urea excretion rate, urea clearance by the kidney, urine flow rate and the ratio of the concentration of urea in urine to that in plasma (urea U: P ratio) were also examined.I I . There were significant correlations between urine flow rate and urea excretion and between plasma urea concentration and urine flow rate.given rations varying in crude protein percentage from 3.5 to 27'3-essentially the same.taken to indicate the quantity of urea degraded in the alimentary tract.