The effects of dietary nitrate on DM digestion, rumen volatile fatty acid concentrations, microbial protein outflow, rumen water kinetics, and methane production were studied. Eight rumen-cannulated sheep were acclimated to a diet consisting of chaffed oaten hay supplemented with either 4% KNO3 or 0% KNO3 but made iso-nitrogenous by the addition of urea. Nitrate supplementation did not affect blood methaemoglobin concentration, DM intake, whole tract or ruminal DM digestibility and the sheep appeared healthy at all times throughout the acclimation and experimental periods. Nitrate did cause changes in rumen fermentation consistent with its acting as a high-affinity hydrogen acceptor, i.e. there was a tendency towards a lower molar percentage of propionate in the rumen volatile fatty acids, and higher molar ratio of acetate to propionate. Methane yield (MY, L methane/kg DM intake) was reduced by 23% in KNO3-supplemented sheep (P < 0.05) and these sheep tended to have a shorter mean fluid retention time in the rumen (MRT). There was a significant association between MRT and MY, such that a shorter MRT was associated with a lower MY. The results confirmed that the presence of nitrate in the diet lowers enteric methane production even though there was considerable between-animal variation in gut kinetics and methane production.

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Inclusion of nitrate (NO3−) in ruminant diets is a means of increasing non-protein nitrogen intake while at the same time reducing emissions of enteric methane (CH4) and, in Australia, gaining carbon credits. Rumen microorganisms contain intracellular enzymes that use hydrogen (H2) released during fermentation to reduce NO3− to nitrite (NO2−), and then reduce the resulting NO2− to ammonia or gaseous intermediates such as nitrous oxide (N2O) and nitric oxide (NO). This diversion of H2 reduces CH4 formation in the rumen. If NO2− accumulates in the rumen, it may inhibit growth of methanogens and other microorganisms and this may further reduce CH4 production, but also lower feed digestibility. If NO2− is absorbed and enters red blood cells, methaemoglobin is formed and this lowers the oxygen-carrying capacity of the blood. Nitric oxide produced from absorbed NO2− reduces blood pressure, which, together with the effects of methaemoglobin, can, at times, lead to extreme hypoxia and death. Nitric oxide, which can be formed in the gut as well as in tissues, has a variety of physiological effects, e.g. it reduces primary rumen contractions and slows passage of digesta, potentially limiting feed intake. It is important to find management strategies that minimise the accumulation of NO2−; these include slowing the rate of presentation of NO3– to rumen microbes or increasing the rate of removal of NO2−, or both. The rate of reduction of NO3− to NO2− depends on the level of NO3− in feed and its ingestion rate, which is related to the animal’s feeding behaviour. After NO3− is ingested, its peak concentration in the rumen depends on its rate of solubilisation. Once in solution, NO3− is imported by bacteria and protozoa and quickly reduced to NO2−. One management option is to encapsulate the NO3− supplement to lower its solubility. Acclimating animals to NO3− is an established management strategy that appears to limit NO2− accumulation in the rumen by increasing microbial nitrite reductase activity more than nitrate reductase activity; however, it does not guarantee complete protection from NO2− poisoning. Adding concentrates into nitrate-containing diets also helps reduce the risk of poisoning and inclusion of microbial cultures with enhanced NO2−-reducing properties is another potential management option. A further possibility is to inhibit NO2− absorption. Animals differ in their tolerance to NO3− supplementation, so there may be opportunities for breeding animals more tolerant of dietary NO3−. Our review aims to integrate current knowledge of microbial processes responsible for accumulation of NO2− in rumen fluid and to identify management options that could minimise the risks of NO2− poisoning while reducing methane emissions and maintaining or enhancing livestock production.

The effects of dietary nitrate (NO3) and elemental sulfur (S) on nutrient utilisation, productivity, and methane emission of Merino lambs were investigated. Forty-four lambs were randomly allocated to four groups (n = 11) fed isonitrogenous and isoenergetic diets. The basal feed was supplemented with 1% urea + 0.18% S (T1), 1.88% NO3 + 0% S (T2), 1.88% NO3 + 0.18% S (T3), or 1.88% NO3 + 0.40% S (T4). Retention of S was improved by increasing the content of elemental S in the NO3-containing diet (P < 0.001), yet the N retention (g/day) by the animal, and the N and S content of wool (%), were not altered by S supplementation (P > 0.05). Dry matter intake, liveweight gain, and feed conversion ratio did not differ (P > 0.05) between treatments. Replacing urea with NO3 improved the rate of clean wool growth by 37% (P < 0.001, T1 vs T3). Clean wool growth increased by 26% (P < 0.001) when the S content of the NO3-containing diet was increased from 0 to 0.18% (T2 vs T3). Methane production (g/day) and methane yield (g/kg DM intake) were reduced (P < 0.05) by 24% when urea was replaced by NO3 (T1 vs T3). The addition of 0.4% S to a diet containing 1.88% NO3 also reduced methane production (P = 0.021) and methane yield (P = 0.028). In conclusion, the addition of 1.88% NO3 and 0.18% elemental S to a total mixed diet increased clean wool production and reduced methane production. However, there was no evidence of inter-relationships between NO3 and S.

The effects of dietary nitrate and of Propionibacterium acidipropionici (PA) on methane and nitrous oxide emissions, methaemoglobinaemia, volatile fatty acid (VFA) concentration and productivity of sheep were studied. It was hypothesised that PA supplementation would increase the rate of nitrite reduction to ammonia in the rumen and therefore reduce risks of methaemoglobinaemia. Fine-wool Merino wethers (n = 28; 31.8 ± 3.7 kg; 11 months of age) were acclimated to four isonitrogenous and isoenergetic diets based on oaten chaff (1.0 kg/day) supplemented with either urea (1.1% of DM; T1 and T2) or a nitrate source (2.0% of DM; T3 and T4) while T2 and T4 were also supplemented with PA (11.5 × 1010 CFU/day). Replacing urea with nitrate lowered methane production (g/day) by 19% and methane yield (g/kg DMI) by 15%, improved clean wool growth by 12% (P < 0.001) and tended to increase skin temperature (P < 0.1). Nitrate increased ruminal acetate to propionate ratio by 27%, increased plasma nitrite and nitrate concentrations and blood methaemoglobin (MetHb) level up to 45% of total haemoglobin. Nitrous oxide emission from sheep confined in respiration chambers was higher (P < 0.001) when nitrate was fed, lowering the net benefit of methane mitigation on global warming potential (CO2 equivalents/kg DMI) by 18%. In contrast, PA had little effect, decreasing total VFA concentration (P < 0.05), increasing rumen pH (P < 0.05) and clean wool growth (P < 0.05) of urea-fed sheep. This study confirmed the beneficial effects of nitrate on net greenhouse gas reduction and wool growth, but showed that methaemoglobinaemia risks may be higher when diets are fed at a restricted level and contain only low levels of readily fermented carbohydrate. PA supplementation was not effective in reducing methaemoglobinaemia, but did increase clean wool growth of urea-fed sheep.

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Methane yield from ruminants is positively correlated with mean retention time (MRT) of digesta, which is known to be influenced by the hormone triiodothyronine (T3).We hypothesised that a decrease in the MRT in the rumen in response to administration of a T3 solution to sheep would reduce their methane yield. To test this hypothesis, 10 mature Merino wethers were injected with T3 (300 µg) on two different protocols (daily; n = 5 and every second day; n = 5) and the effect on daily methane yield, digesta MRT, DM digestibility, rumen volatile fatty acid concentrations, microbial protein output and plasma T3 concentrations studied. Compared with when injected with saline (control), injection of sheep with T3 every second day resulted in decreased methane yield (P < 0.05) and lower acetate (P < 0.001), butyrate (P < 0.001) and propionate (P < 0.01) concentrations in the rumen. MRT of digesta, derived from faecal excretion of CoEDTA and Cr-mordanted fibre, were reduced in the total tract (P < 0.001) and hindgut (P < 0.01) but not in the rumen (P > 0.05). DM digestibility was not affected by injection of T3 every second day but water intake (P < 0.05) and urine output (P < 0.01) were increased. When sheep were injected with T3 daily, changes were only observed in plasma T3 concentration (P < 0.001) and volume of CO2 produced (P < 0.05). The results indicate that increasing plasma concentration of the thyroid hormone T3 within physiological levels reduces digesta retention time, especially retention time in the hindgut and leads to a reduction in enteric methane yield. Further work is warranted to assess whether plasma T3 concentrations may be indicative of enteric methane yield.

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