Metabolism of Dietary Nitrate and its Safe Use for Mitigating Methane Emissions from Sheep
Supplementing ruminants with dietary nitrate (NO3) is an effective methane mitigation strategy if it can be managed so as to not expose ruminants to any risk of clinical nitrite (NO2) toxicity. The objective of this thesis was firstly to deepen the understanding for NO3 metabolism in sheep and secondly to develop practical strategies to reducing risk of NO2 toxicity in sheep supplemented with dietary NO3.
It has been previously established, that in the rumen NO3 is reduced to NO2 and then to NH3, and that supplementing with excessive amounts of NO3 can expose ruminants to NO2 toxicity due to the absorption of NO2. This thesis reports a series of five investigations of NO3 metabolism by sheep and identifies:
Nitrate, like urea, is ‘recycled’ within the ruminant. Transfer of ruminal 15NO3--N into the blood and transfer of blood NO2-N into the rumen being quantified. Only 20% of rumen NO3-and 30% of blood NO2- were recovered in urine.
That in hourly fed sheep approximately 90% of dietary NO3- was rapidly converted to NH3 in the rumen, with the remainder leaving the rumen by absorption into the bloodstream or passage to the lower gastro-intestinal tract.
Within the rumen, the conversion of NO3-to NH3 is neither simple nor complete. In vitro and in-vivo studies showed NO3-is reduced to gaseous nitrous oxide (N2O) and N2O may be further metabolised to N2 gas by the rumen microbiota. Approximately 0.04% and 3.0% of dosed NO3--N was recovered over 10 h from sheep as N2O and N2 respectively, and this was not affected by whether sheep had prior adaption to NO3- or not, identifying denitrification as a reaction not previously reported from the rumen.
From this understanding and a review of the literature on ruminant NO3 metabolism, eight critical control points for reducing the risk of nitrite toxicity (methaemoglobinaemia), were identified and the potential for manipulating five of these evaluated.
Reducing the rate at which NO3 became available to the rumen biota by coating calcium nitrate with paraffin wax significantly reduced blood methaemoglobin level (MetHb; an indicator of NO2 toxicity) in sheep supplemented with NO3.
The extent of methaemoglobinaemia could also be reduced by the daily ration being consumed at shorter intervals rather than in a single bout, and this established that feed management is pivotal to safe feeding of NO3-containing diets.
Enhancing the rumen’s capacity to reduce potentially toxic NO2 -by supplying Propionibactericum acidicpropionici as a direct fed microbial was ineffective in reducing blood MetHb or NO2-concentration of sheep fed NO3- supplemented diets.
Attempts to increase the rate of removal of NO2-from the rumen by providing a substrate (glycerol) to stimulate NADH availability in the rumen, and accelerate the nitrite reductase enzyme system did not reduce the concentration of NO2 in incubations of rumen contents supplemented with NO3-.
We found no evidence that adapting sheep to dietary NO3- protected them against NO2- toxicity. Indeed, in vitro more NO2- accumulated in incubation when donors where adapted to dietary NO3-. Also, no signs of reduced MetHb were noticed after several weeks of NO3-supplementation in vivo.
Other critical control points such as regulating microbial uptake of NO3 and ruminal absorption of NO3 and NO2 were unable to be assessed in this thesis.
The studies reported here also confirmed the practical impacts of NO3 as an effective supplement for reducing enteric methane emissions and increasing wool growth of sheep. As well as providing a better understanding of NO3-metabolism, studies also showed that the greenhouse gas (GHG) abatement impact of methane mitigation may be partly offset by an associated production of the potent GHG, N2O. Discovery of the production of N2O and N2 from NO3-in the rumen and identification of recycling of blood NO2- to the rumen has expanded our understanding of NO3-metabolism. Coating NO3-to decrease the rapidity of NO3- release in the rumen as a strategy to reduce NO2 toxicity was effective but needs further investigation. The applicability of feed grade NO3-as a commercially available feed additive will also depend on the cost of NO3 and the additional cost of the technology to ensure its safe feeding, compared to the cheaper alternative non-protein nitrogen source, urea.
Quantifying daily methane production of beef cattle from multiple short-term measures using the GreenFeed system
On-farm CH₄ emissions have been identified as the largest contributors to the carbon footprint of livestock production systems. A requirement to quantify on-farm mitigation under commercial production conditions and a desire to establish the phenotype of thousands of ruminants for breeding programs, has fueled the development of techniques to estimate daily methane production (DMP) from short-term measures of methane concentration or methane flux.The accuracy, precision and applicability of these methods has been largely untested and forms the susbtance of this thesis. In assessing the accuracy of short-term emissions measures to estimate DMP, a high level of concordance was observed between DMP measured over 24h in a respiration chamber (RC) and estimated from multiple short-term measurement estimates using the GreenFeed Emission Monitoring system (GEM). Three independent experiments comparing DMP confirmed that estimates between methods differ by 5% to 8% (P>0.05). This implies that multiple short-term measures of emission rates are complementary to and consistent with respiration chamber-derived measures, providing capability to measure a greater number of animals, potentially in their production environment over extended periods of time. Methane yields (MY; g CH₄/kg DMI) were also derived based on multiple short-term emission measures, with results consistently within 10% of those calculated based on 24h RC data. The overall MY of animals consuming roughages was 21.8g CH₄/kg DMI using GEM data, in keeping with the 22.3g CH₄/kg DMI average in the literature. That implies that GEM units can not only accurately estimate DMP of cattle but also support accurate MY estimates that can be used in quantifying livestock emissions for national greenhouse inventory calculations.
Effect of measurement duration in respiration chambers on methane traits of beef cattle
Records on 1043 young Angus heifer and bull progeny from 73 sires, measured for methane production in respiration chambers, were used to evaluate the accuracy of a 1-day measurement relative to 2-day measurement duration. The traits assessed were dry matter intake (DMI, kg/day), methane-production rate (MPR, g/day), methane yield (MY, MPR per unit DMI) and four residual methane (RMP, g/day) traits. The RMP traits were computed as actual MPR minus expected MPR, where the expected MPR were calculated from three widely used equations. The expected MPR for the fourth RMP trait was computed by regressing MPR on DMI, using the data from the study. Variance components, heritability, phenotypic and genetic correlations, and the efficiency of selection using 1-day compared with 2-day measurement were used as assessment criteria. The environmental variance for the 2-day measurement was slightly lower than that of the 1-day measurement for all the traits studied, indicating that the addition of an extra day of data was effective in reducing the amount of unexplained variation in each trait. However, these minor reductions did not have a major impact on accuracy; hence, very high phenotypic (rp of 0.91-0.99) and genetic (rg of 0.99 for each trait) correlations were obtained between the two measurement durations. The very high genetic correlation between the two durations of measurement indicated that, at the genetic level, the 1-day duration is measuring the same trait as the 2-day measurement duration. Any enteric-methane emission-abatement strategy that seeks to reduce MPR per se, may have a detrimental impact on ruminant productivity through a correlated reduction in feed intake; hence, MY and the RMP traits are likely to be the traits of interest for genetic improvement. Efficiency of selection for MY and the RMP traits ranged from 0.96 to 0.99, which implies that there would be less than 5% loss in efficiency by adopting a 1-day relative to a 2-day methane-measurement duration. While the throughput of the respiration-chamber facility can be increased by adopting a 1-day measurement duration, additional resources, such as holding pens, would be required to take advantage of the extra day.
Repeatability of methane emissions from sheep
Breeding of ruminant livestock for low CH₄ emission is an attractive means of mitigating enteric CH₄ emissions. However success requires that the mechanism responsible for among animal variation in emissions is repeatable and heritable and has a negligible negative impact on production and functional traits. This study was designed to estimate repeatability and heritability of the CH₄4 emission trait in sheep, and to determine whether the ranking of sheep based on their CH₄ emissions is maintained over a range of contrasting diets. A flock of 105 ewe lambs (10 months old) of a progeny testing program were screened for their CH₄ yields (i.e., emissions/unit feed dry matter (DM) intake) when a molasses containing grass silage was fed at restricted intake (1.3 x maintained 13-15 d between consecutive measurements (i.e., screening phase). Mean CH₄ yield of lambs was 18.4 ± 0.38 g/kg DM intake during the screening phase, and estimates of repeatability and heritability for CH₄ yield were 0.16 and 0.30, respectively. Methane yield in the screening phase was 7.9% higher for the high versus low ranked sheep (19.2 ± 0.18 versus 17.8 ± 0.26 g/kg DM intake). The 10 lowest (low rank) and the 10 highest (high rank) CH₄ yielding sheep were selected and retained for further study. Two repeated measurements of CH₄ yield were conducted, the first measurement while sheep were fed fresh cut perennial ryegrass pasture (grass), the second with the same sheep fed a 400:600 concentrate:forage (wheat grain:lucerne hay; fresh basis) pelleted diet (pellet). Repeated measurements revealed that rankings were maintained among diets, but that there was a CH₄ rank x diet interaction for CH₄4 yield. When fed the grass diet, the high ranked sheep had 13% higher CH₄4 yield than the low ranked sheep, but when fed the pelleted diet, the high ranked sheep had 36% higher CH₄ yield than the low ranked sheep. Emissions of hydrogen were only measurable when sheep were fed the pelleted diet. This study is the first to report that ranking of sheep for CH₄4 emissions is consistent among diets, although the magnitude of difference among the rankings was affected by diet, suggesting that among animal variation in CH₄ emission could be exploited to breed animals for low CH₄ emission.
Lowering ruminant methane emissions through improved feed conversion efficiency
Improvements in feed conversion efficiency (FCE) can be applied to individual animals as well as to production from land, as in a farm system. Our focus relates mainly to food production from individual animals within any animal population where there is divergence in the efficiency that individuals use ingested feed for maintenance and production; primarily due to differences in digestion and metabolism. Intake variation from the predicted mean for individuals of a similar size and level of production in a population has been termed residual feed intake (RFI), with low values indicating an efficient animal. Efficient animals require less feed than average and can be expected to produce less CH₄ and N₂O per unit product than the population average at a similar level of production. Selection for this trait will lower CH₄ emissions per animal, unless more animals are kept to eat the feed not required by efficient animals. There are few published evaluations of CH₄ yields from animals with divergent RFI and there is little evidence that efficient animals have a different CH₄ yield expressed as CH₄/kg dry matter (DM) intake. Of equal or greater importance than RFI is the need to select high producing animals, as this will reduce emissions/unit of product, referred to as emissions intensity (Ei). Research should identify productive individuals that have a low RFI to minimise Ei and maintain food production. The extent to which CH₄ can be reduced by selection for RFI will depend on the heritability of efficiency, dispersal of efficient animals through all populations and their resilience in a production system (i.e., robustness). The benefit of RFI to lowering greenhouse gas (GHG) emissions is its application, irrespective of farming system (i.e., confined, intensive, extensive grazing), especially because efficient animals are likely to increase farm profitability. Efficient animals are already in all herds and flocks and research must identify and remove inefficient individuals, while retaining and ensuring efficient ones are fit to purpose. However, the biggest benefits to reducing emissions and increasing production will be associated with good animal management practice (e.g., appropriate genetics, reproductive performance, longevity) with efficient animals superimposed. Good animal systems management will improve profitability, and apply to both intensive and extensive systems to increase food production and lower Ei. One dilemma for agriculturists will be the practice of feeding grains to ruminants, as gains in animal efficiency, especially in reduction of Ei, are likely to be biggest with high energy density rations, but feeding grain to ruminants may become an unsustainable practice if food supplies for humans are limited.
Genetic and nutritional regulation of lamb growth and muscle characteristics
Combined actions of nutrition and genetic regulation of the growth rate of lambs as well as the physical, biochemical, and eating quality characteristics of their skeletal muscle were assessed in a major field experiment. Data arising were collated and integrated to consolidate findings made at the farm, animal, tissue, cellular, and gene expression levels. At the farm level, increased nutrient availability significantly increased the growth rate of crossbred lambs and increased the growth advantage resulting from the use of sires with high estimated breeding values (EBV) for growth. In contrast, the extra depth of the 'M. longissimus thoracis et lumborum' (EMD) arising from sires with a higher EBV for this trait was constant irrespective of nutrition. Ewe liveweight and body condition were critical in determining the pre-weaning nutrition and growth of lambs, with the LOW plane of nutrition causing stunting of forelimb bones and changes to the allometric growth coefficients for carcass lean and fat. The EBV of the sire for muscling (PEMD) influenced several non-muscling traits, and interactions with nutrition suggested that on HIGH nutrition, absorbed nutrients were partitioned away from wool and fat accretion and towards protein accretion in lambs having superior muscling genetics. Expression levels of known myogenic factors in muscle, together with a suite of peptides and proteins whose identity and levels were determined by proteomic screening, contributed to improved understanding of the mechanisms underpinning nutritional and genetic regulation of skeletal muscle development. The study revealed the need for caution in the use of indirect markers of growth or composition, with their usefulness being constrained if the localisation of the response to selection is specific to parts of the carcass where the selection had initially been concentrated. As well, the possibility that much of the variation explained by potential physiological markers can be accounted for by non-invasive measures of growth and fatness currently used by Australian farmers must be considered.
Associations among methane emission traits measured in the feedlot and in respiration chambers in Angus cattle bred to vary in feed efficiency
The objective of the study was to evaluate associations among animal performance and methane emission traits under feedlot conditions and in respiration chambers in Angus cattle bred to vary in residual feed intake (RFI), which is a measure of feed efficiency. Fifty-nine cattle were tested for feedlot RFI, of which 41 had methane production recorded on an ad libitum grain-based ration in the feedlot, 59 on a restricted grain-based ration in respiration chambers, and 57 on a restricted roughage ration in respiration chambers. The cattle became older and heavier as they went through the different phases of the experiment, but their feed intake (expressed as DMI) and daily emission of enteric methane (methane production rate; MPR) did not increase proportionally, as feed offered was restricted in the respiration chamber tests. Methane emissions by individual animals relative to their DMI were calculated as methane yield (MY; MPR/DMI) and as 2 measures of residual methane production (RMPJ and RMPR), which were calculated as the difference between measured MPR and that predicted from feed intake by 2 different equations. Within each test regime, MPR was positively correlated (r = 0.28 to 0.61) with DMI. Phenotypic correlations for MY, RMPJ, and RMPR between the feedlot test and the restricted grain test (r = 0.40 to 0.43) and between the restricted grain test and the restricted roughage test were moderate (r = 0.36 to 0.41) and moderate to strong between the feedlot test and the restricted roughage test (r = 0.54 to 0.58). These results indicate that the rankings of animals for methane production relative to feed consumed are relatively stable over the 3 test phases. Feedlot feed conversion ratio and RFI were not correlated with MPR in the feedlot test and grain-based chamber test but were negatively correlated with MPR in the chamber roughage test (r = -0.31 and -0.37). Both were negatively correlated with MY and RMPJ in the feedlot test (r = -0.42 to -0.54) and subsequent chamber roughage test (r = -0.27 to -0.49). Midparent estimated breeding values for RFI tended to be negatively correlated with MY and RMPJ in the feedlot test (r = -0.27 and -0.27) and were negatively correlated with MY, RMPJ, and RMPR in the chamber roughage test (r = -0.33 to -0.36). These results showed that in young growing cattle, lower RFI was associated with higher MY, RMPJ, and RMPR but had no significant association with MPR.
Feed intake, growth, and body and carcass attributes of feedlot steers supplemented with two levels of calcium nitrate or urea
Nitrate supplementation has been shown to be effective in reducing enteric methane emission from ruminants, but there have been few large-scale studies assessing the effects of level of nitrate supplementation on feed intake, animal growth, or carcass and meat quality attributes of beef cattle. A feedlot study was conducted to assess the effects of supplementing 0.25 or 0.45% NPN in dietary DM as either urea (Ur) or calcium nitrate (CaN) on DMI, ADG, G:F, and carcass attributes of feedlot steers (n = 383). The levels of NPN inclusion were selected as those at which nitrate has previously achieved measurable mitigation of enteric methane. The higher level of NPN inclusion reduced ADG as did replacement of Ur with CaN (P < 0.01). A combined analysis of DMI for 139 steers with individual animal intake data and pen-average intakes for 244 bunk-fed steers showed a significant interaction between NPN source and level (P = 0.02) with steers on the high-CaN diet eating less than those on the other 3 diets (P < 0.001). Neither level nor NPN source significantly affected cattle G:F. There was a tendency (P = 0.05) for nitrate-supplemented cattle to have a slower rate of eating (g DMI/min) than Ur-supplemented cattle. When adjusted for BW, neither NPN source nor inclusion level affected cross-sectional area of the LM or fatness measured on the live animal. Similarly, there were no significant main effects of treatments on dressing percentage or fat depth or muscling attributes of the carcass after adjustment for HCW (P > 0.05). Analysis of composited meat samples showed no detectable nitrates or nitrosamines in raw or cooked meat, and the level of nitrate detected in meat from nitrate-supplemented cattle was no higher than for Ur-fed cattle (P > 0.05). We conclude that increasing NPN inclusion from 0.25 to 0.45% NPN in dietary DM and replacing Ur with CaN decreased ADG in feedlot cattle without improving G:F.
Methane Production and Productivity Changes Associated with Defaunation in Ruminants
With increasing world population, global demand for a secure and growing food supply challenges the livestock producers of today to increase output of milk and meat while reducing the environmental impact of animal production. This thesis reports a review of literature and targeted new research assessing the consequences of eliminating rumen protozoa (defaunation) on the performance, digestive function and emissions of the greenhouse gas methane, by livestock.
• Comparative studies of rumen fermentation and animal growth were conducted in growing Merino lambs, crossbred sheep and Brahman cattle. In these studies ruminants were defaunated using coconut oil distillate to suppress protozoa then dosed with sodium 1-(2-sulfonatooxyethoxy) dodecane in a protocol that suppressed feed intake for an average of 10 days but had no detrimental effects on animal health.
• Reflecting the diversity in published literature, these studies found inconsistent effects of defaunation on volatile fatty acid (VFA) concentrations and proportions. Averaged over all experiments conducted, defaunation was associated with a small (5%) reduction in total VFA concentration and an increase (5%) in the ratio of acetate to propionate in the rumen.
• While effects on VFA were not consistent, an average 30% reduction in rumen ammonia concentration and a 16% increase in microbial crude protein outflow (estimated by allantoin excretion) were apparent, suggesting substantial differences in the ruminal degradation and outflow of protein due to defaunation. These changes were associated with an 18% increase in average daily gain (ADG), but surprisingly no increase in wool growth rate.
• Defaunation was associated with a lower enteric methane emission (average 20% reduction) compared to faunated ruminants, with the first studies of daily methane production (DMP) ever made while grazing, made using GreenFeed Emission Monitoring (GEM) units, confirming a 3% lower DMP (non-significant; P > 0.05) and a 9% lower methane yield (MY; CH4/kg DMI; P = 0.06) in defaunated sheep.
• Protozoa affected the rumen response to nitrate, with the nitrate induced reduction in MY being 29% greater in faunated compared to defaunated lambs.
• With dietary coconut oil, no interaction with defaunation was apparent with both coconut oil and defaunation significantly reducing DMP and MY in cattle.
• While defaunation tended to increase average daily gain and reduced enteric methane emissions in cattle by 10%, establishing defaunated cattle proved difficult and is a major constraint to expanding defaunation into commercial herds.
• Assessment of the distribution of protozoa in the forestomaches showed that the number of entodiniomorph protozoa attached to the 'leaves' of the bovine omasum was at least as great as the number attached to the entire surface of the rumen, though all tissue-attached populations are far fewer than the population in the rumen fluid.
• It is concluded that defaunation alone or in combination with dietary supplements of nitrate is effective in decreasing methane emissions, while increasing microbial protein supply and ADG. Commercial implementation of defaunation for cattle will not be able to rely on addition of surfactants to the rumen and it is suggested a bioactive compound distributed through the blood may be needed to remove protozoa residing in the omasum.
Selection for residual feed intake can change methane production by feedlot steers
A 70-day residual feed intake (RFI) test on a barley-based feedlot ration was conducted, over which daily feed intake (FI) and weekly liveweight of 91 Angus steers were recorded. Rate of enteric methane production (MPR) was measured in a series of 5×2-day consecutive measurement periods using a marker-based method with the marker gas (SF₆) released from an intraruminal permeation device. Data for 76 steers with 3 or more valid 2-day methane collections were analysed. The 43 low. RFI (high efficiency) line steers (progeny of 9 sires) and the 24 high-RFI (low efficiency) line steers (5 sires) represented approximately 2.4 generations of divergent selection for postweaning RFI. An additional nine intermediate unselected line steers were included. MPR (g/day) was highly, significantly related to daily FI (kg/day) over the 10-day gas-collection period: MPR=13.0±3.0 (se) × FI+34.9, although FI (P<0.0001) explained only 20% of the variance in MPR. From this relationship MPR over the 10-week RFI test was predicted. MPR predicted for the low-RFI line steers was not significantly lower than for the high-RFI line steers (187±4 v 199±4 g/day; P>0.05). Regression analyses showed MPR to be significantly related to genetic variation in RFI (P<0.05), such that a 1 kg/day reduction in estimated breeding value for RFI would be accompanied by a 13.0±5.1 g/day, or 7%, reduction in methane production. This result supports predictions that reduction in methane emissions should accompany the reduction in FI following from selection for lower RFI.