Context: Research into improving feed efficiency by ruminant animals grazing pastures has historically been restrained by an inability to measure feed intake by large numbers of individual animals. Recent advances in portable breath measurement technology could be useful for this purpose but methodologies need to be developed.

Aims: To evaluate predictive models for metabolisable energy intake (MEI) by free-ranging cattle using multiple short-term breath samples and then apply these to predict MEI by free-ranging cattle in a historic grazing experiment with cattle genetically divergent for residual feed intake (feed efficiency).

Methods: Predictive models for MEI were developed using bodyweight (BW) data, and carbon dioxide production rate (CPR) and methane production rate (MPR) from multiple short-term breath measurements, from an experiment with long-fed Angus steers on a grain-based diet, and an experiment with short-fed Angus heifers on a roughage diet. Heat production was calculated using CPR and MPR. Energy retained (ER) in body tissue gain by steers was calculated from BW, ADG, initial and final subcutaneous fat depths, and for both groups using feeding-standards equations.

Key results: Metabolic mid-test BW (MBW) explained 49 and 47% of the variation in MEI in the steer and heifer experiment, respectively, and for the steers adding ADG and then subcutaneous fat gain resulted in the models accounting for 60 and then 65% of the variation in MEI. In the steer experiment, MBW with CPR explained 57% of the variation in MEI, and including MPR did not account for any additional variation. In the heifer experiment, MBW with CPR explained 50%, and with MPR accounted for 52% of the variation in MEI. Heat production plus ER explained 60, 35 and 85% of the variation in MEI in the steer and the heifer experiments, and in the pooled data from both experiments, respectively.

Conclusions: Multiple short-term breath measurements, together simple BW data, can be used to predict MEI by free-ranging cattle in studies in which animals do not have feed-intake or ADG recorded.

Implications: This methodology can be used for research into improving feed efficiency by farm animals grazing pastures.

Monitoring feeding time in ruminants is one means to quantify feed intake. In grazing cattle offered feed supplement blocks, time spent licking can provide valuable information in estimating the level of blocks being ingested. This current study aimed to 1) assess an ear-tag accelerometer's capability to identify the licking behaviour at supplement blocks in grazing cattle and 2) evaluate the performance of the ear-tag accelerometer and radio-frequency identification (RFID) system to predict the individual time spent licking. Two breed groups of Angus (n = 7) and Brahman (n = 7) beef heifers were kept in two separate yards over 28 days. Each heifer was fitted with an ear-tag containing a tri-axial accelerometer set at 12.5 Hz frequency. Feed supplement blocks were provided through an RFID-equipped automatic supplement weighing unit within each yard, with access to the unit being given daily only from 16:00 h - 20:00 h. The accelerometer classification model developed using support vector machine (SVM) algorithm could distinguish between licking and non-licking behaviours, with an accuracy, sensitivity, F1 score, Cohen's kappa coefficient, and Matthew's correlation coefficient (MCC) of 86%, 93%, 0.88, 0.70, and 0.77 for Angus and 87%, 93%, 0.89, 0.73, and 0.79 for Brahman heifers. Time spent licking predicted by accelerometers were acceptable with a mean absolute error (MAE) of 22% and 11%, modelling efficiency (MEF) of 0.81 and 0.94, concordance correlation coefficient (CCC) of 0.88 and 0.96, and a ratio of root mean square prediction error (RSR) of 0.44 and 0.25, for Angus and Brahman heifers, respectively. However, the RFID system derived predictions of time spent licking in grazing heifers were unacceptable for both breeds. Overall, the ear-tag accelerometer offers the potential to predict individual time spent licking in grazing cattle to estimate block supplement intake.

Establishment ofthe rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs' life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month prelambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet" (2) rumen fluid from donor ewes fed CO" or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Demand for livestock products and methane mitigation is increasingly stimulating a search for technologies capable of increasing animal productivity while lowering enteric methane emissions. Dietary nitrate (NO₃⁻) has shown this capability in sheep on low nitrogen diets. Cysteamine hydrochloride (CSH) has also been shown to have such dual efficacy, but whether it affects rumen fermentation directly or indirectly by modifying digesta kinetics is unknown. It was hypothesized that the administration of CSH to cattle would reduce in-vitro and in-vivo methane production and also increase their average daily liveweight gain (LWG) without affecting their DM intake (DMI). An in-vitro experiment was conducted to study the effects of CSH, NO₃⁻, urea and nitrite, on methane and volatile fatty acid (VFA) production and on the protozoal population. Methane production, production of total VFA and acetate, and acetate:propionate ratio were not affected by CSH (P > 0.05) relative to control incubations, however, pH was reduced while hydrogen accumulation was increased (P < 0.05) by CSH relative to control incubations. Subsequently, a 42-d in-vivo experiment was conducted using a completely randomized design with twelve yearling cattle (236 ± 49 kg liveweight; LW) to assess LWG, methane production and feed conversion ratio (FCR) on a basal roughage/concentrate diet containing either no additives, or 1% NO₃⁻ addition, or 80 mg/kg LW of CSH. Daily methane production rate (DMP; g methane/d) was measured over 2 × 24 h periods in open-circuit calorimetry chambers during both weeks 3 and 6 of the study, with nutrient digestibility determined by collecting faecal samples and using acid insoluble ash as an indigestible marker. Relative to cattle fed the control diet CSH supplemented cattle exhibited no change in LWG or FCR (P > 0.05). While neither DMP nor methane yield (MY; methane/kg DMI) were reduced by CSH (mean 9.2% reduction), methane production rate was significantly reduced (P < 0.05.) for up to 6 h post-feeding relative to control animals. Nitrate reduced MY by 31.1% (15.7 g methane/kg DMI; P < 0.01) relative to when the control diet was fed (22.8 g methane/kg DMI), and increased (P < 0.01) dietary crude protein digestibility. It was concluded that while NO₃⁻ can deliver greater methane mitigation than CSH, CSH has in some studies (though not this study) improved the efficiency of animal production, which, together with the observed short term efficacy in reducing methane emissions suggests CSH may have a role in enabling greater animal production at a reduced environmental cost.

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.