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    The effect of early life nutrition on rumen microbial community development and impact on lifetime performance in ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Cristobal Carballo, Omar
    Manipulation of the rumen microbiota in adult ruminants has been intended to improve animal performance and decrease greenhouse gas emissions, but results have only shown a short- or non-lasting effect after intervention. Changes in the ruminal microbiota during rumen development have recently shown promising results in the short-term. Therefore, the purpose of the present body of work was to determine how dietary management and chemical interventions, during rumen development, modify the ruminal microbial community composition, and whether these changes affect rumen fermentation and development, and consequently, performance in the young ruminants. The objectives of this thesis were to: (i) evaluate the impact of early weaning on rumen development and function in artificially-reared lambs; (ii) characterize the impact of early weaning in lambs on the rumen microbiota in the first 16 weeks of life and examine the relationships between rumen microbiota composition and rumen fermentation profiles, rumen development and blood metabolites; (iii) assess whether contrasting feeding regimes in the first 7 months of life lead to an imprint in the rumen microbial community structure, fermentation profiles and methane emissions in the rumen of calves; (iv) and evaluate the effect of methane inhibitors on the rumen microbial community composition, fermentation pathways, and gas emissions in calves. A series of three experiments were carried out in young ruminants separated from their mothers after colostrum intake, to address the objectives of this thesis. In experiment one, 3-5-day-old lambs were euthanized at weeks 0, 4 and 16 of rearing to investigate objectives (i) and (ii). Early weaning of lambs increased plasma hydroxybutyrate at week 4 of rearing, while dry matter intake, fermentation profiles and rumen morphology were similar between groups. Papillae morphology and muscular thickness differed between ruminal sites at 4 and 16 weeks of rearing, but not between treatments. Diversity and relative abundance of ruminal bacteria was affected by feeding management, whilst the archaea community showed few changes. Changes in the proportions of abundant bacteria genera from Bacteroidetes and Firmicutes were associated with fermentation profiles, rumen morphology and blood metabolites; however, further investigations are required to explain these associations. In experiment two, ~1-week-old calves were reared with two divergent feeding systems and different post-weaning forage quality with a common pasture diet after 7 months of age to investigate objective (iii). Consumption of pre-weaning concentrate compared to forage produced lower methane yields and greater total short chain fatty acids (SCFA) concentrations and propionate proportions; whist ruminal microbes showed greater proportions of saccharolitic bacteria and Methanobrevibacter boviskoreani, but lower hemicellulolytic and cellulolytic bacteria, and Mbb. gottschalkii. Post-weaning, high-quality forage produced greater total SCFA concentration and propionate proportions than low-quality forages, while methane yield was similar. Hemicellulolytic bacteria and Methanosphaera spp. were greater in high-quality forages, while cellulolytic bacteria and Methanomassiliicoccales spp. were greater in low-quality forages. No pre-weaning effect was observed. Finally, the consumption of a common diet after 7 months of age resulted in similar methane emissions, fermentation profiles and microbial communities. In experiment three, ~1-week-old calves fed either concentrate starter diets or starter diets plus methane inhibitors were tested to evaluate objective (iv). Inhibitor intake decreased methane yield, but increased hydrogen yield and the proportion of propionate and had no effect on dry matter intake, total SCFA concentrations or animal growth. Within the abundant bacteria, the proportions of hydrogen utilizing and producing bacteria increased and decreased, respectively. Archaea diversity and proportions were affected during the period of methane inhibitor intake. However, similar gas emissions, fermentation profiles, and microbial communities were observed between groups at 24 and 49 weeks of age. Collectively, these results showed that reducing the age at weaning and introducing the solid feed to lambs at ~1 week of life accelerated some aspects of rumen morphology and function. Dietary management and methanogen inhibitor interventions affected the composition of the ruminal microbiota and fermentation profiles during treatment, however, no permanent changes in the microbial community and resulting ruminal fermentation were observed post-treatment in young ruminants.
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    Use of headspace solid-phase microextraction for the analysis and characterisation of volatile compounds in rumen contents : a thesis presented in partial fulfillment of the requirements for the degree of Masterate of Science in Chemistry at Massey University
    (Massey University, 2005) Lu, Shengyi
    Volatile fatty acids (VFAs), alkyl phenols and indolic compounds are produced by rumen microbes during the fermentation of forages in ruminants. In this study, ruminal fluid obtained from sheep was examined by headspace solid-phase microextraction (SPME) sampling followed by GC-MS analysis. This technique provides a non-invasive, clean and selective method to characterize the volatiles in ruminal fluid from an in vitro fermentation system. The factors which can influence the extraction efficiency were studied and include the SPME fibre, sample volume, pH of sample matrix (rumen fluid) and extraction time by the fibre in the headspace. The optimum experimental conditions for the analytes in question included: polyacrylate fibre to perform the headspace SPME above 20 mL of rumen fluid in a 68 mL vial for 5 min, followed by immediate GC-MS analysis. The pH of the rumen fluid sample greatly influenced VFA extraction efficiency. Quantitative analysis of p-cresol, m-cresol, indole and skatolc with SPME were compared with steam distillation simultaneous extraction. This comparison showed that the HS-SPME method was semi-quantitative. The optimum in vitro system (16 mL of rumen fluid and 4 mL of artificial saliva in a 68 mL vial incubated at 39°C) was utilised to study production of indole, skatolc and p-cresol from the anaerobic fermentation of tryptophan and tyrosine. Spirulina is an abundant source of dietary protein. Therefore, ¹³C labelled spirulina was used to study the metabolism of protein and formation of analytes derived from ruminal metabolism of protein. A series of labelled end products, including toluene, acetic acid, propanoic acid, iso-butyric acid, n-butyric acid, iso-valeric acid, n-valeric acid, p-cresol, indole, skatole, dimethyldisulfide and dimethyltrisulfide were detected by GC-MS. This result indicates that these compounds are the products of ruminal metabolism of spirulina. When applied to the in vitro rumen system the headspace SPME technique provides a fast approach to study metabolism of target compounds and allows the researcher to follow proposed pathways with labelled substrate.
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    Homoacetogenesis as an alternative hydrogen sink in the rumen : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand.
    (Massey University, 2016) Raju, Preeti
    Ruminant livestock contribute significantly to global greenhouse gas emissions. This is due to microorganisms, known as methanogens that generate methane from hydrogen and carbon dioxide during feed fermentation in the rumen. Mitigation strategies are being developed to reduce methane emissions from ruminants. However, inhibiting methane production may cause accumulation of unused hydrogen in the rumen, which may slow down rumen fermentation and affect animal productivity. Homoacetogens, microbes known to reside in the rumen, can use hydrogen and carbon dioxide to form acetate. Homoacetogens could take over the role of ruminal hydrogen disposal following inhibition of methanogens. The aims of this study were to quantify the involvement of alternative hydrogen utilisers, such as homoacetogens, in hydrogen or electron utilisation. Chemical compounds were screened to identify specific inhibitors of methanogens (BES, acetylene), and both methanogens and homoacetogens (chloroform). Homoacetogenesis was measured via incorporation of 13CO2 into 13C-acetate using a short-term in vitro assay. This short-term in vitro assay measured and confirmed the occurrence of homoacetogenesis in sheep rumen fluid, and it accounted for 1.67% of electron utilisation in fresh rumen fluid. Homoacetogenesis increased in the assay when BES was added, suggesting homoacetogens could increase their activity in the absence of methanogens. Homoacetogenesis decreased with the addition of chloroform, which is known to partially inhibit homoacetogens. Methane formation was inhibited by acetylene in an in vitro serial batch fermentation inoculated with sheep rumen fluid. Homoacetogenesis did not increase, but the homoacetogens were able to grow and maintain themselves as the rumen material was repeatedly diluted and supplemented with fresh feed. Their activity accounted for 2.32% of electron utilisation. To study their significance in the rumen, methane formation was inhibited in sheep using acetylene. Homoacetogenesis increased and accounted for 6.53% of electron utilisation. However, propionate appeared to be the major electron sink (58-88%) in the absence of methanogenesis both in vitro and in vivo. In the future, knowledge of these hydrogen-utilising microorganisms could be used to divert hydrogen or electrons into more beneficial end-products, leading to the transition from a normal methane-producing rumen to an equally or even more productive low methane one.
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    Methane emissions from ruminants fed white clover and perennial ryegrass forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2011) Hammond, Kirsty Joan
    Ruminant enteric methane (CH4) emissions account for ~35% of New Zealand’s total greenhouse gas (GHG) emissions and a commitment has been made for their reduction. Previous research suggested lower CH4 yields (g/kg dry matter intake; DMI) from sheep fed white clover (Trifolium repens) compared to perennial ryegrass (Lolium perenne; ryegrass), and the initial focus was to account for that difference. However, measurements undertaken here showed little difference between diets in CH4 yield. The objective of this thesis was amended to better understand causes of variation in CH4 emissions from ruminants fed white clover and ryegrass forages. A database analysis showed greater variation in CH4 yield from sheep fed ryegrass forages with measured intakes using the SF6 technique, compared to respiration chambers (23.4 ± 5.70 vs. 23.1 ± 2.90 g/kg DMI). The composition of ryegrass fed to sheep predicted <2% and 20% of the variation in CH4 yield when derived from SF6 and respiration chamber techniques, respectively. For cattle, the database of CH4 yields determined by SF6 found ryegrass composition accounted for 13% of the variation. Measurements in respiration chambers of CH4 yield from sheep in three experiments reported here, had similar values for white clover and ryegrass (22.6 g/kg DMI), despite higher concentrations of fibre and less crude protein in ryegrass. Feed composition predicted less than 19% of variation in CH4 yield. Measurements of CH4 emissions from sheep fed white clover or ryegrass at multiples of 0.8 to 2.5 the metabolisable energy requirements for maintenance (MEm) showed a decline in CH4 yield of 3.47 g/kg DMI for each multiple of MEm intake above maintenance. Measurements of rumen function and digesta kinetics, suggested the rate of liquid flow through the gastro-intestinal tract, and molar percentages of propionate were the main drivers of a change in CH4 yield with intake. This research has shown minor effects of forage composition on CH4 yield, and has highlighted the importance of digestive function to account for effects of intake and individual variation on methanogenesis. The benefits of high feed intakes for production will be complemented by a low CH4 yield and low emissions per unit of production.