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Item Further development of a cell culture based approach to model the diet-derived impacts on the faecal microbiome and potential host health in the domestic dog : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 2023) Phimister, FrancisGlobally, diets that promote and optimise health and a ‘healthy microbiome’ are becoming increasingly popular for pets. However, the impacts of novel diet ingredients and formulations on the health of the host and their microbiome require testing to ensure there are no unforseen detrimental effects. However, there are currently a very limited number of canine-specific intestinal cell lines and the capacity to utilise these cells to model host-food interactions is limited. Thus, this doctoral project aimed to develop an in vitro model of the canine intestine, using a previously established canine intestinal epithelial cell (cIEC) line. This could then be used to characterise the canine response to dietary challenges to the gut microbiota. As there is limited research that has assessed the interactions of the gut microbiota with the canine intestine, the initial step of this project was to evaluate the current knowledge of the intestinal inflammatory response to bacterial ligands and diet-derived metabolites (Chapter Two). This literature review indicated that prior to investigating the interactions between bacteria and the canine intestine an evaluation of the canine intestinal response to these challenge compounds was required. Building on the knowledge base established in Chapter Two, key microbes associated with health in the dog required identification. Thus, this thesis provided the first meta-analysis of the available literature on the relationship of dietary nutrients and their impact on the gut microbiota in the dog (Chapter Three). The hypothesis of this meta-analysis was that dietary protein and dietary fats would have singificant impacts on the faecal microbiota of the dog and additionally, that this analysis would reveal bacterial genera associated with these dietary macronutrients.In the meta-analysis the novel discovery was made that despite its low relative abundance,Sharpea was the genera most associated with causing the shifts in microbial profiles in response to changes in both crude protein, and crude fats, thus confirming the hypotheses. Early results indicated that the methods required to further refine the existing cIEC line as an in vitro model of the canine intestine were sub-optimal and required further development. These method developments are detailed in Chapter Four. Experiments in this chapter assessed methods to promote cell growth and differentiation in a cellZscope system, which automatically performed barrier integrity assessments, whilst inside a temperature-controlled incubator. The inclusion of 150 nM hydrocortisonein cell culture media during the initial 48 hours of cellular differentiation, and subsequent removal of hydrocortisone after this period successfully enabled the cIEC to differentiate and form confluent monolayers in the cellZscope system. These methods were intended to be used going forwards in an apically anaerobic system that would more closely resemble conditions seen in vivo and would have allowed the simultaneous culture of the oxygen-requiring cIEC and anaerobic bacteria. Work utilising this model was stopped due to complications that arose from the Covid-19 pandemic, but work conducted and experiments that were planned are explored in Chapter Six. Utilising the refined methods from Chapter Four, the inflammatory response of the cIEC to butyrate and bacterial lipopolysaccharides (LPS) were characterised (Chapter Five). This was performed to address gaps in the literature highlighted in Chapter Two.It was hypothesised that the stimulation with bacterial LPS would cause a pro-inflammatory response, whilst the stimulation with butyrate would cause an anti-inflammatory response. Furthermore, it was also hypothesised that the stimulation of the cIEC with both LPS and butyrate would cause the butyrate to reduce the pro-inflammatory response, and the LPS to reduce the anti-inflammatory response. It was observed that LPS induced a pro-inflammatory response in the cIEC, which butyrate was able to mitigate in most instances. Overall, the methods developed and refined in this project will be able to be utilised in future experiments utilising these cells, such as evaluating new pet food ingredients for beneficial effects and exploring how changes in the gut microbiome impact gut health in the dog. It can take the knowledge established in Chapter Three to further investigate the impacts of the bacterial genera on the health of the dog. Futhermore, it can utilise the immune responses observed in Chapter Five to better understand the relationship between inflammation and diet in the dog. Future work can build on the knowledge discovered and presented in this thesis to fully understand the impact of diet changes on the health of the dog, and further define the microbial profile of the ‘healthy microbiome’ for the dogItem Aspects of dietary management and dynamics of the faecal microbiota of horses and ponies (Equus caballus) in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science, Massey University, Palmerston North, New Zealand(Massey University, 2016) Fernandes, Karlette AnneThe aim of this thesis was to explore aspects of the dietary management of horses and ponies in New Zealand, and to investigate the association between dietary management and faecal microbiota. To achieve this aim, a series of observational and intervention studies were conducted. The first two studies were cross-sectional surveys of feeding, health and management practices, which showed that most horses and ponies in New Zealand were managed continuously on pasture all year round, with no seasonal differences in the hours allowed for grazing. In addition to pasture, many owners fed their animals a combination of premixed feeds, cereals (oats) and conserved forages. Most horses and ponies kept on pasture were reported to be healthy. Among nutrition-related health issues reported by the owner, obesity, colic, laminitis and grass staggers were most commonly reported (12-14%). Using a standard body condition scoring chart, 22% of owners indicated that their horse or pony was overweight (scored ≥ 7 on a 1-9 scale). Horses and ponies kept on pasture maintained body weight and a higher body condition (median score of 6 on a 1-9 scale) through spring and autumn. A high proportion of pony breeds were observed in this population, and these ponies remained ‘fat’ despite the seasonal fluctuation in the quantity and quality of pasture. Owners tended to underestimate the body condition of their horses, especially ponies, and this finding indicated why a higher percentage of overweight animals may be present in the Pony Club population. The next two studies were observational investigations that characterised the faecal microbiota of forage-fed horses. The faecal microbiota in a cohort of yearling Thoroughbred horses that were abruptly transitioned from an ensiled chopped forage-based diet to pasture was diet-specific and responded rapidly to dietary change within four days. The faecal microbiota profile was dominated by two phyla, Firmicutes and Bacteroidetes, which comprised of several bacterial genera. The abundance of bacterial genera fluctuated over the three-week observation period, when kept at pasture. Similarly, the faecal microbiota of a cohort of mature adult Thoroughbred and Standardbred horses kept on pasture was diet-specific. The abundances of the bacterial genera were influenced by the nutrient composition of the pasture, which was also correlated with seasonal changes in climate (rainfall and temperature) over the one year observation period. This latter finding indicated that the fluctuations observed in the previous study may also be due to changes in pasture composition. The inclusion of hay in the diet appeared to buffer the changes occurring in the faecal microbiota as a result of the seasonal fluctuations in pasture composition, but there was also a large degree of variation between individual horses. The final study was a randomised controlled trial using adult Thoroughbred horses that were kept in loose boxes and fed four forage-based diets. The first phase of the trial identified that the mean retention time of digesta was associated with the dry matter intake of the feed consumed. There was a significant difference in the quantity of feed consumed by individual horses, which appeared to be driven by the moisture content in the forage diets. The second phase of the trial showed that the population of the faecal microbiota was resilient following abrupt dietary transition between four forage-based diets. These findings indicated why the horses in the previous study may have maintained body weight and condition, despite the seasonal fluctuation in the quantity and quality of pasture. This thesis highlights the complexity of the equine faecal microbiota, and demonstrates that the relationship of dietary dry matter intake and mean retention time of digesta in the gastrointestinal tract influences the population dynamics of the faecal bacterial community.Item The effect of lipid on the digestion of cellulose by the ruminant : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science at Massey University(Massey University, 1969) Kirk, Robin David1.Metabolism of Lipids by Ruminants Many workers have added fats, oils and fatty acids to the rations of sheep, beef cattle and dairy cows to investigate the effects of lipid on either one or more of the following; intake, digestibility end energy utilisation of rations, live weight gains, methane production, ammonia production, N retention, milk production, milk constituents, total VPA production and VFA molar proportions or the value of lipid as a source of energy. These factors will be discussed in subsequent sections of this review. Lipids which have been fed or infused into ruminants include coconut, cod liver, corn, cottonseed, linseed, palm kernel, peanut, soyabean, tung and whale oils, animal fats (lard, tallow and poultry fat) and long chain fatty acids (palmitic, stearic, oleic, linoleic and linolenic). Since the amount of lipid ingested by adult ruminants is substantial, the investigations into the effects of lipids are warranted. A cow consuming 45Kg. of pasture daily will ingest about 500g. of lipids and, under conditions of stall-feeding, pregnant and lactating animals may receive rations which provide up to 1.0Kg. of lipids daily (Garton, 1967).[FROM INTRODUCTION]Item Comparative genomics of rumen methanogens : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2016) Li, YangMethane (CH4) emissions from agriculture represent around 9% of global anthropogenic greenhouse gas emissions. The single largest source of this CH4 is animal enteric fermentation, predominantly from ruminant livestock, where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce CH4 emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop CH4 mitigation technologies. To gain insights into the role of methanogens in the rumen environment, two methanogens have been isolated from ovine rumen and their genomes were sequenced: methanogenic archaeon ISO4-H5 of the order Methanomassiliicoccales and Methanobrevibacter sp. D5 of Methanobrevibacter gottschalkii clade. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M (CoM), and requires external supply of CoM to survive. Methanobrevibacter sp. D5 is a hydrogenotrophic methanogen predicted to utilise CO2 + H2 and formate as substrates. Comparisons between the available Methanobrevibacter genomes has revealed a high conservation in energy metabolism and characteristics specific to each clade. The coexistence of different Methanobrevibacter species in the rumen may be partly due to the physical association Methanobrevibacter species with different microorganisms and host surface, which allow unique niches to be established.Item Relationships between hydrogenation and hydrolysis of dietary fat in the bovine rumen : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science(Massey University, 1968) Silcock, William RossOne of the earliest detailed comparisons of the fatty acid and glyceride composition of the depot fats of ruminants and non-ruminants in relation to the diet fed may be obtained from the work of Banks and Hilditch (1931, 1932). These studies involved beef tallows and sow depot fats respectively, and were part of an investigation of the fatty characteristics of market meat. It had been known for some time that very soft body fats, which have always been undesirable from the consumer's point of view, were produced when pigs were fed diets containing high levels of unsaturated fats. Banks and Hilditch (1932) found that the characteristic fatty acids of animal and vegetable fats when fed appeared in sow depot fats. In this case, the sow was fed a diet containing 7% of fish meal, which contains some 20% of unsaturated C20 and C22 fatty acids together with a similar amount of linoleic acid. All these fatty acids appeared in significant amounts in the depot fats of the sow. In contrast, Banks and Hilditch (1931) found that beef tallows contained much less oleic acid and linoleic acid than was present in the diet. Other studies carried out by Thomas, Culbertson and Beard (1934) and by Edwards and Holley (1939) also showed that the type of fatty diet fed to cattle had little effect on the characteristics of the depot fats. Edwards and Holley (1939) note in their paper however, that a liberal allowance of oil in the diet will tend to soften the depot fat of cattle slightly if fed for a long enough period of time.Item The effect of feeding and management on faecal pH of the New Zealand Thoroughbred racehorse : a thesis presented in partial fulfilment of the requirement for the degree Master of Applied Science in Equine Nutrition at Massey University, Palmerston North, New Zealand(Massey University, 2006) Williamson, AnitaQuantifying the risk factors for hindgut acidosis is the first step in understanding the problems of poor management and feeding practices of horses in race training. A non-invasive measure of hindgut acidosis can be obtained by measuring faecal pH (Davie et al., 2000; Eastwood, 2002; Rowe et al., 1995; Zeyner et aL, 2004; Zeyner et al., 1992; Zeyner. 1993). In this study fourteen 3-year-old Thoroughbreds in regular race training, ten Thoroughbred yearlings aged 13-15 months and 140 Thoroughbred horses of mixed aged in race training were surveyed. Approximately 200g±5g of faeces per horse was obtained from all faecal masses in the horse's stable or yard at the time of collection. Faecal pH was measured using a commercial pH meter. The study was divided into three experiments; experiment one consisted of fourteen Thoroughbreds within the same racing stable and identified that subtle change in diet, management, and workload had no effect on mean faecal pH during an 83 day observation period. Experiment two, identified gender, between day or time of faecal collection and the amount of concentrate offered (kg), total feed weight and roughage to concentrate ratio of the diet had no significant effect on the faecal pH of ten Thoroughbred yearlings undergoing sales preparation. However there was considerable variation in faecal pH between horses. Experiment three surveyed 140 Thoroughbred horses under the management of 16 racehorse trainers. Trainer age, number of years training horses, horse age, horse gender, weeks in raee training or racing class had no effect on mean faecal pH. Acidic faecal pH (pH≤6.32) was associated with small stables (1-12 horses). Trainers from small stables offered more concentrate feed than larger stables (13+ horses). Acidic pH was associated with trainers that offered grain as the only form of concentrated feed, or offered ≤2.25kg hay/day, and horses that were fast eaters. Horses that displayed stereotypic behaviours had more alkaline faecal pH than horses that never expresses stable vices (6.70± 0.35 vs. 6.43± 0.29). The total weight of concentrates offered, feed frequency, bedding type, exercise workload and the number of hour's horses were at pasture had no effect on mean faecal pH.Item The influence of diet and intake level on hepatic ammonia metabolism and ureagenesis by the ovine liver : 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, 2001) Greaney, Kenneth BarryThe New Zealand agricultural industry is based on the efficient utilisation of fresh forages, a characteristic of which is a high soluble protein content. A large proportion of the ingested protein is highly soluble in the rumen. A significant proportion of the ingested N is removed from the rumen as ammonia with the bulk of this ammonia being removed from the venous blood by the liver for detoxification to urea. Hepatic urea-N production, or ureagenesis, typically exceeds the rate of hepatic ammonia-N extraction, consequently it has been suggested that the shortfall in N required for ureagenesis is contributed by amino acid-N (Parker et al. 1995; Lobley et al., 1995). This study tested the hypothesis that elevated hepatic ammonia extraction would require a concomitant increase in hepatic amino acid catabolism to supply the additional N required for ureagenesis. In order to evaluate the level of rumen ammonia production and consequently the rates of hepatic ammonia extraction, ureagenesis and amino acid catabolism. the following feeding regimens were tested in sheep held indoors in metabolism crates in three separate experiments; Firstly, lucerne pellets (Medicago sativa) were compared with fresh white clover (Trifolium repens), secondly fresh white clover was offered at either a low or high intake and finally the daily allowance of fresh white clover was fed in two 2 hour periods per day. In each experiment, silicone based catheters were surgically inserted into the posterior aorta and the mesenteric (2), portal and hepatic veins. Following a ten day dietary adjustment period and a ten day nitrogen balance, the sheep were infused with para-aminohippurate (pAH) and 15NH4Cl via the mesenteric vein. The pAH was infused to allow the blood flow across the splanchnic tissues to be estimated, whilst the 15NH4Cl was infused to trace hepatic ammonia metabolism to urea. Blood samples were collected to determine the ammonia, urea, oxygen and amino acid concentrations in the mesenteric, portal and hepatic veins, as well as the posterior aorta. Despite similar DM intakes, the nitrogen intake of the sheep fed fresh white clover was 60% higher (P < 0.001) than that of the same animals fed lucerne pellets. The difference in rumen protein fermentation in these two contrasting diets resulted in higher (P < 0.001) rumen ammonia production in the animals offered fresh white clover. There was, however, only a trend (P = 0.072) toward elevated hepatic ammonia extraction in these animals and urea production was not significantly different to the animals fed lucerne pellets. Hepatic amino catabolism was not elevated in the sheep fed fresh white clover, nor was there a significant difference in the proportion of ME intake that was utilised for ureagenesis between the two groups. In the second experiment the DM intakes of the two groups were different (P < 0 001), with the sheep offered the low intake of fresh white clover consuming 807 g DM/d whilst the high intake group consumed 1118 g DM/d. Even with these differences in intake, portal vein ammonia and urea concentrations were similar. Therefore the rate of hepatic ammonia extraction and urea production were also similar between the two intake groups. However, hepatic extraction of 15N-ammonia was higher (P = 0.033) in the high intake group compared to the low intake group. There was no evidence to suggest that the level of hepatic amino acid catabolism increased with intake level, consequently the proportion of ME intake attributed to urea synthesis was similar for the two intake groups When the experimental animals were restricted to two 2 hour feeding periods per day the DM and N intake decreased by 31% from that of the low intake group in the second experiment. There was no significant effect of time after the onset of feeding on portal ammonia or urea concentrations, hepatic ammonia extraction or hepatic urea production. However portal ammonia concentration and consequently hepatic ammonia extraction and urea production tended to be higher 4-6 hours after ingestion of fresh white clover. However this trend was not observed when the 15N tracer data was used to calculate the hepatic ammonia transfer rate. The ammonia, urea and amino acid hepatic transfer values in this experiment were largely comparable to those recorded for the low and high intake treatments in the second experiment. In these studies, there was no evidence of elevated hepatic amino acid catabolism occurring in response to elevated rates of hepatic ammonia extraction and hence ureagenesis. Additionally there was no suggestion that ammonia provided both of the N atoms of the urea molecule. It is concluded that the liver adapted to the changes in dietary nitrogen supply without incurring significant increases in the metabolic cost of ammonia detoxification to urea. However the nutritional challenges presented to the liver may not have been severe enough to induce measurable changes in hepatic ammonia metabolism. A possible mechanism to account for these observations may be that the liver adapted to the changes in nitrogen supply by altering the activity of the primary regulator of the rate of ureagenesis, carbamoyl phosphate synthetase (CPSI).