Rumen microbiota enable dairy cattle to breakdown fiber into useable energy for milk production. Rumen bacteria, protozoa, and fungi ferment feedstuff into volatile fatty acids (VFA), the main energy source, while methanogens utilize fermentation by-products to produce methane. Milk fat contains several bioactive rumen-derived fatty acids (FA), including odd-chain FA (OCFA) and branched-chain FA (BCFA), important for maintenance of human health. The overarching dissertation goal was to determine which factors affect rumen methanogen and protozoal community structures and their metabolism products, while defining relationships between rumen microbiota and animal performance. Results presented contribute to the goals of providing new knowledge to dairy farmers, maintaining ruminant health, and enhancing bioactive FA in milk. The first objective was to use next-generation sequencing techniques to determine if lactation stage and dairy breed affect rumen methanogen and protozoal community structures and protozoa cell FA compositions in Jersey, Holstein, and Holstein-Jersey crossbred cows at 3, 93, 183, and 273 days in milk (DIM). A core methanogen community persisted by lactation stage and breed. At 3 DIM, methanogen 16S rRNA gene sequences formed distinct clusters apart from 93, 183, and 273 DIM, reflective of the dietary transition period post-partum. The starch-utilizing protozoal genus Entodinium, was more abundant in Holsteins than in Jerseys and Holstein-Jersey crossbred cows and positively correlated with milk yield. Jerseys had greater iso-BCFA contents in protozoa and milk and protozoa of the genus Metadinium. The second objective was to determine if supplementation of mixed cool-season grasses with annual forages (AF) alters the forage, microbial, and milk FA contents during typical periods of decreased pasture growth in Northeastern US. In short-term grazing (21d) of AF, ruminal VFA and major rumen-derived FA were not altered in bacterial and protozoal cells, suggesting little alteration of biohydrogenation and maintenance of ruminant health. In spring, milk contents of iso-15:0 and 17:0 per serving of whole milk were greater in control (CON)-fed cows, while contents of 12:0 and 14:0 per serving were greater in AF-fed cows. Contents of de novo FA and OCFA per serving of whole milk were greater in summer AF-fed cows than CON-fed cows, while total contents and BCFA did not differ, suggesting post-ruminal FA modifications in adipose tissue and the mammary gland. The third objective was to characterize and relate the rumen microbiota from CON- and AF-fed cows to animal performance. Rumen protozoal taxa were not altered, while less abundant bacterial taxa (< 5%) were different in both periods. The protozoal genus Diplodinium was positively correlated with feed efficiency and milk fat yield. In spring, AF-fed cows had greater abundances of the methanogen species Methanobrevibacter millerae, whereas CON-fed cows had greater abundances of the methanogen species Methanobrevibacter ruminantium, potentially as a result of differences in substrate availability. In conclusion, the work presented identifies several factors that influence rumen microbiota, rumen microbial FA, and milk FA, while providing new information to dairy farmers, researchers, and consumers.

Recent findings have shown an inverse association between the circulating levels of pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) with the risk of pathological development in type 2 diabetes, cardio vascular disease and neurological disorders. From previously published research, it has been said that both these odd chain fatty acids are biomarkers of their dietary intake and are significantly correlated to dietary ruminant fat intake. However, there are profound studies that show the contrary where they do not display this biomarker correlation. Additionally, several astute studies have suggested or shown odd chain fatty acid endogenous biosynthesis, most often suggested via alpha oxidation; the cleavage of a single carbon unit from a fatty acid chain within the peroxisomes. To better understand the correlations and interactions between these two fatty acids with pathological development, the origin of these odd chain fatty acids needed to be determined, along with confirming their association with the disease aetiology. To minimise animal & human experimentation we made use of existing sample sets made available through institutional collaborations, which produced both animal and human interventional study samples suitable for odd chain fatty acid investigations. These sample collaborations allowed us to comprehensively investigate all plausible contributory sources of these odd chain fatty acids; including from the intestinal microbiota, from dietary contributions, and derived from novel endogenous biosynthesis. The investigations included two intestinal germ-free studies, two ruminant fat diet studies, two dietary fat studies and an ethanol intake study. Endogenous biosynthesis was assessed through: a stearic acid infusion, phytol supplementation, and an Hacl1 knockout mouse model. A human dietary intervention study was used to translate the results. Finally, a study comparing circulating baseline C15:0 and C17:0 levels with the development of glucose intolerance. We found that the circulating C15:0 and C17:0 levels were not significantly influenced by the presence or absence of intestinal microbiota. The circulating C15:0 levels were significantly and linearly increased when the C15:0 dietary composition increased; however, there was no significant correlation in the circulating C17:0 levels with intake. Circulating levels of C15:0 were affected by the dietary composition and factors affecting the dietary intake, e.g. total fat intake and ethanol, whereas circulating C17:0 levels were found to be independent of these variables. In our studies, the circulating C15:0 levels were not significantly affected by any expected variations in alpha oxidation caused by pathway substrate inhibition or gene knockout. However, C17:0 was significantly related, demonstrating it is substantially endogenously biosynthesised. Furthermore, we found that the circulating C15:0 levels, when independent of any dietary variations, did not correlate with the progression of glucose intolerance when induced, but the circulating C17:0 levels did significantly relate and linearly correlated with the development of glucose intolerance. To summarise, the circulating C15:0 and C17:0 levels were independently derived; the C15:0 levels substantially correlated with its dietary intake, whilst the C17:0 levels proved to be separately derived from its endogenous biosynthesis via alpha oxidation of stearic acid. C15:0 was found to be minimally endogenously biosynthesised via a single cycle of beta oxidation of C17:0 in the peroxisomes, however, this did not significantly contribute to the circulating levels of C15:0. Additionally, only the baseline levels of C17:0 significantly correlated with the development of glucose intolerance. These findings highlight the considerable differences between both of these odd chain fatty acids that were once thought to be homogeneous and similarly derived. On the contrary, they display profound dietary, metabolic, and pathological differences.