A study of circulating neutrophils and exosomes associated with innate immune function in the periparturient grazing dairy cow : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand

Listed in 2017 Dean's List of Exceptional ThesesDairy cows are at greatest risk of infectious and metabolic disease during the periparturient period. This period of three weeks either side of calving is also known as the transition period due to the transition into lactation. This thesis had several aims; one was to characterise innate immune function during the transition period in grazing dairy cows by investigating molecular changes in circulating neutrophils and to assess if common on-farm management strategies (pre-calving feeding level and body condition at calving) were able to influence these molecular changes. Next, metabolic stress on neutrophil function was assessed by establishing a model of cows divergent in metabolic health status. This model was further utilised with the aim to investigate nanoparticles (exosomes), which are regulators of innate immune function and indicators of disease state. To address these aims blood was collected from pasture-fed transition dairy cows. Cellular and molecular methods used included cell and exosome isolation, reverse transcriptase (RT)-quantitative PCR, RNA sequencing, cell culture, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that grazing dairy cows experience a change in innate immune function during the transition period, reflective of reduced functional capacity of the immune system to overcome infectious agents. This altered function was similar to that experienced by housed cows fed a total mixed ration, which adds evidence to support that the dysfunction is a natural part of the transition into lactation at calving. These results also indicated that the functional changes could be influenced by nutrition status, feeding level, and metabolic stress. Analysis of exosomes isolated from the blood of transition cows indicated that these particles carried cargo indicative of metabolic state during the transition period and that they had the ability to alter target cell processes (gene expression, protein expression, and cell proliferation). The conclusions from this thesis increase our understanding of transition cow immune function and how it is influenced by nutrition and cow metabolism. These data are particularly relevant for grazing dairy cows and the findings will contribute to on-farm recommendations and the improvement in animal health and well-being. ii