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Simple Summary  Dairy cows experience immune suppression around calving, which can result in the development of uterine diseases. White blood cells, and more specifically polymorphonuclear leukocytes (PMN), are important components of the immune system. Although it is regarded as a common research approach to study PMN isolated from blood, it would be interesting to know more about the viability and functionality of uterine PMN. We developed a method to isolate PMN from the bovine postpartum uterus to perform viability and functionality tests. We also evaluated whether we could identify uterine PMN using a specific antibody. Uterine cells were recovered using an adapted medical brush, and PMN were successfully isolated and identified. The percentage of viable intra-uterine PMN in postpartum cows (9–37 days in milk) roughly ranged from 10 to 80%, indicating that the viability of the uterine PMN is highly dynamic. We could also identify PMN that ingested labeled particles, which lets us conclude that uterine PMN are functional. Using the presently described methods, further research can be performed to unravel the role of uterine PMN viability and functionality in bovine uterine health.    Abstract  Postpartum dairy cows experience impaired peripheral polymorphonuclear leukocyte (PMN) functionality, which has been associated with reproductive tract inflammatory diseases. However, it has not been elucidated yet whether endometrial PMN functionality is (equally) impaired. We developed a method for endometrial PMN isolation and flow cytometric assessment of their viability and functionality. We also evaluated PMN immunolabeling, using a specific bovine granulocyte marker, CH138A. Blood and endometrial cytobrush samples were collected in duplicate from seventeen clinically healthy Holstein-Friesian cows between 9 and 37 days in milk. The proportion of viable, apoptotic, and necrotic PMN in endometrial samples roughly ranged from 10 to 80%, indicating highly dynamic endometrial PMN populations in the postpartum uteri. Endometrial PMN functionality testing revealed that PMN immunolabeling increased the accuracy, although this protocol might influence the median fluorescence intensity of the sample. Phagocytosis seemed the most stable and reliable endometrial PMN function and could be assessed satisfactorily without prior CH138A immunolabeling. However, the interpretation of oxidative burst and intracellular proteolysis tests remains challenging. The correlation between peripheral and endometrial PMN functionality was poor. Further research is warranted to unravel the role of uterine PMN viability and functionality in bovine uterine health.

Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows