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

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.

Bovine neutrophils kill the sexually-transmitted parasite Tritrichomonas foetus using trogocytosis

“…Using previously established centrifugation protocols (Roth and Kaeberle 1981 ), we isolated Peripheral Blood Mononuclear Cells (PBMC), and PMNs. After staining our fractionated PBMC or PMN cell fractions using an anti- bovine granulocyte monoclonal antibody (GR-1) (Etchevers et al 2023 ; Lietaer et al 2021 ; Lopes et al 2021 ; Oliveira et al 2020 ), we found that our PMN fraction was viable, and had a PMN identity, as anti-GR-1 reactivity was very high in all of the PMN cells, but few of the PBMC cells (Fig. 1 A).…”

“…Isolated leukocytes were stained with anti- bovine GR-1 antibody to confirm PMN identity, as previously described (Etchevers et al 2023 ; Lietaer et al 2021 ; Lopes et al 2021 ; Oliveira et al 2020 ). Briefly, cells were stained with mouse anti-bovine granulocyte (GR-1) monoclonal IgM primary antibody (clone-CH138A, Kingfisher Biotech), or isotype control (clone-COLIS52A2, Kingfisher Biotech) at a concentration of 1.0 ug/mL, followed by a FITC goat anti-mouse IgM polyclonal secondary antibody (ThermoFisher) at a concentration of 1.5 ug/mL.…”

Maladaptation to the transition period and consequences on fertility of dairy cows

“…Our research group has recently developed a technique to flow cytometrically to evaluate the viability and functionality of uterine neutrophils collected via the cytobrush technique (Lietaer et al, 2021, 2022). In new, unpublished data collected from 32 dairy cows at 9, 21, and 36 postpartum, we found that uterine neutrophils in the fifth week postpartum are mostly necrotic in healthy cows, but viable and functional in cows with clinical endometritis.…”