Flow cytometric assessment of the viability and functionality of uterine polymorphonuclear leukocytes in postpartum dairy cows

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

Quantitative and functional dynamics of circulating and endometrial polymorphonuclear leukocytes in healthy peripartum dairy cows

The aim of the present study was to assess the counts, viability, and functionality of circulating and endometrial polymorphonuclear leukocytes (PMN) isolated from fourteen clinically and metabolically healthy multiparous dairy cows in the peripartum period. For this, blood samples were collected at -5, +9, +21 and + 37 days (d) relative to calving. Cytology samples were collected from the vagina, cervix, and uterus at +9, +21 and + 37 d, using the cytobrush technique. Additional vaginal samples were collected at -5 d. Cytology smears were prepared and the PMN-to-all nucleated cell proportions (PMN%) were calculated. The endometrial cytobrush samples were also used for flow cytometric assessment of endometrial PMN (ePMN) viability and functionality. Functionality tests for circulating PMN (cPMN) included phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation. For ePMN, we evaluated PC only. The effect of day relative to calving on PMN viability and functionality were fitted in linear regression models, accounting for repeated measures. The endometrial PMN% were higher at +9 d (23.5 +/- 0.4%; least-squares means +/- standard error) and +21 d (8.5 +/- 0.3%) than at +37 d (1.4 +/- 0.3%). No changes in PMN% were found on either vaginal or cervical cytology along the peripartum period. The cPMN counts were higher pre- (6.2 +/- 0.4 x 10(6)/mL) than postpartum (4.9 +/- 0.4 x 10(6)/mL). Upon viability analysis, only the percentage of viable cPMN tended to be lower at -5 d (90.1 +/- 1.5%) than at +37 d (94.1 +/- 1.4%), and no other changes in the percentage of apoptotic and necrotic cPMN, nor in their functionality were found during the peripartum period. Analysis of ePMN viability showed that the percentage of viable ePMN did not change over time. In marked contrast, the percentage of apoptotic ePMN was higher at +9 d (37.8 +/- 5.1%) than at +21 d (20.9 +/- 5.1%) and +37 d (11.9 +/- 5.3%), while the percentage of necrotic ePMN was lower at +9 d (27.0 +/- 6.3%) than at +37 d (54.9 +/- 6.6%). The percentage of ePMN PC was higher at +9 d (27.5 +/- 3.4%) than at +37 d (13.3 +/- 4.9%). In conclusion, during the peripartum period ePMN in the healthy postpartum uterus are highly dynamic in terms of counts, viability, and functionality compared to their circulating counterparts. (C) 2021 Published by Elsevier Inc

Quantitative and functional dynamics of circulating and endometrial polymorphonuclear leukocytes in healthy peripartum dairy cows

The aim of the present study was to assess the counts, viability, and functionality of circulating and endometrial polymorphonuclear leukocytes (PMN) isolated from fourteen clinically and metabolically healthy multiparous dairy cows in the peripartum period. For this, blood samples were collected at -5, +9, +21 and + 37 days (d) relative to calving. Cytology samples were collected from the vagina, cervix, and uterus at +9, +21 and + 37 d, using the cytobrush technique. Additional vaginal samples were collected at -5 d. Cytology smears were prepared and the PMN-to-all nucleated cell proportions (PMN%) were calculated. The endometrial cytobrush samples were also used for flow cytometric assessment of endometrial PMN (ePMN) viability and functionality. Functionality tests for circulating PMN (cPMN) included phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation. For ePMN, we evaluated PC only. The effect of day relative to calving on PMN viability and functionality were fitted in linear regression models, accounting for repeated measures. The endometrial PMN% were higher at +9 d (23.5 +/- 0.4%; least-squares means +/- standard error) and +21 d (8.5 +/- 0.3%) than at +37 d (1.4 +/- 0.3%). No changes in PMN% were found on either vaginal or cervical cytology along the peripartum period. The cPMN counts were higher pre- (6.2 +/- 0.4 x 10(6)/mL) than postpartum (4.9 +/- 0.4 x 10(6)/mL). Upon viability analysis, only the percentage of viable cPMN tended to be lower at -5 d (90.1 +/- 1.5%) than at +37 d (94.1 +/- 1.4%), and no other changes in the percentage of apoptotic and necrotic cPMN, nor in their functionality were found during the peripartum period. Analysis of ePMN viability showed that the percentage of viable ePMN did not change over time. In marked contrast, the percentage of apoptotic ePMN was higher at +9 d (37.8 +/- 5.1%) than at +21 d (20.9 +/- 5.1%) and +37 d (11.9 +/- 5.3%), while the percentage of necrotic ePMN was lower at +9 d (27.0 +/- 6.3%) than at +37 d (54.9 +/- 6.6%). The percentage of ePMN PC was higher at +9 d (27.5 +/- 3.4%) than at +37 d (13.3 +/- 4.9%). In conclusion, during the peripartum period ePMN in the healthy postpartum uterus are highly dynamic in terms of counts, viability, and functionality compared to their circulating counterparts. (C) 2021 Published by Elsevier Inc