Viability and function dynamics of circulating versus endometrial polymorphonuclear leukocytes in postpartum dairy cows with subclinical or clinical endometritis.

We aimed to compare the viability of circulating polymorphonuclear leukocyte (cPMN) and endometrial PMN (ePMN) and their function dynamics in postpartum dairy cows with subclinical (SCE) or clinical endometritis (CE). To do so, blood samples from 38 Holstein cows were collected at -7, 9, 21, and 36 d relative to calving, and endometrial cytology samples from 32 Holstein cows were harvested at 9, 21, and 36 d postpartum. Uterine health status was assessed at 36 d postpartum, and cows were classified as healthy (absence of abnormal vaginal discharge and ≤5% ePMN), SCE (absence of abnormal vaginal discharge and >5% ePMN), or CE (mucopurulent or purulent vaginal discharge and >5% ePMN). Viability (viable, apoptotic, and necrotic) and function parameters phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation were evaluated for cPMN via flow cytometry. For ePMN, only viability and PC were evaluated. The association of cPMN and ePMN viability and functional parameters with reproductive tract health classification were fitted in mixed linear regression models, accounting for repeated measures, sampling day, and interactions of reproductive tract status and day. Cows with CE had a lower proportion of cPMN viability (84.5 ± 2.1%; least squares means ± standard error) and a higher proportion of apoptosis (14.4 ± 2.0%) than healthy (92.4 ± 1.3 and 6.7 ± 1.3%, respectively) or SCE (95.3 ± 2.4 and 3.8 ± 2.3%, respectively) at 9 d postpartum. Interestingly, cPMN intracellular proteolytic degradation was lower [6.2 ± 0.1 median fluorescence intensity (MFI)] in SCE compared with healthy (6.7 ± 0.08 MFI) or CE (6.8 ± 0.1 MFI) at d 9 postpartum. No other differences in cPMN function were found among experimental groups. The proportion of necrotic ePMN was higher for healthy (49.6 ± 5.1%) than SCE (27.4 ± 7.3%) and CE (27.7 ± 7.3%) cows at 36 d postpartum. Also, at 36 d postpartum, the proportion of ePMN performing PC was higher in CE (47.0 ± 8.6%) than in healthy (18.4 ± 7.6%) cows, but did not differ from SCE cows (25.9 ± 8.7%). Results of the present study suggest that cPMN viability and function at 9 d postpartum are associated with the development of uterine disease. Furthermore, ePMN at 36 d postpartum are mostly necrotic in healthy cows but viable and functional in cows with CE, probably due to active uterine inflammation. Remarkably, ePMN in cows with SCE at 36 d postpartum are also mostly viable but seem to display a numerically lower proportion of PC compared with ePMN in CE cows.

Studying the pre-implantation uterine microbiota in cattle using transabdominal laparoscopic low-volume lavage: Aiming for zero-contamination.

Recent studies have suggested that bacteria associated with the female reproductive tract - the uterine microbiota - may be important for reproductive health and pregnancy success. Therefore, uterine microbiome research gained much interest in the last few years. However, it is challenging to study late postpartum uterine samples, since they hold a low microbial biomass. Next-generation sequencing techniques are very sensitive for microbial identification, but they cannot make a distinction between actual microbiota and contaminant bacteria or their DNA. Our aim was to test a new method to sample the bovine uterine lumen in vivo, while minimizing the risk of cross-contamination. In order to evaluate this method, we performed a descriptive assessment of the microbial composition of the obtained samples. Transabdominal, laparoscopic sampling of the uterine lumen was conducted in five Holstein-Friesian cows. Uterine fluid from the uterine horns was collected by low-volume lavage. DNA from the samples was extracted using two different DNA extraction methods, and negative controls (sampling blank controls and DNA extraction blank controls) were included. Bacteria were identified using 16S rRNA gene amplicon sequencing. In this proof-of-concept study, no evidence for authentically present uterine microbiota could be found. During laparoscopic sampling, some practical challenges were encountered, and the reliability of low-volume-lavage for the collection of a low microbial biomass could be questioned. By comparing two DNA extraction methods, a significant contamination background could be noticed originating from the DNA extraction kits.

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 106/mL) than postpartum (4.9 ± 0.4 x 106/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.

The Composition of the Microbiota in the Full-Term Fetal Gut and Amniotic Fluid: A Bovine Cesarean Section Study.

The development of a healthy intestinal immune system requires early microbial exposure. However, it remains unclear whether microbial exposure already begins at the prenatal stage. Analysis of such low microbial biomass environments are challenging due to contamination issues. The aims of the current study were to assess the bacterial load and characterize the bacterial composition of the amniotic fluid and meconium of full-term calves, leading to a better knowledge of prenatal bacterial seeding of the fetal intestine. Amniotic fluid and rectal meconium samples were collected during and immediately after elective cesarean section, performed in 25 Belgian Blue cow-calf couples. The samples were analyzed by qPCR, bacterial culture using GAM agar and 16S rRNA gene amplicon sequencing. To minimize the effects of contaminants, we included multiple technical controls and stringently filtered the 16S rRNA gene sequencing data to exclude putative contaminant sequences. The meconium samples contained a significantly higher amount of bacterial DNA than the negative controls and 5 of 24 samples contained culturable bacteria. In the amniotic fluid, the amount of bacterial DNA was not significantly different from the negative controls and all samples were culture negative. Bacterial sequences were identified in both sample types and were primarily of phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with some individual variation. We conclude that most calves encounter in utero maternal-fetal transmission of bacterial DNA, but the amount of bacterial DNA is low and viable bacteria are rare.

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.