Fig 1.
Experimental design of the clinical trial.
Postpartum dairy cows (19–23 DIM) were randomly assigned to receive either pegbovigrastim (rG-CSF; recombinant granulocyte colony-stimulating factor) or saline (Control). Cows were examined and sampled immediately prior to treatment (Day 0) and subsequently on Days 3, 6, 10, and 21 post-treatment. Uterine cytobrush samples from a subset of cows were collected on Days 0, 3, 6, 10, and 21. Clinical examinations, including vaginoscopy to assess vaginal discharge and transrectal ultrasonography to detect intrauterine fluid accumulation, were performed to diagnose clinical endometritis.
Fig 2.
Temporal changes in total leukocyte (A), neutrophil (B), and monocyte (C) counts (Mean ± SE) in the peripheral blood of pegbovigrastim (rG-CSF, recombinant granulocyte colony-stimulating factor, n = 12) and saline-treated (Control, n = 11) postpartum cows.
Blood samples were collected from postpartum cows (19-23 DIM) just prior to treatment (Day 0) and on Days 3, 6, 10, and 21 post-treatment. Significant difference (P < 0.05) between the groups on the respective sampling days are indicated by asterisks (*).
Fig 3.
Effect of parenteral administration of pegbovigrastim on granulocyte phagocytic activity.
(A) Forward (FSC) and side scatter (SSC) properties in combination with CH138A–allophycocyanin labeling (APC) were used to identify the granulocyte population; these were further gated FITC-labeled S. aureus internalization. (B) Comparison of granulocyte phagocytic activity between treatment groups on different days post-treatment, expressed as the proportion (%) of granulocytes positive for internalized FITC-labeled Staphylococcus aureus. Postpartum cows were treated with pegbovigrastim (rG-CSF, recombinant granulocyte colony-stimulating factor; n = 12) or saline (Control; n = 11). Blood samples were collected at 19–23 DIM just prior to treatment (Day 0) and on Days 3, 6, 10, and 21 post-treatment. Significant differences between treatment groups on specified sampling days are indicated by P < 0.05 in panel B.
Fig 4.
Effect of parenteral administration of pegbovigrastim on granulocyte phagocytic capacity.
(A) Forward scatter (FSC) and side scatter (SSC) properties in combination with CH138A–allophycocyanin (APC) labeling were used to identify granulocyte population; these were further gated FITC-labeled S. aureus internalization and assessed for fluorescent intensity. (B) Comparison of granulocyte phagocytic capacity between groups on different days post-treatment, expressed as the median fluorescent intensity (MFI) of the FITC signal. This measure reflects the average number of internalized FITC-labeled S. aureus per cell. Postpartum cows were treated with pegbovigrastim (rG-CSF, recombinant granulocyte colony-stimulating factor, n = 12), or saline (Control, n = 11). Blood samples were collected from postpartum cows (19-23 DIM) just prior to treatment (Day 0), and then on Days 3, 6, 10, and 21 post-treatment. Significant differences between treatment groups on specified sampling days are indicated by P < 0.05 in panel B.
Fig 5.
Effect of parenteral administration of pegbovigrastim on oxidative burst capacity of peripheral granulocytes.
(A) Representative flow cytometry plots show cell selection based on characteristic forward scatter (FSC) and side scatter (SSC) properties in combination with CH138A–allophycocyanin labeling to identify target cell population. (B) Comparison of granulocyte oxidative burst capacity between groups on different days post-treatment, expressed as the median fluorescent intensity (MFI) of rhodamine 123 signal. Postpartum cows were treated with pegbovigrastim (rG-CSF, recombinant granulocyte colony-stimulating factor, n = 12), or saline (Control, n = 11). Significant differences between treatment groups on specified sampling days are indicated by P < 0.05 in panel B.
Fig 6.
Proportions (Mean ± SE) of neutrophils (A), macrophages (B) and epithelial cells (C) in the uterus of pegbovigrastim (rG-CSF, n = 8) and saline-treated (Control, n = 7) postpartum cows.
Uterine cytobrush samples were collected from postpartum cows (19-23 DIM) and cytosmears were prepared just prior to treatment (Day 0), and then from a subset of cows on Days 3, 6, 10, and 21 post-treatment. One hundred cells were counted in each quadrant of a cytosmear. Significant differences (P < 0.05) between groups on specified sampling days are indicated by asterisks (*) in the respective panels.