Flow cytometric evaluation of leukocyte subpopulations in dairy cows with subacute ruminal acidosis (SARA) under continuous reticulorumen pH monitoring

Subacute Ruminal Acidosis (SARA) is common in periparturient dairy cows. While alterations in innate immunity have been described, the impact on adaptive immunity remains poorly characterised. This study aims to evaluate peripheral leukocyte subsets in cows classified based on acidosis events. Fifty-four periparturient dairy cows were equipped with indwelling reticulorumen pH sensors for continuous monitoring. K3EDTA-anticoagulated blood samples were collected at four time points (T0 −5 days (d), T1 +5 d, T2 +30 d, T3 +60 d relative to parturition). Peripheral leukocyte subsets were analysed by flow cytometry. Cows were classified as controls (no acidosis events), SARA6, SARA5.8, or SARA5.5 (pH <6.0, <5.8 and <5.5 for ≥300 min, respectively); controls and SARA6 were considered healthy, and SARA5.8–5.5 were categorised as diseased. Data were analysed using a linear mixed-effects model with time, acidosis category (healthy vs. diseased), and their interaction included as fixed effects; post-hoc comparisons used estimated marginal means (p<0.05). P-values were adjusted using the Benjamini–Hochberg False Discovery Rate method. A significant time effect was observed for some leukocyte subsets (P<0.001): γδ T- lymphocytes (WC1⁺CD4⁻) increased between T2 and T3 (p<0.001), T-helper lymphocytes decreased between T0 and T1 (p=0.041) and increased between T2 and T3 (p<0.001). Monocytes decreased between T0 and T1 (p=0.015). No effects of acidosis category or time × category interaction were observed. Changes in peripheral leukocyte subsets were associated with time but not with SARA classification, suggesting that periparturient physiological changes rather than SARA may drive the observed immune variations.