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Both ruminal microbial structure and functionality might play a role in inter-individual variation in susceptibility for subacute rumen acidosis (SARA) observed in dairy cows. The aims of this study were to determine whether differences between cows with distinct SARA susceptibility were reflected in distinct (1) ruminal microbial communities, (2) salivary bacterial communities, and (3) fermentative capacity of ruminal microbiota assessed in vitro. To test this hypothesis, rumen samples were collected via an esophageal tube on 21 d postpartum from 38 multiparous Holstein cows, which were classified into 4 groups differing in median and mean time of reticular pH below 6 as well as area under the curve of pH below 6.0. During the 21 d postpartum, all cows within a group fulfilled following criteria: susceptible (S, n = 10; mean or median ≥180 min/d), moderately susceptible (MS, n = 7; 60 min/d < mean time of pH below 6 < 180 min/d, and median time of pH below 6 <180 min/d), moderately unsusceptible (MU, n = 11; 10 min/d < mean < 60 min/d, and median time of pH below 6 ≤30 min/d), or unsusceptible (U, n = 10; median = 0 min/d, and mean <10 min/d). Groups did not differ in total daily dry matter intake nor in total, roughage, or concentrate intake during daily 6-h time intervals. Rumen bacterial α-diversity did not differ among groups, but β-diversity varied and bacterial 16S rRNA gene copy numbers were lower in S compared with U cows. The relative abundance of genera Streptococcus, Sharpea, Prevotellaceae_YAB2003, Succinivibrionaceae_UCG-001, Ruminococcus, and Ruminococcaceae_UCG-001 were higher in S compared with U cows. In contrast, Lachnospiraceae_ND3007 and Oscillospiraceae_V9D2013 were more abundant in U cows. Although pH-associated, inter-animal differences were also observed in the salivary bacteria, common differences in ruminal and salivary bacterial genera were limited. The functionality of the rumen microbiota was evaluated in vitro through exposure of the microbial inoculum of S and U cows to an anaerobic buffer at pH 5.8 and 6.8, in the presence of sterile supernatant of their own and of dry cows' rumen fluid (2 × 2 design). Generally, the S inoculum produced more volatile fatty acids, except at low pH with dry cows' supernatant, where volatile fatty acid production was completely impaired and lactate accumulation was highest. Compared with the microbes of U cows, microbes of S cows showed less fermentative activity in situations with 2 stress factors (low pH and an unfamiliar environment, i.e., rumen fluid supernatant of dry cows).

The measurement of pH in the reticulorumen in combination with a time-pH threshold has been widely applied in research to diagnose subacute ruminal acidosis. However, other pH metrics also have biological value. In this study, 44 animals were monitored during the transition period using reticuloruminal pH boluses. Traditional and more complex pH characteristics were calculated to characterize the reticuloruminal pH profile: time pH <6, slope of a logistic cumulative pH curve (β 0 ), and deviations [squared error (SqEr)] from pH predictions based on a harmonic static model. In this study, we aimed to examine the associations between those pH metrics and metabolic health parameters, feed intake, and activity. Finally, to describe the reticuloruminal pH dynamically, we also constructed a dynamic linear model. The results of this model were studied in relation to feed intake. All pH parameters were mutually correlated (particularly β 0 and SqEr; mean Pearson correlation of −0.52). pH patterns, rather than time pH <6, were associated with metabolic health and feed intake: high variation in daily pH (β 0 parameter) was reflected in higher blood concentrations of nonesterified fatty acids. Moreover, pH deviations of the harmonic model were negatively associated with feed intake and rumination behavior. This research confirms the biological importance of pH metrics focusing on pH variation and pH deviations and provides deeper insight into its associations with metabolic health status, feed intake, and activity during early lactation.

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.

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