Decomposing variation in ex vivo leukocyte responses and measures of metabolic status in multiparous holstein cows during the transition period

For the commercial dairy cow, the transition period, from dry off through 30 days in milk, represents a time of elevated metabolic and physiologic stress. This metabolic and physiologic stress is attributable to multiple sources, including the metabolic demands of lactation, the physiologic stress of parturition, and multiple regroupings and pen moves during the transition period. The stressors also contribute to generalized immunosuppression during the transition period. As a consequence of these stressors and this generalized immunosuppression, the transition period is characterized by increased incidences of metabolic diseases including ketosis, fatty liver, displaced abomasum, milk fever, and retained placenta, and infectious diseases such as mastitis and metritis. Disease incidence during the transition period, however, is highly variable both among and within dairy herds. A data simulation was conducted with the intent of illustrating the utility and robustness of some common effect size statistics and two studies were conducted with the aim of decomposing variation in ex vivo leukocyte responses and measures of metabolic status in multiparous Holstein cows during the transition period. In the first study, the effects of herd, parity, and the herd x parity interaction on ex vivo leukocyte responses and measures of metabolic status and hematology were investigated. Eight commercial dairy herds in western Texas and eastern New Mexico were included in the study. Peripheral blood samples were collected from 30 clinically healthy multiparous cows per herd. Cows were starting their 2nd, 3rd, or ≥ 4th lactation and were 2-8 days in milk. Leukocyte responses evaluated included neutrophil surface expression of L-selectin, neutrophil oxidative burst capacity when co-cultured with an environmental Escherichia coli, and the secretion of tumor necrosis factor-α and interferon-γ when diluted whole blood was co-cultured with lipopolysaccharide and phytohemagglutinin-P, respectively. Plasma concentrations of glucose, urea nitrogen, non-esterified fatty acids, β-hydroxybutyrate, and haptoglobin were measured. Total leukocyte count and differential were also measured. Effect size estimates for herd, herd x parity interaction, and residual variation were calculated as omega-squared and the intraclass correlation coefficient, and estimates for parity were calculated only as omega-squared. Effect size estimates were classified based on magnitude as either very small, small, moderate, or large based on previous recommendations. Generally, herd, parity, and herd x parity interaction explained a small or very small portion of the total variation in plasma metabolites and hematology, with the exception of plasma β-hydroxybutyrate concentration, where herd explained a moderate amount of the total variation. Parity and herd x parity interaction also only explained a small or vary small portion of total variation in the leukocyte responses that were evaluated. Contrastingly, herd explained a moderate or large portion of the total variation in most leukocyte responses, excepting neutrophil expression of L-selectin. Residual variation, representing variability among cows within herd x parity cohorts, was large for all response variables evaluated. These data suggest that leukocyte responses are more sensitive to herd-level factors than indicators of metabolic status or hematology. Large residual variation also suggests that a better understanding of what contributes to variation among cows in these cohorts is needed, and may lead to improvements in health of periparturient cows. In the second study, 24 multiparous Holstein cows from a single herd were sampled throughout the transition period in order to estimate effect sizes associated with variation between sampling days, variation between cows, and residual, within-cow variation for ex vivo leukocyte responses and measures of metabolic status. Peripheral blood was collected from cows at dry off (approximately -60 days in milk), move to close up pen (approximately -30 days in milk), calving, and 14 and 28 days in milk. Leukocyte responses evaluated included neutrophil surface expression of L-selectin, neutrophil oxidative burst capacity when co-cultured with an environmental Escherichia coli, and the secretion of tumor necrosis factor-α and interferon-γ when diluted whole blood was co-cultured with lipopolysaccharide and phytohemagglutinin-P, respectively. Plasma concentrations of glucose, urea nitrogen, non-esterified fatty acids, β-hydroxybutyrate, and haptoglobin were measured. Total leukocyte count and differential were also measured. Effect size estimates for between-cow variation and residual, and estimate of within-cow variation, were estimated as omega-squared and the intraclass correlation coefficient, while estimates for between-day variation were estimated only as omega-squared. Estimates were classified as described in the previous study. Effect size estimates for between-day variation were large for all responses evaluated with the exception of whole blood secretion of tumor necrosis factor-α and interferon-γ from stimulated cultures and lymphocyte count, suggesting that these responses are not as sensitive to the physiologic and metabolic stresses of the transition period as other responses evaluated in the study. Between-cow variation was large for whole blood secretion of tumor necrosis factor-α and interferon-γ from stimulated cultures, neutrophil oxidative burst capacity, and total leukocyte and lymphocyte counts, while between-cow variation was small for neutrophil L-selectin expression, neutrophil count, and ratio of neutrophils to lymphocytes. Effect size estimates for between-cow variation were moderate or large for plasma concentrations of non-esterified fatty acids, β-hydroxybutyrate, and urea nitrogen, and were small for plasma haptoglobin concentration. These data suggest that some leukocyte responses may be more largely influenced by genetic and epigenetic variation than others, resulting in larger between-cow variation. Residual variation, representing heterogeneity of response patterns among cows throughout the transition period, was large for all response variables measured. Further identification of factors that contribute to heterogeneity of responses during the transition period may allow for more effective management of cows during this time.