Effects of parity and early pregnancy on peripheral blood leukocytes in dairy cattle.

Subfertility remains a major problem in the dairy industry. Only 35% to 40% of high-yielding dairy cows and 55% to 65% of nonlactating heifers become pregnant after their first service. The immune system plays a critical role in the establishment of pregnancy. However, it can also create challenges for embryo survival and contribute to reduced fertility. We conducted 2 separate experiments to characterize changes in subsets of peripheral blood leukocytes (PBL) and their phenotype over the estrous cycle and early pregnancy in heifers and cows. We used flow cytometry and reverse transcription-quantitative PCR to assess protein and mRNA expression of molecules important for immune function. We observed that CD14+ monocytes and CD3+ T cells tended to be affected by pregnancy status in heifers, whereas CD8B+ lymphocytes and NCR1+ natural killer cells were affected during early pregnancy in cows. Changes in expression of immune function proteins appeared to be greater in heifers than cows. To compare the most striking differences between heifers and cows observed in the initial experiments, we conducted a third experiment where PBL sampled from heifers and cows were simultaneously collected and analyzed under the same experimental conditions. Our results indicate that, compared with heifers, cows had greater mRNA expression of proinflammatory cytokines (IFN-γ and IL6) and AHR protein along with greater percentage of MM20A+ neutrophils and myeloid cells expressing SIRPA, ITGAM, and ITGAX. Moreover, animals that failed to become pregnant showed altered expression of anti-inflammatory molecules compared with cyclic and pregnant animals. Overall, these findings support the hypothesis that early pregnancy signaling alters the phenotype of immune cells in the peripheral blood and that differences in the peripheral immune response to pregnancy between cows and heifers exist. Because cows have lower conception rates than heifers, it is possible that a more proinflammatory immune status in peripheral blood may play a role in embryo loss.