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

Abstract

Subfertility remains a major problem in the dairy industry. Only 35-40% of high-yielding dairy cows and 55-65% 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 RT-qPCR to assess protein and mRNA expression of molecules important for immune function. We observed that monocytes and T cells were most affected by pregnancy status in heifers, whereas, CD8⁺ lymphocytes and natural killer (NK) cells were most affected during early pregnancy in cows. Changes in immune parameters measured appeared to be greater in heifers than cows including changes in expression of numerous immune function molecules. To test the hypothesis, we conducted a third experiment to simultaneously analyze the immunological responses to pregnancy between cows and heifers. We observed that cows had greater expression of proinflammatory cytokines and molecules associated with leukocyte migration and phagocytosis compared to heifers. Moreover, animals that failed to become pregnant showed altered expression of anti-inflammatory molecules. Overall, these findings support the hypothesis that early pregnancy signaling alters the proportions and functions of peripheral blood immune cells and differences between cows and heifers may yield insight into the reduced fertility of mature lactating dairy cows.

Keywords: bovine; cytokines; immune; leukocytes; pregnancy.

Figure 1:. Percentage of leukocytes and protein expression of immunoregulatory molecules in heifers from experiment 1.

Changes in the percentage of (A) monocytes and (B) total T cells in cyclic (white triangle, N = 7) versus pregnant (black triangle, N = 7) animals. (C) Changes in percentage of leukocyte subsets over days of early pregnancy. Protein expression/cell of (D) CD8B, (E) CR2, (F) NCR1, (G) CD14, (H) MM20A, (I) CD47, and (J) SIRPA in cyclic compared to pregnant animals. (K) Protein expression/cell of immunoregulatory molecules over days of pregnancy.

Figure 2:. Percentage of leukocytes and protein expression of immunoregulatory molecules in cows from experiment 2.

Percentage of (A) CD8B+ T cells and (B) NK cells in cyclic (white circle, N = 6) versus pregnant (black circle, N = 8) animals. (C) Changes in percentage of leukocyte subsets over days of early pregnancy. Protein expression/cell of (D) MM20A and (E) CD47. (F) Protein expression/cell of immunoregulatory molecules over days of pregnancy.

Figure 3:. Plasma progesterone (P4) in cyclic, pregnant, and bred-nonpregnant (A–C) heifers and (D–F) cows from experiment 3.

Note that 7 of 10 heifers showed low P4 or regressing/regressed corpus luteum prior day 18 of estrous cycle.

Figure 4:. Percentage of leukocytes and immunoregulatory molecules differentially expressed between heifers and cows from experiment 3.

Changes in percentage of (A) neutrophils, (B) SIRPA⁺ cells, (C) ITGAM⁺ cells, and (D) ITGAX⁺ cells. mRNA fold change of (E) CD3E, (F) IFNG, and (G) IL6. Protein expression/cell of (H) AHR and mRNA expression of (I) AHR and its target gene (J) CYP1A2. The mRNA fold change was calculated relative to cyclic heifers. When parity was a significant source of variation, the effect of status was assessed within parity using values of mRNA fold change relative to their respective cyclic animals. Legends: day 18 of estrous cyclic (D18C); day 18 of pregnancy (D18P); day 18 of bred-nonpregnant (D18BNP); a, b when p < 0.05 and x, y when 0.5 > p < 0.10 for effect of status.

Figure 5:. Percentage of leukocytes and immunoregulatory molecules differentially expressed in bred-nonpregnant animals from experiment 3.

mRNA fold change of (A) IL10, and (B) IL Percentage of (C) PPARG⁺ cells and (D) protein abundance/cell and (E) mRNA expression of PPARG and its target gene (F) FABP Proportion of (G) IDO1⁺ cells and (H) protein abundance/cell and (I) mRNA expression of IDO1. The mRNA fold change was calculated relative to cyclic heifers. When parity was a significant source of variation, the effect of status was assessed within parity using values of mRNA fold change relative to their respective cyclic animals. Legends: day 18 of estrous cyclic (D18C); day 18 of pregnancy (D18P); day 18 of bred-nonpregnant (D18BNP); a, b when p < 0.05 and x, y when 0.5 > p < 0.10 for effect of status.