1. Introduction
The role of leukocytes in the defense of the mammary gland is well known [
1,
2], and differential leukocyte count (DSCC) is considered important information on udder immune response and to improve mastitis diagnosis [
3,
4,
5,
6]. Indeed, somatic cell counts (SCC), the tool currently applied for this aim, is not able to completely describe the defense mechanisms within the udder [
7]. Until recently, investigations on milk DSCC were limited by the available investigation techniques: direct microscopical and flow-cytometry analysis. Both these techniques have poor reproducibility, high costs, and are labor-intensive, thus limiting their application to studies with a relatively small sample size. These technical aspects and small sample sizes, in addition to different designs of experiments and sampling procedures could explain why there are very large and overlapping ranges among studies on leukocyte proportions [
4,
7,
8,
9,
10,
11]. Moreover, the problems previously mentioned reduce the chance to analyze DSCC in cows with different physiological, pathological, and lactation stages. Indeed, SCC vary in relation to parity, days in milk (DIM), health, and welfare status [
12], and these factors very likely have an influence on DSCC.
The recent availability of a high-throughput tool to perform DSCC opens the way to explore these factors in an economically sustainable way, allowing proper sample sizes and compositions [
13]. This new tool allows to identify within a milk sample the macrophages (MAC) and the combination of polymorphonuclear neutrophils (PMN) and lymphocytes (LYM). Diagnostic characteristics and performances were described by Damm et al., 2017 [
13]. DSCC is expressed as the combined proportion (%) of PMN and LYM on the overall count of milk cells.
To the best of our knowledge, studies on leukocyte patterns throughout lactation and parities are scarce and the few available are focused on specific aspects, such as the association with SCC or the values at the end of lactation [
14,
15,
16,
17]. Moreover, studies on the variability of the total amount of different leukocytes in milk are scarcer [
4,
18], and based on a very small sample size. We hypothesized that DSCC alone do not fully describe the pattern of leukocytes in milk.
This study was designed to describe DSCC and total amount of different cells in milk in cows during the course of lactation with different parity and levels of SCC.
4. Discussion
In this study, herd selection was not random, constrained by the necessity to have DSCC data over at least a 12 months-period; however, herd size, milk yield, and SCC are similar to the ones observed in previous larger studies on the same area [
16,
24,
26].
As expected, primiparous cows were the prevalent group among parities (40%), as well as 73% samples were with ≤200,000 cells/mL. Samples with ≤50,000 cells/mL had a frequency of 39%, confirming the high prevalence of cows with very low SCC [
16,
26].
To the best of our knowledge, this is the first time that the total number of cells was considered to analyze leukocytes pattern in milk. Our data showed that the milk collected during a whole milking contains an average of 1 × 10
9 PMN and LYM. Cows in HS have a P + LT average between 5.0 × 10
8 and 3.0 × 10
9 cells (
Table 3), while in the IS group, the values were in the range of 1.6 × 10
10 and 2.5 × 10
10 cells (
Table 6).
Despite the applied technique not being able to differentiate PMN from LYM, previous data suggested that PMN is the predominant cell type when SCC is largely below ≤100,000 cells/mL [
27], and that LYM is in the range of 1%–19%, independent of the SCC of the samples [
13]. Therefore, the changes in DSCC and in P+LT in relation to SCC levels should high likely be related to variation in PMN concentrations.
The presence of a well-defined inflammatory process (samples in IS subsets) increased the overall amount of PMN and LYM of 1 log, from 1 × 109 to 1 × 1010 or, in other words, of about 1 billion cells, most of which are presumably PMN. This change is the result mainly of overall increase in SCC, rather than changes in DSCC proportions. Indeed, in the latter case, the increase is in the range of 15–25%, when DSCC proportions were compared in samples with the lowest SCC (≤50,000 cells/mL) and the highest SCC (>800,000 cells/mL). The assessment of total amount of PMN and LYM, to our knowledge, has never been reported in scientific literature; the value reported may be proposed as a benchmark for studies on immune response in the mammary gland.
The availability of a sample size larger than the ones reported even in recent studies on DSCC [
14,
15,
16,
24,
28] and the period of time during which samples were collected allows to investigate the pattern of cell types during the course of the whole lactation. The statistical model applied for evaluating factors affecting DSCC variability in a healthy subset showed that, among random factors, the herd has a very low influence, while all fixed factors considered, as well as their interactions showed a statistically significant influence (
Table 2). These results were consistent among the four different SCC thresholds considered, similar to what was observed in studies on SCC [
12]. The increase in SCC levels were associated with an increase in DSCC as expected, but our data showed a decrease of DSCC values as parity increased (
Table 4). The trend analysis showed a decrease of DSCC with increasing DIM when SCC was ≤50,000 cells/mL, but no significant trends were observed at higher SCC levels (
Table 7). These data did not agree with the results of studies applying flow-cytometry [
29], neither with the ones from a recent study applying the same instrument as in this study [
14]. However, in the latter case, data were not ranked by SCC levels.
When P + LT were considered, a different scenario appears. Indeed, cows in first and second lactation have a significant lower amount of PMN and LYM, when compared to cows in third and higher lactation (
Table 3). However, these differences are numerically not very large (7%), and they suggest that, in healthy animals, the number of immune cells is kept as constant as possible, as previously suggested [
30], and a reference value of 1.4 × 10
9 for P + LT in healthy cows can be proposed. Therefore, primiparous cows cannot be considered to have a different level of cell immunity compared to older cows, as suggested by the higher DSCC values observed. Indeed, the DSCC peak observed at the beginning of lactation in cows smoothen when P + LT is considered (
Figure 1 and
Figure 2). Very likely, the higher proportion of PMN and LYM are needed to compensate both the lower SCC levels and yield, generally observed in primiparous cows, in order to keep PMN and LYM at the proper level. These data also confirm that cows with very low SCC are not at greater risk of developing mastitis [
31], since there are not large differences in total milk PMN + LYM, when compared to older cows.
Often dilution or concentration effects are suggested as explanation of abnormal changes in SCC in healthy cows [
32]. The data of our study did not support this hypothesis in healthy cows. Indeed, trends are generally positive for P + LT as DIM increases, suggesting that the overall amount of PMN and LYM released into milk increases when milk yield generally decreases, irrespective of the number of lactations. These increases may be related to the prolonged exposure to bacteria during lactation, thus stimulating epithelial cells, which release pro-inflammatory mediators [
33,
34] and increasing the recruitment of leukocytes from blood circulation.
The same analysis applied to the cows with >200,000 cells/mL (IS) showed that the role of herd, as random factor, has proportionally a relatively higher influence on DSCC and P + LT variance, in comparison with HS (
Table 2). These data suggest that the level of inflammation, herd management and, very likely, bacteria prevalence play an important role in modulating cellular immune response in diseased animals. These observations are supported by the absence of an influence from the interaction between SCC and parity or DIM.
The overall mean values for both DSCC and P + LT in IS are largely higher than the values in HS (
Table 6 and
Figure 3 and
Figure 4). An increase in DSCC with rise in SCC was also observed in this group, but the influence of parity on DSCC was less consistent. Significant differences were observed, but without a clear trend, while P + LT showed patterns similar to the ones observed in HS with a large increase in cell concentration from third parity and over (
Table 6).
The analysis of trends based on DIM showed that both DSCC and P + LT show a significant negative trend (
Table 7), with a few exceptions for DSCC (primiparous cows and samples >800,000 cells/mL). These data suggest that in this group, the presence of high SCC as lactation proceeds is associated with a progressive increase in the number of macrophages. An increase in the proportion of macrophages in late lactations have been reported in previous studies on quarter milk samples [
17,
35], but these studies involved both healthy cows and with subclinical mastitis. In our study, reduction in the proportion and correspondent increase in overall number of macrophages was observed only in cows with a clear inflammatory process; these results may be related to the increase frequency of chronic mastitis in late lactation [
36,
37].