Milk Yield and Composition
According to the cut-off of SCC for diagnosis of mastitis, 33 cows with SCC ≥ 500,000 ml–1, 22 cows with 200,000 ≤ SCC < 500,000 ml–1, and 23 cows with SCC < 200,000 ml–1 were defined as high (HSCC), middle (MSCC), and low (LSCC), respectively. These classifications were not based on mastitic signs, but are reflective of mastitis risk.
Milk yield was lower (P < 0.01), but milk protein content was higher (P = 0.02) in cows with HSCC than in LSCC cows (Table 1). Content of milk urea nitrogen content was lower in the HSCC cows than in LSCC cows (P < 0.01). The average SCC was 77.6, 346.0, and 1460.5 103/ml–1 in the HSCC, MSCC, and LSCC cows, respectively.
Serum Biochemical Variables
During the whole experimental period, HSCC cows had higher serum concentrations ofβ-hydroxybutyric acid (BHBA) than LSCC cows (P < 0.01, Figure 1a), with an interaction effect (P < 0.01) of health status and week. Serum concentration of non-esterified fatty acid (NEFA) in HSCC cows was greater than in LSCC cows (P < 0.01, Figure 1b) at all the sampling weeks, except week 4. The MSCC cows had higher BHBA concentrations than the LSCC cows did at weeks –4, –3 and week 1 (P < 0.01).
Cows with HSCC had lower activity of superoxide dismutase (SOD) (P < 0.01, Figure 1c) and glutathione peroxidase (GSH-Px) (P < 0.01, Figure 1d) compared with LSCC cows during the whole period, except week –2. The HSCC cows had greater reactive oxygen species (ROS) (P < 0.01, Figure 1e) at weeks 1 and 2, and greater activity of malondialdehyde (MDA) (P < 0.01, Figure 1f) during the whole period, than LSCC cows. The effects of interaction between health status and week were not found (P < 0.01, Figure 1) on serum SOD, GSH-Px, ROS, and MDA.
Blood variables Relative to Health
Numbers of lymph (P < 0.01, Figure 2a) was lower in cows with HSCC than in LSCC cows, while the PMN, platelet, NLR and PLR were greater in HSCC cows than in LSCC cows (P < 0.05, Figure 2b, 2c, 2e, 2f) during the whole period. The cows with HSCC had a lower concentration of white blood cell (WBC) throughout the study (P = 0.01, Figure 2b) than LSCC cows. The NLR had an effect of interaction between health status and week (P = 0.03, Figure 2e). The MSCC cows had lower NLR (P < 0.05) at week 4 and lower lymph concentration (P < 0.01) at weeks –4 and –3 than the LSCC cows. At week –4, cows with MSCC had greater PLR than LSCC cows (P < 0.01, Figure 2f).
Serum Cytokines
Concentration of CD8 was lower in HSCC cows than in LSCC cows (P < 0.01, Figure 3a), with lower values in MSCC cows than in LSCC cows at week 1 (P = 0.03) and 2 (P = 0.01) postpartum. Serum CD4 concentration was lower in HSCC cows than in LSCC cows during the whole period except week –2 and 2 (P < 0.01, Figure 3b), with higher value at week 4 in MSCC cows than in LSCC cows (P < 0.01). The content of IL–6 (P < 0.01, Figure3c) was higher in the HSCC cows during the whole period. These cows also had higher TNF-α at week –4, –3, 1, 1, and 4 (P < 0.05, Figure 3d) than the LSCC cows. The content of IL–6 was higher (P < 0.05) in the MSCC cows than in LSCC cows at week –4, –3, 1, 1 and week 4, but the TNF-α was not different between the two groups (P > 0.05). The content of IL–10 was lower (P < 0.05, Figure 3e) in HSCC cows than in LSCC cows at week –3, –1, 1, 2, and 4. At week 2 and 4, the IL–10 content was lower (P = 0.03) in MSCC cows than in LSCC cows. Serum PSGL–1 content was higher (P < 0.01, Figure3f) in HSCC cows than in LSCC cows during the whole experimental period except at week –2 and 2, with higher value (P < 0.01) in the MSCC cows than in LSCC cows from week –4 to 4 relative to calving.
Correlation Analysis
Correlations between serum BHBA or NEFA levels and milk SCC are shown in Figure 4. Overall, milk SCC were positively correlated with pre-partum serum BHBA (r = 0.51, P < 0.01, Figure 4a) and serum NEFA (r = 0.45, P < 0.01, Figure 4b), respectively. Serum BHBA concentration was positively correlated to SCC, with coefficients of 0.65 (week –4, Figure S1a), 0.73 (week –3, Figure S1b) and 0.72 (week –1, Figure S1c). The coefficients of correlation between serum NEFA and SCC in cows overall were 0.75 (week –4, Figure S2a), 0.72 (week –3, Figure S2b), and 0.75 (week –1, Figure S2c). Furthermore, serum NEFA content at week –4 and SCC exhibited a stronger correlation compared to other weeks.
Overall, milk SCC were positively associated with the NLR (r = 0.73, P < 0.01, Figure 5a) and PLR (r = 0.74, P < 0.01, Figure 5b) before calving. Milk SCC had positive correlations with NLR (P < 0.01) at week –4 (r = 0.81, Figure S3a), –3 (0.79, Figure S3b), and –1 (r = 0.74, Figure S3c), and with the PLR (P < 0.01) at week –4 (r = 0.77, Figure S4a), –3 (r = 0.74, Figure S4b), and –1 (r = 0.72, Figure S4c).
The receiver-operator characteristic (ROC) curve of NLR and PLR showed that the area under the curve (AUC) of the NLR in HSCC cows were 0.880 (week –4, Figure S5a), 0.853 (week –3, Figure S5b), and 0.883 (week –1, Figure S5c), and the AUC of the PLR were 0.848 (week –4, Figure S5a), 0.733 (week –3, Figure S5b), and 0.765 (week –1, Figure S5c). The AUC of the NLR in the MSCC cows were 0.765 (week –4, Figure S6a), 0.625 (week –3, Figure S6b), and 0.740 (week –1, Figure S6c), and the AUC of PLR were 0.850 (week –4, Figure S6a), 0.583 (week –3, Figure S6b), and 0.635 (week –1, Figure S6c). The ROC curve analysis results for BHBA, NEFA (Figure S7, S8), SOD, GSH-Px, ROS, MDA (Figure S9, S10) in HSCC cows and LSCC cows and in MSCC and LSCC cows are shown in the supplementary figures.