Prism ver 8

The pH data were summarized 1 day before the sample collection to minimize the influence of opening the cannula stopper on the ruminal pH. The normality of the distributions of variables was assessed using the Shapiro–Wilk test. Significant differences in ruminal pH, duration of time where pH was <5.6 and 5.8, VFA components, lactic acid concentration, LPS activity, and peripheral LBP concentration among the early, middle, and late stages were evaluated using paired t-tests for normally distributed variables and the Wilcoxon rank-sum test for non-normal variables (Prism ver. 8.10; GraphPad Software, La Jolla, CA, USA). The microarray data were analyzed using paired Student’s t-tests with the Benjamini–Hochberg FDR multiple testing correction (FDR corrected P < 0.05) and summarized using GeneSpring 12.0 (Agilent Technologies). Fold changes were calculated by comparing the middle and early (first period), late and middle (second period), and late and early (total period) stages. The PCA plot coordinates were calculated using the devtools package with microarray data in R ver. 3.3.2 (http://www.r-project.org; R Foundation for Statistical Computing, Vienna, Austria). Significant differences were determined with a threshold of P < 0.05, while trends that suggested possible significance were identified at 0.05 < P < 0.10.

Statistical analysis was conducted using commercial software (Prism ver. 8, GraphPad, San Diego, CA, USA). The hemoglobin concentration data, optical pathlengths, SO₂, and age were analyzed by one-way ANOVA with post hoc Tukey’s multiple comparison test. The effect size and the 95% confidence interval (CI) of difference were also used to confirm the findings. The gender ratio was checked by the chi-squared test. The correlation between the NIR-TRS data and the medication dosage was examined using Spearman correlation coefficients.

All statistical analyses were performed using Prism ver.8 (GraphPad, La Jolla, CA, USA). We used parametric tests (Fig 2A–2D, Fig 3B and 3C, and S1C Fig) and nonparametric tests (Fig 4A, 4B and 4C and S3C and S3D Fig). See S1 Data for more details (for example, exact sample sizes and P-value).